Compositions and methods for chimeric antigen receptors specific for the B cell receptor

Abstract

The present invention relates to compositions and methods for treating or preventing hematological cancers or autoimmune diseases in mammals using anti-BCR CARs. One aspect includes modified T cells and pharmaceutical compositions comprising the modified cells for adoptive cell therapy and for treating cancers or autoimmune diseases associated with B cells that contain enriched stereotypic BCRs. TIFF2024532851000044.tif108170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 234,514, filed August 18, 2021, which is incorporated by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (046483-7330WO1-02992 Sequence Listing.xml; size: 108,648 bytes; and creation date: August 17, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] 2. Background of the Invention Malignant or pathogenic B cell clones in lymphomas, leukemias, myelomas, or other hematological cancers can be identified based on the expression of specific, recurrent, or enriched stereotypic B cell receptors (BCRs) that may be involved in the pathogenesis of B cell malignancies. In addition, some autoimmune disorders are characterized by the enrichment of specific B cell receptors that are believed to be involved in the pathogenesis of autoimmune diseases. Existing therapies that target B cells associated with cancer or autoimmune diseases have drawbacks because they cannot specifically target pathogenic clones, but rather deplete the entire B cell compartment. For example, chimeric antigen receptors (CARs) directed against CD19 target all patient B cells, often resulting in B cell aplasia and patients requiring long-term IgG infusions to prevent recurrent infections. Furthermore, without B cells, patients do not respond well to vaccines.

[0004] There remains an unmet need for effective therapies that target pathogenic B cell clones associated with hematological cancers or autoimmune diseases, while sparing normal B cells or plasma cells. Summary of the Invention

[0005] [Brief description of the drawings]

[0006] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which: For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, it being understood, however, that the invention is not limited to the exact arrangements and instrumentalities of the embodiments shown in the drawings.

[0007] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0008] [Figure 1] Figure 1 shows the percentage of patients with various hematological malignancies that have B cell receptors containing immunoglobulin heavy region (IGHV) 4-34. In patients affected by primary vitreoretinal lymphoma (PVRL), over 60% of these patients have this specific BCR, whereas in primary central nervous system lymphoma (PCNSL), activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and hairy cell leukemia variant (HCLv), approximately 30-40% of patients have this specific BCR in their malignant tumor cells. In contrast, BCRs containing IGHV4-34 are found in only a small number of normally healthy B cells (<5%) (Belhouaci et al, Blood Advances, 2020). PVRL: primary vitreoretinal lymphoma; PCNSL: primary central nervous system lymphoma; DLBCL-ABC: diffuse large B-cell lymphoma-activated B-cell-like; DLBCL-GCB: diffuse large B-cell lymphoma-germinal center-like; BL: Burkitt's lymphoma; CLL: chronic lymphocytic leukemia; MCL: mantle cell lymphoma; SMZL: splenic marginal zone lymphoma; OAMZL: ocular adnexal marginal zone lymphoma; CBL-MZ: clonal B-cell lymphocytosis of marginal zone origin; HCLc: hairy cell leukemia-classical; HCLv: hairy cell leukemia-variant. [Figure 2A]Figure 2A shows the enrichment of specific IGHVs in a large cohort of chronic lymphocytic leukemia (CLL) patients. Red bars indicate enrichment of the following IGHV regions in 5–12% of CLL patients, respectively: IGHV1-69, IGHV3-23, and IGHV4-34. [Figure 2B] Figure 2B shows that approximately 17–18% of patients in a separate CLL cohort harbor IGLV3-21 BCRs containing mutations at the R110 site (Maity et al, PNAS 2020; Muggen et al, Immunity & Ageing 2019). [Diagram 3] Figure 3 is a schematic diagram showing the development of CARs to target enriched stereotype BCRs. Chimeric antigen receptor T cells against enriched stereotype BCRs specifically kill tumor clones while sparing normal B cells; thus eliminating B cell aplasia, eliminating the need for intravenous (IV) immunoglobulin for survival, and reducing opportunistic infections. Furthermore, signaling downstream of BCRs is known to drive tumor cell survival and proliferation. Thus, unlike targeting CD19, which is not required for tumor B cell survival and proliferation, targeting enriched stereotype BCRs also means targeting the "Achilles heel" of leukemia / lymphoma cells (L / L), i.e., the BCR they require to survive. Thus, this strategy may have the potential to reduce antigen-negative escape (e.g., CD19-negative relapse). IV: intravenous; L / L: leukemia / lymphoma cells; StBCR: stereotype / enriched BCR. [Figure 4]Figure 4 is a schematic diagram showing the development of CAR T cells to specifically target tumor B cell clones. This strategy disclosed herein has several advantages over CD19-directed CAR T cell therapy, including: (i) CD19-directed CAR T cell therapy results in the death of all mature B cells (B cell aplasia), but by targeting only B cells with disease-specific "stereotyped" or "enriched" BCRs, tumor B cell clones are eliminated while all other polyclonal B cells with diverse BCRs are preserved. These polyclonal B cells can then actively proliferate in patients after anti-BCR CAR T cell infusion and protect patients from life-threatening opportunistic infections; and (ii) CD19-directed CAR T cell therapy initially kills tumor B cells with CD19, but CD19 is not a molecule required for tumor B cell survival or proliferation, so CD19-negative tumor cells generally develop even in the presence of CD19-directed CAR T cells. In contrast, given the critical role of the BCR in promoting B cell signaling, survival, and proliferation, it is believed that BCR-negative tumor B cells are unlikely to be abundant. BCR: B cell receptor. [Figure 5-1] Figure 5 is a schematic diagram showing an exemplary embodiment of a CAR construct. The CAR disclosed herein comprises a single chain variable fragment (scFv) derived from an antibody that recognizes a specific immunoglobulin heavy region (IGHV). This portion of the CAR is responsible for directing the CAR T cell to pathogenic B cells expressing a specific BCR. Each construct was cloned with antigen binding domains (scFv) in both light to heavy (L2H) and heavy to light (H2L) configurations. The various scFvs that recognize specific BCRs were paired with the 41BB-CD3z intracellular signaling domain that has been used previously in anti-CD19 CARs. scFv: single chain variable fragment. 41BB: intracellular domain of 41BB. [Figure 5-2] See description of Figure 5-1. [Figure 6-1]Figure 6 is a schematic diagram showing the approach disclosed herein to develop tumor B cell lines expressing stereotypic BCRs. As a first step, endogenous immunoglobulin heavy (IGH) and light (IGL) chains were knocked out using CRISPR / Cas9, followed by lentiviral transduction with immunoglobulin heavy and light chains (IGLV3-21 R110; IGHV1-69; IGHV4-34; IGHV3-23) encoding stereotypic or enriched BCRs. The sequences encoded by these BCRs were derived from patients with hematological malignancies. CRISPR: clustered regularly interspaced short palindromic repeats; LV: lentivirus; IgM C: IgM constant region; IgG1 C: IgG1 constant region. [Figure 6-2] See description of Figure 6-1. [Figure 7A-1] Figures 7A-7B relate to anti-BCR CART production for CAR T cells with various CARs capable of recognizing stereotypic or enriched BCRs. Figure 7A shows (i) total T cell counts after stimulation with CD3 / CD28 coated magnetic beads; cell volume of T cells after stimulation (which shows an initial expansion followed by a period of contraction in cell size); and population doubling of T cells after stimulation. Figure 7B shows successful expression of anti-BCR CARs at day 8 after stimulation. AVA=anti-3-21 IGLV; hG6.3=anti-1-69 IGHV; 9G4=anti-IGHV4-34; 3C9=anti-3-23 IGLV. [Figure 7A-2] See legend to Figure 7A-1. [Figure 7A-3] See legend to Figure 7A-1. [Figure 7B-1] See legend to Figure 7A-1. [Figure 7B-2] See legend to Figure 7A-1. [Figure 7B-3] See legend to Figure 7A-1. [Figure 8A-1]Figures 8A-8B show an in vitro assay for the efficacy of anti-BCR CAR T cells. Figure 8A shows that culturing tumor B cells with WT BCR, tumor B cells with stereotypic BCR, or tumor B cells depleted for CD19 with CAR T cells specific for either CD19, VL3-21-R110, VH1-69, or VH4-34 results in a specific reduction in the number of viable tumor cells in the expected conditions: CART19 T cells result in depletion of all tumor B cells except those that are CD19-KO; CART3-21 results in depletion of Jeko1 VL3-21* but not other tumor B cells; CART1-69 results in depletion of Jeko1 VH1-69 but not other tumor B cells; CART4-34 results in depletion of Jeko1 VH4-34 but not other tumor B cells. These data clearly demonstrate the specificity of the anti-BCR CAR T cells of the present invention. Figure 8B shows the proliferation of different CAR T cells after 6 days of culture with different tumor B cells. These results demonstrate that CAR T cells respond and proliferate specifically when they encounter an antigen (CD19 or stereotyped / enriched BCR) that they can recognize via the CAR they express. [Figure 8A-2] See legend to Figure 8A-1. [Figure 8B-1] See legend to Figure 8A-1. [Figure 8B-2] See legend to Figure 8A-1. [Figure 9]Figure 9 shows the specific anti-tumor effect of the CAR T cells of the present invention against tumor cells from patients. In this experiment, B cells from a healthy patient, or tumor B cells from a CLL patient expressing IGHV1-69+BCR, or tumor B cells from a CLL patient expressing IGHV4-34+BCR were co-cultured with CART19, or CART1-69 or CART4-34. The data show that, as expected, CART19 exhibits strong cytotoxicity against all types of B cells, including healthy B cells and tumor B cells from both patients. In contrast, CART1-69 exhibits specific cytotoxicity against IGHV1-69+CLL tumor B cells while sparing normal healthy B cells. Similarly, CART4-34 exhibits specific cytotoxicity against IGHV4-34+CLL tumor B cells while sparing normal healthy B cells. ND580 B cells: normal / healthy donor B cells. [Figure 10] Figure 10 shows the in vitro cytotoxicity of CART3-23 CAR T cells against a diffuse large B cell lymphoma (DLBCL) cell line (OCI-Ly18) expressing an IGHV3-23+BCR, but no appreciable cytotoxicity against another DLBCL cell line, HBL1, expressing an IGHV4-34+BCR. These results demonstrate the specific nature of CART3-23 to specifically target B cells with an IGHV3-23+BCR. [Figure 11-1] Figure 11 shows the ability of CART4-34 to control tumors in vivo. A cell line (Mec1, CLL) was engineered by deleting its endogenous immunoglobulin heavy and light chains, followed by overexpressing a BCR containing the IGHV4-34 region. This IGHV4-34+ cell line was injected intravenously into mice on day 0, followed by intravenous injection of CART19 T cells or CART4-34 T cells on day 13 of the experiment (untransduced (UTD) T cells served as negative controls). This figure shows that both CART19 and CART4-34 can suppress tumor growth to similar levels. [Figure 11-2] See description of Figure 11-1. [Figure 12]Figure 12 shows a heatmap of IGHV gene frequency and BCR subtypes in autoimmune diseases. IGHV4-34 BCR is enriched in systemic lupus erythematosus; IGHV1-69 is enriched instead in Behcet's disease; and IGHV1-69 is enriched in thrombotic thrombocytopenic purpura. TTP: thrombotic thrombocytopenic purpura; SLE: systemic lupus erythematosus; CD: Crohn's disease; BD: Behcet's disease; EGPA: eosinophilic polyangiitis granulomatosis; TTP: thrombotic thrombocytopenic purpura; AAV: ANCA-associated vasculitis; IgAV: IgA vasculitis; IgAV: IgA vasculitis. Figure modified from Bashford-Rogers, RJM et al., Nature, 574(7776):122-1261-29 (2019). [Figure 13] Figure 13 is a map showing the epitope specificity of anti-ADAMTS13 scFv based on information from patients with thrombotic thrombocytopenic purpura (TTP). Figure reproduced from Ostertag, EM et al., Transfusion 56, 1763-1774 (2016). [Figure 14] Figure 14 shows some of the characteristics of stereotypic BCR in chronic lymphocytic leukemia (CLL) patients: B cell receptor sequences are shared among chronic lymphocytic leukemia patients; 41% of CLL patients have malignant B cells with stereotypic BCR; and a subset of CLL patients with stereotypic BCR exhibit poor prognosis. CLL subset 2: IGHV3-21 and IGLV3-21; unique HCDR3 sequence; poor prognosis associated with BCR with IGLV3-21. Figures reproduced from Maity, PC et al., Proc. Natl. Acad. Sci. USA, 117:4320-4327 (2020), Stamatopoulos, B. et al., Clin Cancer Res, 24:5048-5057 (2018), and Agathangelidis, A. et al. Blood 119, 4467-4475 (2012). [Figure 15]Figure 15 shows that the sequence logos of stereotypic BCRs in subsets 2, 4, and 6, corresponding to VL 3-21, VH 4-34, and VH 1-69, respectively, are enriched in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Figure reproduced from Agathangelidis, A. et al. Blood 119, 4467-4475 (2012). [Figure 16] Figure 16 is a schematic diagram showing the development of CAR to target stereotype BCR. Chimeric antigen receptor T cells against stereotype BCR specifically kill tumor clones while sparing normal B cells; thus, it is believed that B cell aplasia is eliminated, the need for IV immunoglobulin for survival is eliminated, and opportunistic infections are reduced. Furthermore, targeting stereotype BCR also means targeting the "Achilles heel" of CLL cells, i.e., the BCR that they need to survive (unlike CD19); thus, this strategy is contemplated herein to reduce antigen-negative escape (e.g., CD19-negative relapse). "R110 BCR" is IGLV3-21 with R110 mutation. [Figure 17-1] FIG. 17 is a schematic (example) of the vector map of the pTRPE AVA L2H CAR and the corresponding sequence of the CAR (SEQ ID NO: 47) according to one embodiment of the CAR. [Figure 17-2] See description of Figure 17-1. [Figure 18] Figure 18 is a graph showing in vitro killing experiment: specific killing of VL 3-21. Legend: UTD-non-transduced T cells; CART19-anti-CD19 CAR T cells; 3.21*CAR-anti-IGLV3-21 R110 BCR CAR T cells; and 1.69 CAR-anti-IGHV1-69 BCR CAR T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description The present invention relates to a strategy of adoptive cell transfer of cells (e.g., immune cells, e.g., T cells) transduced to express chimeric antigen receptors (CARs). CARs are molecules that combine specificity based on antigen binding domains for target antigens with T cell receptor activating intracellular domains to generate chimeric proteins that exhibit specific anti-target cell immune activity. In some aspects, the present invention includes a type of cell therapy in which T cells are genetically modified to express CARs for stereotypic B cell receptors (e.g., CARs for VH4-34 BCR, VL3-21 BCR, VH3-23 BCR, or VH1-69 BCR), and the CAR T cells can be infused into recipients in need thereof. The infused cells can target enriched / stereotypic B cells in the recipient, thereby targeting malignant or pathogenic B cell clones, such as in lymphomas, leukemias, myelomas, other hematological malignancies, or autoimmune diseases, while protecting the recipient's normal B cells.

[0010] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice to test the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0011] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0012] Moreover, the experiments described herein use conventional molecular and cell biological techniques and immunological techniques that are within the skill of those skilled in the art, unless otherwise specified.Such techniques are well known to those skilled in the art and are fully described in the literature.See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008) (including all supplements), Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J.Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0013] Methods and techniques using T cells with chimeric antigen receptors (CAR T cells) are described, for example, in Ruella, M. et al., Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J. Clin. Invest., 126(10):3814-3826 (2016) and Kalos, M. et al., T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia, Science Translational Medicine, 3 (95), 95ra73:1-11 (2011), the contents of which are incorporated herein by reference in their entireties.

[0014] Unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of potential ambiguity, the definitions provided herein take precedence over any definitions in dictionaries or elsewhere herein. Unless otherwise indicated by context, singular terms shall include the plural, and plural terms shall include the singular. Unless otherwise indicated, the use of "or" means "and / or." The use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0015] In general, the terms used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are generally carried out according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise specified. Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications, as commonly performed in the art, or as described herein. The terms used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and their laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0016] In order that this disclosure may be more readily understood, selected terms are defined below.

[0017] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0018] The term "about" as used herein in referring to measurable values ​​such as amounts and time periods is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0019] "Activated" as used herein refers to a state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers specifically to T cells undergoing cell division.

[0020] As used herein, the term "adapter molecule" refers to a polypeptide having a sequence that allows for interaction with two or more molecules and, in certain embodiments, promotes the activation or inactivation of cytotoxic cells.

[0021] The term "antibody" as used herein means an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0022] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0023] "Antibody heavy chain," as used herein, means the larger of the two types of polypeptide chains present in all antibody molecules in their native conformations.

[0024] "Antibody light chain" as used herein refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their native conformation. Kappa light chain and lambda light chain refer to the two main antibody light chain isotypes.

[0025] The term "synthetic antibody," as used herein, refers to an antibody made using recombinant DNA techniques, e.g., an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody made by synthesis of a DNA molecule encoding the antibody (which DNA molecule expresses the antibody protein) or an amino acid sequence specifying the antibody, which DNA sequence or amino acid sequence was obtained using synthetic DNA or synthetic amino acid sequence techniques that are available and well known in the art.

[0026] The term "anti-cancer effect" as used herein refers to a biological effect that may be manifested by a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, and / or an improvement in various physiological symptoms associated with a cancerous condition (e.g., blood cancer). An "anti-cancer effect" may also be manifested, among other things, by the ability of the peptides, polynucleotides, cells, and / or CARs of the present invention in preventing the development of cancer.

[0027] The term "autoimmune disease" as used herein is defined as a disorder caused by autoimmune response. Autoimmune disease is the result of inappropriate or excessive response to self-antigens. Examples of autoimmune disease include, among others, systemic lupus erythematosus (SLE), Crohn's disease (CD), Behcet's disease (BD), eosinophilic granulomatosis with polyangiitis (EGPA), thrombotic thrombocytopenic purpura (TTP), ANCA-associated vasculitis (AAV), immunoglobulin A vasculitis (IgA vasculitis), IgA vasculitis (IgAV), Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotid arthritis, and pulmonary tuberculosis. These conditions include, but are not limited to, adenitis, diabetes mellitus (type I), epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0028] As used herein, the term "autologous" is intended to mean any material derived from an individual that is to be later reintroduced into that same individual.

[0029] "Allogeneic" means a graft derived from a different organism of the same species.

[0030] "Xenogeneic" refers to a graft derived from an organism of a different species.

[0031] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial T cell receptor that is expressed on immune effector cells and engineered to specifically bind to an antigen. CARs can be used as therapeutics by adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific for a particular form of antigen. CARs can also include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen binding region. In some aspects, CARs include a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody fused to a transmembrane domain and an intracellular domain.

[0032] The term "chimeric intracellular signaling molecule" refers to a recombinant receptor that contains one or more intracellular domains of one or more costimulatory molecules. The chimeric intracellular signaling molecule substantially lacks an extracellular domain. In some embodiments, the chimeric intracellular signaling molecule contains additional domains, such as a transmembrane domain, a detectable tag, and a spacer domain.

[0033] As used herein, the term "conservative sequence modification" is intended to mean an amino acid modification that does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, for example, one or more amino acid residues in the complementarity determining regions (CDRs) of an antibody or antigen-binding fragment thereof can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for antigen-binding ability using the functional assays described herein.

[0034] The term "cytotoxic" or "cytotoxicity" means killing or damaging a cell. In one embodiment, the modified cell has increased cytotoxicity, e.g., increased cytolytic activity of a T cell.

[0035] A "disease" is an animal's health condition in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate if the disease is not ameliorated.In contrast, an animal's "disorder" is an animal's health condition in which the animal is able to maintain homeostasis, but the animal's health condition is less favorable than would be expected in the absence of the disorder.If left untreated, the disorder does not necessarily cause the animal's health condition to deteriorate further.

[0036] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-cancer activity as measured by any method suitable in the art.

[0037] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties attributed thereto. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to code for the protein or other product of the gene or cDNA.

[0038] As used herein, "endogenous" means any substance that is derived from or produced within an organism, cell, tissue, or system.

[0039] As used herein, the term "exogenous" means any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0040] The term "expand" as used herein means to increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number compared to the number initially present in the culture. In another embodiment, T cells expanded ex vivo are increased in number compared to other cell types in the culture. The term "ex vivo" as used herein means cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0041] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0042] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include any vector known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0043] "Homologous" as used herein means subunit sequence identity between two polymer molecules, for example between two nucleic acid molecules, for example between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomeric subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the DNA molecules are homologous at that position. The homology between two sequences is a direct function of the number of positions that match or are homologous, for example, if half of the positions in two sequences (e.g., 5 positions in a polymer 10 subunits long) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous. When applied to nucleic acids or proteins, "homologous" as used herein means sequences that have about 50% sequence identity. More preferably, the homologous sequences have about 75% sequence identity, and even more preferably at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0044] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) (e.g., complementarity determining region 1 (CDR1) and / or complementarity determining region 2 (CDR2) and / or complementarity determining region 3 (CDR3)) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody also optimally contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0045] "Fully human" refers to an immunoglobulin, such as an antibody or antigen-binding fragment thereof, where the entire molecule is of human origin or consists of an amino acid sequence identical to that of a human version of the antibody or antigen-binding fragment thereof.

[0046] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, for example, if a position in each of the two polypeptide molecules is occupied by arginine, then the amino acid sequences are identical at that position. Identity, or the degree to which two amino acid sequences have the same residue at the same position in an alignment, is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that are matched or identical, for example, if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0047] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even higher, 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0048] Typically, sequence identity is measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, the BLAST program, the BESTFIT program, the GAP program, or the PILEUP / PRETTYBOX program). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Typically, conservative substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to measuring the degree of identity, the BLAST program may be used, where e -3 ~e -100 A probability score of 0.05 indicates closely related sequences.

[0049] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells may be referred to as BCR (B cell receptor) or antigen receptor. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that, upon exposure to allergens, causes the release of mediators from mast cells and basophils, resulting in immediate hypersensitivity reactions.

[0050] The term "immune response," as used herein, is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0051] As used herein, "instructional material" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be attached to a container containing the nucleic acid, peptide, and / or composition of the invention, or may be shipped together with a container containing the nucleic acid, peptide, and / or composition. Alternatively, the instructional material may be shipped separately from the container, with the intention that the recipient will use the instructional material and the compound cooperatively.

[0052] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as a host cell.

[0053] " Lentivirus " as used herein refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; it can deliver a significant amount of genetic information into the DNA of host cells, and is therefore one of the most efficient methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0054] The term "modified" as used herein means that the state or structure of a molecule or cell of the invention has been altered. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified through the introduction of nucleic acids.

[0055] The term "modulate" as used herein means to cause a detectable increase or decrease in the level of a response in a subject, compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses disrupting and / or affecting the natural signal or response, thereby achieving a beneficial therapeutic response in a subject, preferably a human.

[0056] In the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" means adenosine, "C" means cytosine, "G" means guanosine, "T" means thymidine, and "U" means uridine.

[0057] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, since nucleotide sequences encoding proteins can contain introns in some forms.

[0058] The term "functionally linked" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which allows the expression of the heterologous nucleic acid sequence.For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.For example, a promoter is functionally linked to a coding sequence when it affects the transcription or expression of the coding sequence.Usually, functionally linked DNA sequences are adjacent and, if necessary, are in the same reading frame to link two protein coding regions.

[0059] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc) injection, intravenous (iv) injection, intramuscular (im) injection, intrasternal injection, or infusion techniques.

[0060] "Single chain antibody" refers to an antibody formed by recombinant DNA technology, in which the heavy and light fragments of immunoglobulin chains are linked to the Fv region via an engineered linker consisting of amino acids.Various methods for producing single chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0061] The term "specifically binds" as used herein with respect to an antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antibody or antigen-binding fragment thereof as specific. In another example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen may also bind to various allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody or antigen-binding fragment thereof as specific. In some examples, the term "specific binding" or "specifically binds to" can be used in reference to the interaction of an antibody or antigen-binding fragment thereof, a protein, or a peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody or antigen-binding fragment thereof recognizes and binds to a particular protein structure, rather than to a protein in general. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and an antibody or antigen-binding fragment thereof will reduce the amount of labeled A bound to the antibody or antigen-binding fragment thereof.

[0062] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. A "subject" or "patient" as used herein may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0063] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. Substantially purified cells also refer to cells that are separated from other cell types with which they are normally mixed in their natural state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that are separated from cells with which they are normally mixed in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0064] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, amelioration, or eradication of a disease state.

[0065] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0066] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder (e.g., cancer, autoimmune disease) experienced by a subject.

[0067] A "vector" is a composition that contains isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell.A number of vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides combined with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term "vector" includes self-replicating plasmids or viruses.This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds and liposomes.Examples of viral vectors include but are not limited to adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors.

[0068] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0069] explanation The present invention includes compositions and methods for the treatment or prevention of B cell cancers (including but not limited to lymphomas, myelomas, leukemias) and / or B cell-mediated autoimmune diseases. According to the present invention, immune cells, including but not limited to T cells (including but not limited to natural killer T (NKT) cells and γδ T cells), natural killer (NK) cells, and macrophages, are modified for adoptive cell (e.g., T cell) therapy by expressing a CAR that contains an antigen-binding domain that specifically recognizes and binds to a disease-specific or stereotypic B cell receptor expressed by a cell (e.g., a cancerous B cell of a chronic lymphocytic leukemia (CLL) patient). The modified cells (e.g., modified T cells) of the present invention are specific for a disease-associated or stereotypic B cell receptor and have improved cytotoxicity and efficacy against B cells with a stereotypic B cell receptor.

[0070] For example, many B-cell non-Hodgkin's lymphomas express limited or "stereotyped" immunoglobulin variable heavy and light chain genes in their B-cell receptors (BCRs). One-third of CLL patients express stereotyped receptors, and stereotyped receptors are found in a significant proportion of other B-cell non-Hodgkin's lymphoma patients, including 33.9% of diffuse large B-cell lymphoma (DLBCL) patients, 46.3% of mantle cell lymphoma (MCL) patients, and 45.8% of splenic marginal zone lymphoma (SMZL) patients. Without wishing to be bound by theory, these findings suggest that antigens may play a role in the selection and expansion of neoplastic clones. For example, VL 3.21 (including VL 3.21 with R110 mutation), VH 1.69, VH 4.34, and VH 3.23 are significantly enriched in these diseases. These conserved antigens serve as novel and tumor-selective antigens for CAR therapy.The CAR of the present invention that targets such antigens is more tumor-selective cell therapy than, for example, CAR that targets CD19, which targets all mature B cells and thus requires patients to receive lifelong IgG infusions to prevent infection.In some embodiments, these BCR isoform CARs of the present invention target a portion of the B cell population and therefore have reduced extratumoral toxicity.

[0071] In other embodiments, the CAR T cells of the present invention can also be used to specifically eliminate autoimmune B cell clones. Without wishing to be bound by theory, some autoimmune disorders are characterized by the enrichment of specific BCRs that are believed to be involved in the pathogenesis of the disease. Specifically, VH 1.69 antibodies are enriched in thrombotic thrombocytopenic purpura (TTP) and Behcet's disease, and VH 4.34 is enriched in systemic lupus erythematosus (SLE), eosinophilic granulomatosis with polyangiitis (EGPA), and Crohn's disease (CD). Thus, in some embodiments, the CAR T cells of the present invention can specifically deplete B cells and plasma cells that express and produce antibodies that are involved in the pathogenesis of such autoimmune diseases.

[0072] Data reported from CD19, CD20, and CD22 CAR T cell therapies in B cell malignancies suggest that resistance to this class of therapeutic agents may result from antigen escape, i.e., the emergence of tumors with reduced or downregulated target antigens.

[0073] Thus, in yet another embodiment, the CAR cells (e.g., CAR T cells) of the present invention can be used, alone or in combination with other treatments, to reduce or eliminate antigen escape by B cell cancers (e.g., cancerous B cells in CLL patients) by targeting a protein essential for the survival of B cell leukemias and lymphomas, i.e., the BCR.

[0074] Chimeric antigen receptors (CARs) In one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain specifically binds to an enriched stereotypic B cell receptor (BCR).

[0075] Antigen-binding domain The antigen-binding domain of CAR is the extracellular region of CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids.The antigen-binding domain can include any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof.

[0076] In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single chain variable fragment (scFv).

[0077] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoded linker that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain comprises an scFv with a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain comprises an scFv with a VL-linker-VH configuration from the N-terminus to the C-terminus. Those skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0078] In some embodiments, the linker is rich in glycine for flexibility and rich in serine or threonine for solubility. The linker can link the heavy and light chain variable regions of the extracellular antigen binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including glycine serine (GS) linkers, such as (GS) n , (GSGGS) n (SEQ ID NO: 1), (GGGS) n (SEQ ID NO: 2), and (GGGGS) n (SEQ ID NO: 3), where n represents an integer of at least 1. Exemplary linker sequences include, but are not limited to, The antigen-binding domain of the present invention may comprise an amino acid sequence including, but not limited to, TIFF2024532851000002.tif19160 and the like. One skilled in the art will be able to select an appropriate linker sequence for use in the present invention. In one embodiment, the antigen-binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL are separated by a linker sequence having the amino acid sequence of SEQ ID NO: 11, which is a nucleic acid sequence represented by the nucleic acid sequence of SEQ ID NO: 12. It can be coded by TIFF2024532851000003.tif4163.

[0079] Single chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0080] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion; for example, digestion of an antibody with the enzyme papain produces two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0081] As used herein, "F(ab')2" refers to an antibody fragment generated by pepsin digestion of a full-length IgG antibody, in which the fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains (a portion of a heavy chain and a light (L) chain linked by an S-S bond for binding to an antigen), with the remaining portions of the heavy chain linked to each other. The "F(ab')2" fragment can be split into two individual Fab' fragments.

[0082] In other embodiments, the antigen-binding domain comprises an antibody mimetic protein, such as, for example, an artificial ankyrin repeat protein (DARPin), an affibody, an adnectin, or anticalin. Constructs with specific binding affinity can be generated using DARPin libraries, for example as described in Seeger, MA et al., Design, construction, and characterization of a second generation DARPin library with reduced hydrophobicity, Protein Sci., 22:1239-1257 (2013).

[0083] In some embodiments, the antigen binding domain can be derived from the same species in which the CAR will ultimately be used. For example, for human use, the antigen binding domain of the CAR can comprise a human antibody or fragment thereof, as described elsewhere herein.

[0084] "Enriched stereotype" BCR, or "enriched stereotype" with respect to BCR, refers to BCRs that have similar primary structures (e.g., length, amino acid composition, and unique amino acid residues of recombinant junctions) defined by highly similar Ig V regions in H and L chains, and in some embodiments, have different H and L CDR3 configurations. These enriched stereotype BCRs can be grouped into various subsets of enriched stereotype BCRs, each of which is conventionally designated by a consecutive number (e.g., subsets 2, 4, or 6) and contains antigenic targets for the antigen-binding domain of the CAR of the present invention. These enriched stereotype BCRs are directly involved in pathogenesis, since they have the unique ability to signal and activate B cells. For example, Stamatopoulos, B. et al., Clin. Cancer Res., 24:5048-5057 (2018), Rovida et al., Clin. Cancer Res., 27(3):729-739 (2021), Hacken, ET et al., Leukemia, 33(2):287-298 (2019), Messmer, BT et al., J. Exp. Med., 200(4): 519-525 (2004), and Rossi, D. & Gaidano, G., Haematologica, 95(12):1992-1995 (2010), each of which is incorporated by reference in its entirety, describe multiple distinct sets of enriched stereotypic receptors.

[0085] In one embodiment, the BCRs of subset 1 can be characterized by the use of IGHV1-5-7, IGHD6-19, IGHJ4, and / or IGKV1-39; the BCRs of subset 2 can be characterized by the use of IGHV3-21, IGHJ6, and / or IGLV3-21; the BCRs of subset 4 can be characterized by the use of IGHV4-34, IGHD5-5 or D4-17, IGHJ6, and / or IGKV3-30; The BCRs of subset 5 can be characterized by the use of IGHV1-69, IGHD3-10, IGHJ6, and / or IGKV1-33 or IGLV3-21; the BCRs of subset 6 can be characterized by the use of IGHV1-69, IGHD3-16, IGHJ3, and / or IGLV3-20; and the BCRs of subset 8 can be characterized by the use of IGHV4-39, IGHD6-13, IGHJ5, and / or IGKV1-39.

[0086] In some embodiments, the enriched stereotypic BCR is an enriched stereotypic BCR of subset 1, 2, 4, 5, 6, or 8. In one embodiment, the antigen binding domain of the CAR of the invention specifically binds to an enriched stereotypic BCR of subset 1, 2, 4, 5, 6, or 8.

[0087] In other embodiments, the enriched stereotypic BCR is an enriched stereotypic BCR of subset 2, 4, or 6. In another embodiment, the antigen binding domain of the CAR of the invention specifically binds to an enriched stereotypic BCR of subset 2, 4, or 6.

[0088] In some embodiments, the enriched stereotypic BCRs are characterized as autonomously active BCRs.

[0089] In one embodiment, an autonomously active BCR can be driven by autonomous BCR signaling, e.g., by binding via HCDR3 to an internal epitope of another BCR, resulting in BCR-BCR binding, aggregation, and / or activation (see, e.g., Duhren-von Minden, M. et al., Nature, 489(7415):309-12 (2012); see also, e.g., U.S. Patent Application Publication No. 2020 / 0199225, which is incorporated by reference in its entirety).

[0090] For example, U.S. Patent Application Publication No. 2020 / 0199225 describes a CLL B cell receptor subset 2 variant region associated with receptor autonomously active functionality characterized by the amino acid sequences KLTVLRQPKA (SEQ ID NO: 13) and VAPGKTAR (SEQ ID NO: 14) of the light chain, and a subset 4 variant region associated with receptor autonomously active functionality characterized by the amino acid sequence KLTVLRQPKA (SEQ ID NO: 14) of the variable portion of the heavy chain. This explains that it is defined by TIFF2024532851000004.tif13145.

[0091] In other embodiments, the enriched stereotype BCRs are Contains the amino acid sequence of TIFF2024532851000005.tif12156.

[0092] In another embodiment, the antigen binding domain of the CAR of the present invention specifically binds to an enriched stereotype BCR comprising a light chain variable region (VL) comprising the amino acid sequence of KLTVLRQPKA (SEQ ID NO: 13) and / or VAPGKTAR (SEQ ID NO: 14).

[0093] In another embodiment, the antigen binding domain of the CAR comprises: It specifically binds to enriched stereotype BCRs containing a heavy chain variable region (VH) containing the amino acid sequence of TIFF2024532851000006.tif12141.

[0094] In another embodiment, the antigen binding domain comprises: It specifically binds to enriched stereotype BCR containing the sequence of TIFF2024532851000007.tif48165.

[0095] In another embodiment, the antigen binding domain comprises: It specifically binds to enriched stereotype BCR containing the sequence TIFF2024532851000008.tif63165.

[0096] In some embodiments, the antigen binding domain of the CAR of the present invention is a binding domain specific for an enriched stereotype BCR, comprising a VH and a VL.

[0097] In one embodiment, the antigen binding domain comprises a VH comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence of GFSLTSYG (SEQ ID NO: 25), CDR2 comprises the sequence of IWRGGGT (SEQ ID NO: 26), and CDR3 comprises the sequence of ARSRYDEEESMNY (SEQ ID NO: 27).

[0098] In one embodiment, the antigen binding domain comprises a VL comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence of GNIHSY (SEQ ID NO: 28), CDR2 comprises the sequence of NAKT (SEQ ID NO: 29), and CDR3 comprises the sequence of QHFWNTPPT (SEQ ID NO: 30).

[0099] In one embodiment, the antigen binding domain comprises: Contains VH containing the sequence of TIFF2024532851000009.tif85166.

[0100] In one embodiment, the antigen binding domain comprises: The VH encoded by the sequence comprising TIFF2024532851000010.tif202165.

[0101] In one embodiment, the antigen binding domain comprises: Contains VL with sequence TIFF2024532851000011.tif88165.

[0102] In one embodiment, the antigen binding domain comprises: Contains the VL encoded by the sequence comprising TIFF2024532851000012.tif179166.

[0103] In some embodiments, the antigen binding domain comprises an scFv having, from N-terminus to C-terminus, the configuration VH-linker-VL or VL-linker-VH, where the linker comprises the sequence of SEQ ID NO:11.

[0104] In yet another embodiment, the scFv comprises: Contains the amino acid sequence of TIFF2024532851000013.tif158166TIFF2024532851000014.tif128165.

[0105] In a further embodiment, the scFv comprises TIFF2024532851000015.tif77165TIFF2024532851000016.tif223166TIFF2024532851000017.tif223165TIFF2024532851000018.tif223165TIFF2024532851000019.tif19163.

[0106] Acceptable variants of antigen-binding domains that maintain specific binding to enriched stereotype BCRs are known to those of skill in the art. For example, in some embodiments, the antigen-binding domain comprises an amino acid sequence having 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%, at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 25-30, 31-32, 35-36, 39-42, 64, 66, 68, 69, 80, and 81.

[0107] In other embodiments, the antigen binding domain is encoded by a nucleic acid sequence having 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%, at least 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 33-34, 37-38, 43-46, 65, 67, 70, 71, 82, and 83.

[0108] The antigen binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, both of which are described elsewhere herein. In one embodiment, the nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a transmembrane domain and a nucleic acid encoding an intracellular domain.

[0109] An antigen binding domain described herein, for example, an antibody or fragment thereof that specifically binds to an enriched stereotype BCR, can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that can be included in a CAR.

[0110] For example, in some embodiments, the CAR comprises: Contains the following sequences: TIFF2024532851000020.tif19164TIFF2024532851000021.tif223165TIFF2024532851000022.tif223165TIFF2024532851000023.tif48165

[0111] In some embodiments, the CAR comprises an amino acid sequence having 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%, at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 47-50 and 72-75.

[0112] In other embodiments, the CAR comprises: TIFF2024532851000024.tif120165TIFF2024532851000025.tif223165TIFF2024532851000026.tif223166TIFF2024532851000027.tif223165TIFF2024532851000028.tif223166TIFF2024532851000029.tif223165TIFF2024532851000030.tif223165TIFF2024532851000031.tif92165

[0113] In some embodiments, the CAR is encoded by a nucleic acid sequence comprising a nucleotide sequence having 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%, at least 99% sequence identity to any one of the sequences set forth in SEQ ID NOs: 84-91.

[0114] Transmembrane domain In some embodiments, CAR comprises a transmembrane domain fused to an extracellular domain.In one embodiment, CAR comprises a transmembrane domain that is naturally associated with one of the domains of the CAR.In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domain of the same or different surface membrane protein, so as to minimize interaction with other members of the receptor complex.

[0115] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one embodiment, the transmembrane domain may be synthetic, in which case it will contain primarily hydrophobic residues such as leucine and valine. In one aspect, a triad of phenylalanine, tryptophan, and valine is present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, 2-10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine (GS) dyads are particularly suitable linkers.

[0116] In other embodiments, a spacer domain may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular or cytoplasmic domain in a polypeptide chain. A spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0117] In some embodiments, a spacer domain before the transmembrane domain can be used, including a CD8 or human Ig (immunoglobulin) hinge or a glycine-serine linker. In one embodiment, a hinge N-terminal to the transmembrane domain (e.g., a CD8 alpha hinge) is included in the CAR between the extracellular domain and the transmembrane domain of the CAR, for example, between the antigen binding domain and the transmembrane domain of the CAR.

[0118] Examples of hinge and / or transmembrane domains include those of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD10 3, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM 1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, C These include, but are not limited to, the hinge and / or transmembrane domains of D100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0119] In one embodiment, the CAR comprises a transmembrane domain, such as, but not limited to, TIFF2024532851000032.tif4128 or an amino acid sequence having 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% sequence identity thereto.

[0120] In some embodiments, the CD8 alpha transmembrane domain is TIFF2024532851000033.tif26165 or a nucleic acid sequence having 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% sequence identity to SEQ ID NO: 60 or 61.

[0121] In some embodiments, the CAR comprises: The CD8 alpha hinge comprises a sequence of TIFF2024532851000034.tif4153 or an amino acid sequence having 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% sequence identity thereto.

[0122] In some embodiments, the CD8 alpha hinge region is TIFF2024532851000035.tif19165 or a nucleic acid sequence having 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% sequence identity thereto.

[0123] Cytoplasmic domain The cytoplasmic domain or otherwise intracellular signaling domain of the CAR of the present invention is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used instead of the intact chain, provided that it transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0124] Examples of intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that work in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capability.

[0125] It is known that the signal generated through TCR alone is insufficient to fully activate T cells, and that secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0126] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0127] Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3 zeta (CD3ζ).

[0128] In one embodiment, the cytoplasmic signaling sequence derived from CD3 zeta is The amino acid sequence of TIFF2024532851000036.tif19164 or an amino acid sequence having 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% sequence identity thereto.

[0129] In another embodiment, the nucleic acid sequence encoding the CD3 zeta signaling domain is a nucleic acid sequence having 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% sequence identity thereto; or TIFF2024532851000038.tif49166, or a nucleic acid sequence having 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% sequence identity thereto.

[0130] In another embodiment, the cytoplasmic domain of the CAR comprises a CD3 zeta signaling domain, alone or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. By costimulatory signaling region is meant the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte response to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, and the like. Thus, the present invention is exemplified primarily using 4-1BB as a costimulatory signaling element, although other costimulatory elements are also within the scope of the present invention.

[0131] In one embodiment, the intracellular signaling domain of 4-1BB is The amino acid sequence of TIFF2024532851000039.tif4147 or an amino acid sequence having 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% sequence identity thereto.

[0132] In another embodiment, the nucleic acid sequence encoding the intracellular signaling domain of 4-1BB is The sequence of TIFF2024532851000040.tif19164 or a nucleic acid sequence having 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% sequence identity thereto.

[0133] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specific order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the linkage. Glycine-serine duplexes are particularly suitable linkers.

[0134] In one embodiment, the cytoplasmic domain comprises the signaling domain of CD3 zeta and the intracellular signaling domain of 4-1BB, hi another embodiment, the cytoplasmic domain comprises the signaling domain of CD3 zeta and the signaling domain of CD28.

[0135] In one embodiment, the signaling domain of CD28 is The present invention relates to an amino acid sequence having 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% sequence identity thereto.

[0136] In another embodiment, the nucleic acid sequence encoding the signaling domain of CD28 is TIFF2024532851000042.tif19165 or a nucleic acid sequence having 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% sequence identity thereto.

[0137] In yet another embodiment, the cytoplasmic domain comprises the signaling domain of CD3 zeta and the intracellular signaling domains of 4-1BB and CD28.

[0138] In one embodiment, the cytoplasmic domain in a CAR of the invention comprises the intracellular signaling domain of 4-1BB and the signaling domain of CD3 zeta, wherein the intracellular signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:59 and the signaling domain of CD3 zeta comprises the amino acid sequence set forth in SEQ ID NO:56.

[0139] Other domains In some embodiments, the CAR comprises a signal peptide (e.g., a CD8 signal peptide). In some embodiments, the signal peptide is responsible for translocating the receptor to the T cell surface.

[0140] In some embodiments, the signal peptide is A CD8 signal peptide comprising the amino acid sequence of TIFF2024532851000043.tif4128 or an amino acid sequence having 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% sequence identity thereto.

[0141] Nucleic Acid / Vector In another aspect, the present invention provides a recombinant nucleic acid molecule comprising a sequence encoding a CAR of the present invention having an antigen-binding domain that specifically binds to an enriched stereotypic BCR.

[0142] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening libraries derived from cells expressing the gene, by obtaining it from a vector known to contain the gene, by screening against a pathogenic target (e.g., ADAMTS13), or by isolating it directly from cells and tissues containing the gene, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0143] The present invention also provides, in some embodiments, a vector into which the DNA of the present invention is inserted. A vector derived from a retrovirus, such as a lentivirus, is a suitable tool for achieving long-term gene transfer, since it allows long-term and stable integration of transgenes and their transmission to daughter cells. Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses, such as mouse leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In other embodiments, the CAR is introduced into cells (e.g., T cells) using, for example, a transposon system (e.g., PiggyBac™), liposomes, nanoparticles, lipid nanoparticles, or mRNA. In yet other embodiments, the CAR of the present invention is introduced into cells (e.g., T cells) in vivo.

[0144] In brief summary, expression of natural or synthetic nucleic acid encoding CAR is typically achieved by functionally linking the nucleic acid encoding CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector.These vectors can be suitable for replication and integration in eukaryotes.Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that are useful for regulating the expression of desired nucleic acid sequences.

[0145] The expression construct of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocol.The method for gene delivery is known in the art.See, for example, U.S. Patent No. 5,399,346, U.S. Patent No. 5,580,859, U.S. Patent No. 5,589,466, the entirety of which is incorporated herein by reference.In another embodiment, the present invention provides a gene therapy vector.

[0146] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0147] Furthermore, expression vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.In general, suitable vectors include a replication origin functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0148] Several virus-based systems have been developed for gene transfer into mammalian cells.For example, retroviruses provide a convenient platform for gene delivery systems.Selected genes can be inserted into vectors and placed into retroviral particles using techniques known in the art.The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo.Some retroviral systems are known in the art.In some embodiments, adenoviral vectors are used.Some adenoviral vectors are known in the art.In one embodiment, lentiviral vectors are used.

[0149] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even when the positions of elements are reversed or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart, after which activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or separately to activate transcription.

[0150] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuL V promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which it is operably linked when such expression is desired or turn off expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0151] To evaluate the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cell can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0152] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that code for a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is evaluated at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (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 obtained commercially. Usually, the construct with the minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate transcription driven by the promoter.

[0153] Methods for introducing and expressing genes into cells are known in the art.In relation to expression vectors, the vectors can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art.For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0154] The physical method for introducing polynucleotide into host cell includes calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation.Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art.See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).The preferred method for introducing polynucleotide into host cell is calcium phosphate transfection.

[0155] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA vectors and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362. In other embodiments, CARs are introduced into cells (e.g., T cells) using, for example, transposon systems (e.g., PiggyBac™), liposomes, nanoparticles, lipid nanoparticles, or mRNA. In yet other embodiments, CARs of the present invention are introduced into cells (e.g., T cells) in vivo.

[0156] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0157] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acid into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with lipid. The nucleic acid associated with lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule that is bound to both the liposome and the oligonucleotide, trapped in the liposome, complexed with the liposome, dispersed in a solution that includes lipid, mixed with lipid, combined with lipid, contained in suspension in lipid, contained with or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in the dissolved state. For example, they may be in a bilayer structure as a micelle or with a "folded" structure. They may simply be dispersed in the solution, possibly forming aggregates that are not uniform in size or shape.Lipids are fatty substances that can be natural lipids or synthetic lipids.For example, lipids include the fatty droplets that naturally occur in cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0158] Lipids suitable for use can be obtained from commercial suppliers. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vehicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures in solution that differ from normal vesicular structures are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0159] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitor of the present invention, various assays can be carried out to confirm the presence of recombinant DNA sequence in host cells.Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assay described herein for identifying agents within the scope of the present invention.

[0160] Source of T cells Prior to the expansion and genetic modification of the T cells of the present invention, a source of T cells is obtained from a subject. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, placental tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumor, and through differentiation of T cells from induced pluripotent stem cells (iPSCs) or other stem cell sources. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from a blood bolus taken from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In one preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells harvested by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution may lack calcium and lack magnesium, or lack many, but not all, divalent cations. Also surprisingly, performing the initial activation step in the absence of calcium results in enhanced activation. As one of skill in the art will readily appreciate, the washing step may be accomplished by methods known to those of skill in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells may be washed with, for example, phosphate buffered saline (PBS), or phosphate buffered saline (PBS). 2+ Mg-free 2+ The cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions with or without buffering agents. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0161] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation on a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells, e.g., CD3 + T cells, CD28 + T cells, CD4 + T cells, CD8 + T cells, CD45RA + T cells, and CD45RO +T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e. 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient for positive selection of the desired T cells. In one embodiment, the period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or longer and all integer values ​​therebetween. In a further embodiment, the period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours and can extend to 14 days. In one preferred embodiment, the incubation period is 24 hours. Longer incubation times, such as 24 hours, can be used to isolate T cells from leukemia patients to increase cell yield. Longer incubation times can be used to isolate T cells in any situation where there are few T cells compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immune-compromised individuals. Furthermore, by using longer incubation times, the efficiency of capturing CD8+ T cells can be increased. Thus, subpopulations of T cells can be preferentially selected or eliminated at the beginning of culture or at other times during this process by simply shortening or lengthening the time that T cells are bound to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein). Furthermore, subpopulations of T cells can also be preferentially selected or eliminated at the beginning of culture or at other desired times by increasing or decreasing the ratio of anti-CD3 antibodies and / or anti-CD28 antibodies on beads or other surfaces. Those skilled in the art will recognize that multiple rounds of selection can also be used in the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process.The "unselected" cells can also be subjected to additional rounds of selection.

[0162] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to the cells being negatively selected. One method is cell sorting and / or cell selection via negative magnetic immunoadhesion or negative flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the cells being negatively selected. For example, negative selection can be used to enrich for CD4 + When enriching cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 + It may be desirable to enrich or positively select for regulatory T cells that typically express. Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

[0163] To isolate desired cell populations by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed (i.e., increase cell concentration) to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 million or 150 million cells / ml may be used. Using a high concentration may increase cell yield, cell activation, and cell expansion. Furthermore, using a high cell concentration allows more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, from samples with many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. For example, using a high cell concentration may allow more efficient capture of CD8 T cells, which normally have weak CD28 expression. + This allows for more efficient selection of T cells.

[0164] In a related embodiment, it may be desirable to use a low concentration of cells. By significantly diluting the mixture of T cells and a surface (e.g., a particle such as a bead), the interaction between the particles and the cells is minimized. This allows the selection of cells that express large amounts of the desired antigen that binds to the particles. For example, CD4 + T cells expressed higher levels of CD28 and, at dilute concentrations, CD8 +T cells are captured more efficiently than T cells. In one embodiment, the cell concentration used is 5×10 6 In other embodiments, the concentration used is about 1×10 5 / ml~1×10 6 / ml, and any integer value therebetween.

[0165] In other embodiments, cells can be incubated on a rotator at various speeds for various lengths of time at either 2-10° C. or room temperature.

[0166] T cells for stimulation can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps result in a more homogenous product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO or 31.25% PlasmaLyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media, including, for example, Hespan and PlasmaLyte A, and then the cells are frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled freezing performed immediately at -20°C or in liquid nitrogen, may be used.

[0167] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to sit at room temperature for 1 hour prior to activation using the methods of the invention.

[0168] It is also contemplated in the present invention to collect blood samples or apheresis products from subjects at a time period prior to when the expanded cells described herein may be needed. Thus, the source of cells to be expanded can be collected at any time required, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that may benefit from T cell therapy, such as those described herein. In one embodiment, blood samples or apheresis samples are collected from generally healthy subjects. In certain embodiments, blood samples or apheresis samples are collected from generally healthy subjects who are at risk of developing disease but have not yet developed disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and used later. In certain embodiments, samples are collected from patients shortly after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood or apheresis sample from the subject prior to any number of relevant treatment modalities, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993).In further embodiments, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously with, or after) bone marrow or stem cell transplantation; T cell depletion therapy using either chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells can be isolated prior to B cell depletion therapy, such as an agent reactive with CD20, e.g., Rituxan, and frozen for later use for subsequent treatment.

[0169] In a further embodiment of the present invention, T cells are obtained from a patient immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of T cells obtained may be optimal or improved in terms of their ability to expand ex vivo immediately after treatment during the period when patients would normally be recovering from treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for promoting engraftment and in vivo expansion. Thus, it is contemplated in the present invention to harvest blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that favor the repopulation, recirculation, regeneration, and / or expansion of certain cell types, particularly during a defined time frame after treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0170] T cell activation and expansion Either before or after genetic modification of the T cells to express a desired CAR, the T cells can be engineered to express a CAR using methods described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,0 The cells can be activated and expanded, typically using the methods described in U.S. Patent Application Publication No. 20060121005, and in which the cells are cultured.

[0171] Typically, the T cells of the present invention are expanded by contacting with a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells are bound. In particular, the T cell population can be stimulated as described herein, for example, by contacting with surface-immobilized anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies, or by contacting with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, the T cell population can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. CD4 + T cells or CD8 +To stimulate the proliferation of any of T cells, anti-CD3 antibody and anti-CD28 antibody. Examples of anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0172] In certain embodiments, the primary and costimulatory signals for T cells can be provided by various protocols. For example, the agents providing each signal can be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" form) or to separate surfaces (i.e., in a "trans" form). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, these agents can be in soluble form and then crosslinked to a surface, for example, a cell expressing an Fc receptor or an antibody or other binding agent that will bind to these agents. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding T cells in the present invention.

[0173] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment, CD4 + A 1:1 ratio is used for each antibody bound to the beads for T cell expansion and T cell proliferation. In a particular aspect of the invention, a ratio of anti-CD3 and anti-CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed compared to the expansion observed when a 1:1 ratio is used. In one particular embodiment, an increase of about 1 to about 3 fold is observed compared to the expansion observed when a 1:1 ratio is used. In one embodiment, the ratio of CD3 and CD28 antibodies bound to the beads ranges from 100:1 to 1:100 and all values ​​therebetween. In one aspect of the invention, more anti-CD28 antibodies than anti-CD3 antibodies are bound to the particles, i.e., the ratio of CD3 to CD28 is less than 1. In a particular embodiment of the invention, the ratio of anti-CD28 and anti-CD3 antibodies bound to the beads is greater than 2:1. In one particular embodiment, a CD3:CD28 ratio of 1:100 is used for the antibodies bound to the beads. In another embodiment, a CD3:CD28 ratio of 1:75 is used for the antibody bound to the beads. In a further embodiment, a CD3:CD28 ratio of 1:50 is used for the antibody bound to the beads. In another embodiment, a CD3:CD28 ratio of 1:30 is used for the antibody bound to the beads. In a preferred embodiment, a CD3:CD28 ratio of 1:10 is used for the antibody bound to the beads. In another embodiment, a CD3:CD28 ratio of 1:3 is used for the antibody bound to the beads. In yet another embodiment, a CD3:CD28 ratio of 3:1 is used for the antibody bound to the beads.

[0174] Particle to cell ratios of 1:500 to 500:1 and any value therebetween can be used to stimulate T cells or other target cells. As one of skill in the art can readily appreciate, the particle to cell ratio can vary depending on the particle size relative to the target cells. For example, small sized beads can only bind a few cells, while larger beads can bind many cells. In certain embodiments, the cell to particle ratio ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments, the ratio includes 1:9 to 9:1 and any integer value therebetween, and can also be used to stimulate T cells. The ratio of anti-CD3-bound particles and anti-CD28-bound particles to T cells that results in T cell stimulation can vary as described above, but certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least one particle per T cell. In one preferred embodiment, a particle-to-cell ratio of 1:1 or less is used. In one particular embodiment, the preferred particle-to-cell ratio is 1:5. In a further embodiment, the particle-to-cell ratio can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 10:1 on day 1, and then additional particles are added to the cells every day or every other day for up to 10 days, with a final ratio of 1:1 to 1:10 (based on the number of cells on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on day 1 of stimulation, and adjusted to 1:5 on days 3 and 5 of stimulation. In another embodiment, particles are added every day or every other day to a final ratio of 1:1 on day 1 of stimulation, and 1:5 on days 3 and 5 of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on day 1 of stimulation, and adjusted to 1:10 on days 3 and 5 of stimulation. In another embodiment, particles are added every day or every other day to a final ratio of 1:1 on day 1 of stimulation, and 1:10 on days 3 and 5 of stimulation.Those of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, the ratios will vary depending on the particle size and the size and type of cells.

[0175] In a further embodiment of the present invention, cells such as T cells are mixed with beads coated with an agent, then the beads and cells are separated, and then the cells are cultured.In an alternative embodiment, before culturing, the beads coated with an agent and the cells are not separated, but are cultured together.In a further embodiment, the beads and cells are first concentrated by applying a force such as a magnetic force, which increases the ligation of cell surface markers, thereby inducing cell stimulation.

[0176] As an example, cell surface proteins may be ligated by contacting the T cells with anti-CD3 and anti-CD28 conjugated paramagnetic beads (3×28 beads). In one embodiment, the cells (e.g., 10 4 ~10 9T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1:1) are mixed in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Again, one skilled in the art can readily appreciate that any cell concentration can be used. For example, the target cells may be very rare in the sample and constitute only 0.01% of the sample, or the entire sample (i.e., 100%) may constitute the target cells of interest. Thus, any cell number is within the scope of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed (i.e., increase the cell concentration) to ensure maximum contact of the cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, more than 100 million cells / ml is used. In further embodiments, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used. Using high concentrations can increase cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations allows more efficient capture of cells that may weakly express the target antigen of interest, such as CD28 negative T cells. Such cell populations may have therapeutic value, and in certain embodiments, it is desirable to obtain them. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which normally have weaker CD28 expression.

[0177] In one embodiment of the invention, the mixture may be cultured for a period of several hours (about 3 hours) to about 14 days, or any hourly integer value therebetween. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired so that the culture period of the T cells can be 60 days or more. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of hormones and / or cytokines in sufficient amounts for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0178] Non-activated T cells or T cells exposed to various stimulation times can display different characteristics. For example, a typical blood or apheresized peripheral blood mononuclear cell product may contain either cytotoxic T cell populations or suppressor T cell populations (T C , CD8 + ) than the helper T cell population (T H , CD4 + Ex vivo expansion of T cells by stimulating the CD3 and CD28 receptors primarily results in T cells prior to about day 8-9. H The T cell population consists of T cells, whereas after about days 8-9, the T cell population consists of a growing number of T C Therefore, depending on the purpose of the treatment, mainly T H It may be advantageous to inject a population of T cells containing T C When an antigen-specific subset of cells has been isolated, it may be beneficial to expand this subset to a greater extent.

[0179] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers also change significantly, but largely reproducibly, during the cell expansion process, thus enabling the ability to tailor activated T cell products for specific purposes.

[0180] Therapeutic applications In other aspects, the present invention encompasses cells (e.g., T cells) transduced by lentiviral vectors (LV) or other suitable vectors.For example, in some embodiments, the LV encodes the CAR of the present invention, which comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain specifically binds to enriched stereotype BCR.Thus, in other embodiments, the transduced T cells can induce CAR-mediated T cell responses against specific malignant and / or pathogenic B cell clones, for example, in lymphoma, leukemia, or autoimmune disease.

[0181] The present invention provides the use of CARs to redirect the specificity of immune cells (e.g., primary T cells) to antigens present on enriched stereotype BCRs. Thus, the present invention also provides a method for stimulating a T cell-mediated immune response against a target B cell population in a mammal, comprising administering to the mammal T cells expressing a CAR of the present invention.

[0182] In one embodiment, the present invention includes a type of cell therapy in which immune cells (e.g., T cells) are genetically modified to express a CAR (e.g., CARs against VL3-21, VH3-23, and VH1-69 VH4-34) and the CAR immune cells (e.g., CAR T cells) are infused into a recipient in need thereof. The infused cells can target enriched stereotypic B cells in the recipient, for example, to target malignant or pathogenic B cell clones in lymphoma, leukemia, or autoimmune disease, while protecting normal B cells. In some embodiments, unlike antibody therapy, CAR immune cells (e.g., CAR T cells) can replicate in vivo and thus persist long term, thereby resulting in sustained tumor control.

[0183] In one embodiment, the CAR T cells of the present invention can undergo vigorous in vivo T cell expansion and can persist for a long period of time.In another embodiment, the CAR T cells of the present invention evolve into specific memory T cells that can be reactivated to inhibit any new tumor formation or tumor growth.Without being bound to any particular theory, CAR T cells can differentiate into a central memory-like state in vivo when they encounter target cells that express surrogate antigens and subsequently eliminate them.

[0184] The cancer that can be treated or prevented includes blood cancer, such as blood or bone marrow cancer.Examples of hematological (or hematopoietic) cancer include leukemia, including but not limited to acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), or splenic marginal zone lymphoma (SMZL).

[0185] In other embodiments, the CAR cells (e.g., CAR T cells) of the present invention are used alone or in combination with other treatments to reduce or eliminate antigen escape by pathogenic B cells (e.g., cancerous B cells of CLL patients).Thus, in one embodiment, the present invention provides a method for reducing or eliminating antigen escape by B cell cancer in a subject in need thereof, comprising administering to the subject an effective amount of the genetically modified cells disclosed herein.In some embodiments, the B cell cancer is a CD19-negative cancer.In other embodiments, the B cell cancer is a CD19-negative cancer.

[0186] Some specific BCRs (e.g., VH4-34, VH1-69, etc.) are also enriched in autoimmune diseases, such as systemic lupus erythematosus, Crohn's disease, Behcet's disease, eosinophilic polyangiitis granulomatosis, and thrombotic thrombocytopenic purpura (TTP). See, for example, Bashford-Rogers, RJM et al., Nature, 574(7776):122-1261-29 (2019) and Ostertag, EM et al., Transfusion 56:1763-1774 (2016), each of which is incorporated herein by reference in its entirety. In some embodiments, CAR T cells can be developed for patients with B cell-mediated autoimmune diseases. In other embodiments, the advantage of this approach compared to other approaches for autoimmune diseases (e.g., CAART) is that this approach uses a CAR that includes an antigen-binding domain (e.g., scFv) rather than an Ab targeting protein.

[0187] In some embodiments, autoimmune diseases that may be treated or prevented include, but are not limited to, systemic lupus erythematosus (SLE), Crohn's disease (CD), Behcet's disease (BD), eosinophilic granulomatosis with polyangiitis (EGPA), thrombotic thrombocytopenic purpura (TTP), ANCA-associated vasculitis (AAV), immunoglobulin A vasculitis (IgA vasculitis), and immunoglobulin A vasculitis (IgAV).

[0188] The CAR modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with diluents and / or other components, such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may comprise the target cell population described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline and phosphate buffered saline; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants, such as aluminum hydroxide; and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0189] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration are determined based on factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0190] When an "anticancer effective amount," "cancer inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account the individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and pathology. The pharmaceutical compositions comprising the T cells described herein can be administered in doses of 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6It can be generally stated that the T cell composition may be administered at a dosage of 1000 cells / kg body weight (including all integer values ​​within these ranges). The T cell composition may also be administered multiple times at these dosages. The cells can be administered by using injection techniques that are generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting the treatment accordingly.

[0191] In certain embodiments, it may be desirable to administer the activated T cells to a subject, followed by redrawing blood (or performing apheresis), activating T cells derived therefrom in accordance with the present invention, and reinfusing these activated and expanded T cells back into the patient. This procedure can be performed multiple times, every few weeks. In certain embodiments, T cells can be activated from a blood draw of 10cc to 400cc. In certain embodiments, T cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc. Without wishing to be bound by theory, using this multiple blood draw / multiple reinfusion protocol can be useful in selecting specific populations of T cells.

[0192] The administration of the composition of the present invention can be carried out in any convenient manner, including aerosol inhalation, injection, oral ingestion, transfusion, implantation, or transplantation.The composition described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally.In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection.In another embodiment, the T cell composition of the present invention is preferably administered by iv injection.

[0193] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously, or after) with any number of relevant treatment modalities. In further embodiments, the T cells of the invention can be used in combination with plasmapheresis, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapeutics, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cell composition of the present invention is administered to a patient in combination with (e.g., before, simultaneously with, or after) T cell depletion therapy using either bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B cell depletion therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0194] The dosages of the above therapeutic agents administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to practices accepted in the art.

[0195] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0196] Although the present invention has been described in relation to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that other suitable modifications and alterations of the methods described herein may be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Although specific embodiments have been described in detail above, the same will be more clearly understood by reference to the following examples. The examples are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0197] Experimental Example The present invention will be further described in detail by referring to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.Therefore, the present invention should not be interpreted as being limited to the following examples in any way, but rather as embracing any and all variations that become apparent as a result of the teachings provided herein.

[0198] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the methods of the claims. The following examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0199] Example 1 Anti-IGLV3-21 R110 BCR CAR T cells, anti-IGHV1-69 BCR CAR T cells, and anti-IGHV4-34 BCR CAR T cells specifically kill lymphoma cells expressing VL3-21 R110 BCR, VH1-69 BCR, and VH4-34 BCR, respectively Jeko1 cells were generated that were depleted of endogenous BCR and transduced with the appropriate vector to express the following individual enriched stereotype BCRs: VL3-21 R110 BCR ("Jeko1 VL3-21*"), VH1-69 ("Jeko1 VH1-69"), or VH4-34 ("Jeko1 VH4-34"). Jeko1 cells with endogenous BCR ("Jeko1 WT BCR") and CD19 knockout cells ("Jeko1 CD19KO") served as controls.

[0200] Anti-IGLV3-21 R110 BCR CAR T cells ("CART3-21*") were prepared by transducing T cells with the pTRPE AVA L2H CAR vector shown in Figure 5 to express the CAR anti-IGLV3-21 R110 BCR CAR-T cells comprising the amino acid sequence of SEQ ID NO: 47, which contains an scFv (SEQ ID NO: 35) that specifically binds to VL3-21 R110, a CD8 hinge (SEQ ID NO: 54), a CD8 transmembrane domain (SEQ ID NO: 51), an intracellular domain of the costimulatory molecule 4-1 BB (SEQ ID NO: 59), and a CD3 zeta signaling domain (SEQ ID NO: 56).

[0201] When preparing anti-IGHV1-69 BCR CAR T cells ("CART1-69"), the CAR-encoding sequence of the vector pTRPE AVA L2H CAR was replaced with the CAR-encoding sequence of SEQ ID NO: 50 and transduced into T cells to obtain anti-IGHV1-69 BCR CAR-T cells ("CART1-69").

[0202] If anti-IGHV4-34 BCR CAR T cells ("CART4-34") were to be prepared, the CAR-encoding sequence of the vector pTRPE AVA L2H CAR was replaced with the CAR-encoding sequence of SEQ ID NO: X and transduced into T cells to obtain anti-IGHV4-34 BCR CAR-T cells ("CART4-34").

[0203] If anti-IGHV3-23 BCR CAR T cells ("CART3-23") were to be prepared, the CAR-encoding sequence of vector pTRPE AVA L2H CAR was replaced with the CAR-encoding sequence of SEQ ID NO: X and transduced into T cells to obtain anti-IGHV3-23 BCR CAR-T cells ("CART3-23"). Anti-CD19 CAR T cells ("CART19") and untransduced T cells ("UTD") served as positive and negative controls, respectively.

[0204] The cytotoxicity of each Jeko1 cell (Jeko1 WT BCR, Jeko1 VL3-21*, Jeko1 VH1-69, Jeko1 VH4-34, Jeko1 CD19KO) was measured after co-culturing the cells with transduced or non-transduced T cells, as shown in Figure 6. T cells transduced with anti-IGLV3-21 R110 BCR CAR-T cells ("CART3-21*") specifically killed Jeko1 VL3-21* cells expressing VL3-21 R110 BCR while sparing other cells (Figure 6, top graph), and CART3-21* T cells proliferated over time in response specifically to Jeko1 VL3-21 cells (Figure 6, bottom graph). T cells transduced with anti-IGHV1-69 BCR CAR T cells (CART1-69) specifically kill Jeko1 VH1-69 cells expressing the VH1-69 BCR while sparing other cells, and CART1-69 T cells specifically respond to Jeko1 VH1-69 cells and proliferate over time. T cells transduced with anti-IGHV4-34 BCR CAR T cells specifically kill Jeko1 VH4-34 cells expressing the VH4-34 BCR while sparing other cells, and CART4-34 T cells specifically respond to Jeko1 VH4-34 cells and proliferate over time.

[0205] Enumerated Aspects The following listed aspects are provided, but the numbering should not be construed as indicating a degree of importance.

[0206] Embodiment 1 provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain specifically binds to a B cell receptor (BCR). Embodiment 2 provides the CAR of embodiment 1, wherein said antigen binding domain is an antibody or an antigen-binding fragment thereof. Embodiment 3 provides the CAR of embodiment 1 or 2, wherein said antigen-binding fragment is a single chain variable fragment (scFv), an antigen-binding fragment (Fab), or a single domain antibody. Aspect 4 provides the CAR of any one of aspects 1 to 3, wherein the antigen-binding fragment is an scFv.

[0023] Aspect 5 provides a CAR according to any one of aspects 1-4, wherein said enriched stereotype BCR is a membrane-bound protein on B cells or plasma cells.

[0023] Aspect 6 provides a CAR of any one of aspects 1-5, wherein the enriched stereotype BCR comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13-17, 18-20, and 21-24; or an amino acid sequence having at least 85% identity to a sequence set forth in any one of SEQ ID NOs: 13-17, 18-20, and 21-24. Embodiment 7 provides a CAR according to any one of embodiments 1 to 6, wherein the antigen-binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 25 to 30, 31 to 33, 37 to 39, and 43 to 48; or an amino acid sequence having at least 85% identity to a sequence as set forth in any one of SEQ ID NOs: 25 to 30, 31 to 32, 35 to 36, 39 to 42, 64, 66, 68, 69, 76, 78, 80, and 81. Embodiment 8 provides the CAR of any one of embodiments 1 to 7, wherein said transmembrane domain comprises a CD8 transmembrane domain. Embodiment 9 provides the CAR of any one of embodiments 1 to 8, wherein said transmembrane domain comprises the amino acid sequence of SEQ ID NO:51; or an amino acid sequence having at least 85% identity to the sequence depicted in SEQ ID NO:51. Embodiment 10 provides the CAR of any one of embodiments 1-9, wherein said intracellular signaling domain comprises a CD3 zeta signaling domain. Embodiment 11 provides the CAR of any one of embodiments 1 to 10, wherein said intracellular signaling domain comprises an amino acid sequence of SEQ ID NO:56; or an amino acid sequence having at least 85% identity to the sequence depicted in SEQ ID NO:56. Embodiment 12 provides the CAR of any one of embodiments 1 to 11, further comprising an intracellular domain of a costimulatory molecule. Example 13 provides the CAR of Example 12, wherein said costimulatory molecule is 4-1BB.

[0023] Embodiment 14 provides a CAR of embodiment 12 or 13, wherein said intracellular domain comprises an amino acid sequence of SEQ ID NO:59; or an amino acid sequence having at least 85% identity to the sequence shown in SEQ ID NO:59. Embodiment 15 provides a CAR according to any one of embodiments 1 to 14, further comprising a CD8 alpha hinge. Embodiment 16 provides a CAR of embodiment 15, wherein said CD8 alpha hinge comprises the amino acid sequence of SEQ ID NO:54; or an amino acid sequence having at least 85% identity to the sequence depicted in SEQ ID NO:54. Embodiment 17 provides a CAR according to any one of embodiments 1 to 16, further comprising a CD8 signal peptide. Embodiment 18 provides the CAR of embodiment 17, wherein said CD8 signal peptide comprises the amino acid sequence of SEQ ID NO:63; or an amino acid sequence having at least 85% identity to the sequence shown in SEQ ID NO:63. Embodiment 19 provides a CAR according to any one of embodiments 1 to 14, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 47-50; or an amino acid sequence having at least 85% identity to a sequence as set forth in any one of SEQ ID NOs: 47-50 and 72-75. Embodiment 20 provides a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR of any one of embodiments 1 to 19. Embodiment 21 provides a vector comprising the nucleic acid of embodiment 20. Embodiment 22 provides a genetically modified cell comprising a CAR of any one of embodiments 1 to 21. Embodiment 23 provides the genetically modified cell of embodiment 22, wherein said cell is a human T cell. Embodiment 24 provides a pharmaceutical composition comprising a CAR according to any one of embodiments 1 to 19, a nucleic acid molecule according to embodiment 20, a vector according to embodiment 21, or a genetically modified cell according to embodiment 22 or 23, and a pharma- ceutically acceptable excipient. Embodiment 25 provides a method for specifically eliminating enriched stereotypic B cells in a subject in need thereof, the method comprising administering to the subject an effective amount of the genetically modified cells of embodiment 23 or 23. Embodiment 26 provides a method for treating or preventing hematological cancer in a subject in need thereof, comprising administering to the subject an effective amount of a genetically modified cell of embodiment 22 or 23. Example 27 provides the method of Example 26, wherein the hematological cancer is leukemia. Example 28 provides the method of Example 26 or 27, wherein said hematological cancer is chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, or chronic lymphocytic leukemia. Embodiment 29 provides a method for treating or preventing an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of a genetically modified cell of embodiment 22 or 23. Example 30 provides the method of example 29, wherein said autoimmune disease is systemic lupus erythematosus (SLE), Crohn's disease (CD), Behcet's disease (BD), Eosinophilic granulomatosis with polyangiitis (EGPA), Thrombotic thrombocytopenic purpura (TTP), ANCA-associated vasculitis (AAV), IgA vasculitis (IgAV), or IgA vasculitis (IgAV). Example 31 provides the method of any one of Examples 25 to 30, wherein the subject is a human. Embodiment 32 provides the use of a genetically modified cell of embodiment 22 or 23 for the manufacture of a medicament for the treatment or prevention of a hematological cancer or an autoimmune disease in a subject in need thereof.

[0207] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0208] While the present invention has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

[Claim 1] The invention described in the specification of this application.

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