Bispecific chimeric antigen receptors targeting cd19 and bcma

Abstract

The invention relates to a bispecific chimeric antigen receptor (CAR) targeting CD19 and BCMA, the CAR comprising an extracellular antigen-binding domain comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds to CD19 and the second antigen-binding domain binds to BCMA.

Description

Bispecific chimeric antigen receptor targeting CD19 and BCMA Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a bispecific chimeric antigen receptor targeting CD19 and BCMA and its use. Background Art

[0002] CD19 is a 95 kDa transmembrane glycoprotein expressed on the surface of B-cell lineage cells. As a member of the immunoglobulin (Ig) superfamily, it is a component of the B-cell surface signaling complex, participating in the regulation of B-cell receptor signaling and playing a crucial role in B lymphocyte activation. In addition to multipotent hematopoietic stem cells and plasma cells, CD19 is expressed at all stages of B lymphocyte differentiation, even during neoplastic transformation. CD19 is also expressed on the surface of most B-cell lymphomas, mantle cell lymphomas, ALLs, CLLs, and some acute myeloid leukemia cells. CD19 is not expressed on hematopoietic stem cells, a feature that reduces the risk of autoimmune disease and irreversible spinal cord injury with CD19-targeting drugs. Therefore, CD19 is considered a promising immunotherapy target for the treatment of CD19-associated tumors.

[0003] B cell maturation antigen (BCMA), also known as CD269, is a member of the tumor necrosis factor receptor superfamily member 17 (TNFRS17). It consists of 185 amino acid residues and is a type III transmembrane protein with a cysteine-rich extracellular domain that is somewhat conserved. As a cell surface protein expressed exclusively on B cells, BCMA, along with two other members of the TNF receptor superfamily: B cell activation factor (BAFF) and its receptor (BAFF-R) and transmembrane activator and calcium modulator and cyclophilin ligand (TACI), plays a crucial role in regulating B cell proliferation, survival, maturation, and differentiation into plasma cells. Unlike BAFF-R and TACI, BCMA is primarily expressed on the surface of plasma cells or malignant plasma cells. BCMA expression can also be detected on the surface of some memory B cells and plasmacytoid dendritic cells that can differentiate into malignant plasma cells. BCMA is largely unexpressed in naive B cells, pluripotent stem cells, and non-lymphoid tissues. However, it is expressed in almost all multiple myeloma (MM) cell lines, and its expression is higher in malignant plasma cells than in normal plasma cells. This evidence makes it a very promising target for CAR T cell therapy in MM.

[0004] Currently, the first CAR-T drug to be marketed is Kymariah, which targets CD19 and has shown significant long-term efficacy in treating patients with refractory / relapsed B-cell acute lymphoblastic leukemia. The BCMA CAR-T drug, Ikiorensay injection, has an overall response rate (ORR) of 96%.

[0005] Although CD19 CAR-T can effectively target most B cells, it cannot eliminate terminally differentiated plasma cells. BCMA CAR-T can effectively eliminate plasma cells but cannot target naive B cells. The indications for these two single-target CAR-T cell therapies are limited, and tumor cells are prone to immune escape due to reduced target protein expression. Therefore, the development of a chimeric antigen receptor that can simultaneously target both CD19 and BCMA and reduce tumor immune escape caused by reduced target protein expression is the key to solving these problems. Summary of the Invention

[0006] The present application provides a chimeric antigen receptor that can simultaneously target CD19 and BCMA, which can specifically bind to and immune-recognize two different antigens, CD19 and BCMA, and the chimeric antigen receptor can produce an immune response by binding to either CD19 or BCMA, or can simultaneously bind to CD19 and BCMA to produce an immune response.

[0007] Compared with chimeric antigen receptors targeting a single antigen, the chimeric antigen receptor provided in the present application can target CD19 and BCMA. The cell activity of immune cells expressing the chimeric antigen receptor is enhanced, and it has a stronger killing effect. It can effectively kill cells expressing CD19 and / or BCMA. The CD19 and BCMA targets have additive or synergistic effects and have a wider therapeutic range. Simultaneously targeting CD19 and BCMA on the cell surface can reduce the possibility of antigen escape caused by downregulation or deletion of a single surface antigen.

[0008] The chimeric antigen receptor targeting CD19 and BCMA provided in the present application can have one or more effects selected from the following: (1) excellent expression efficiency and appropriate copy number; (2) cells expressing the chimeric antigen receptor have significant target cell killing effect; (3) cells expressing the chimeric antigen receptor have sustained target cell killing effect; (4) cells expressing the chimeric antigen receptor have significant cell activation ability; and (5) cells expressing the chimeric antigen receptor have significant cytokine secretion ability.

[0009] In one aspect, the present application provides a chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain, wherein the extracellular antigen binding domain comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds CD19, the second antigen binding domain binds BCMA, and:

[0010] 1) the first antigen-binding domain comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3; and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and

[0011] 2) The second antigen-binding domain comprises a second heavy chain variable region (VH), and the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2.

[0012] In certain embodiments, the extracellular antigen-binding domain further comprises a linker, which connects the first heavy chain variable region, the first light chain variable region and the second heavy chain variable region.

[0013] In certain embodiments, the linker comprises a β-stranded linker and / or a G4S linker.

[0014] In certain embodiments, the G4S linker comprises a G4S3 linker and / or a G4S2 linker.

[0015] In certain embodiments, the β-stranded linker comprises the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0016] In certain embodiments, the G4S3 linker comprises the amino acid sequence shown in SEQ ID NO:4.

[0017] In certain embodiments, the G4S2 linker comprises the amino acid sequence shown in SEQ ID NO:5.

[0018] In certain embodiments, the extracellular antigen binding domain comprises from N-terminus to C-terminus:

[0019] 1) a first heavy chain variable region, a linker, a first light chain variable region, a linker, and a second heavy chain variable region;

[0020] 2) a first heavy chain variable region, a linker, a second heavy chain variable region, a linker, and a first light chain variable region;

[0021] 3) a second heavy chain variable region, a linker, a first light chain variable region, a linker, and a first heavy chain variable region;

[0022] 4) a second heavy chain variable region, a linker, a first heavy chain variable region, a linker, and a first light chain variable region;

[0023] 5) a first light chain variable region, a linker, a second heavy chain variable region, a linker, and a first heavy chain variable region; or

[0024] 6) A first light chain variable region, a linker, a first heavy chain variable region, a linker, and a second heavy chain variable region.

[0025] In certain embodiments, the extracellular antigen-binding domain comprises, from N-terminus to C-terminus:

[0026] 1) a first light chain variable region, a G4S2 linker, a second heavy chain variable region, a G4S2 linker, and a first heavy chain variable region;

[0027] 2) a first light chain variable region, a G4S3 linker, a second heavy chain variable region, a G4S3 linker, and a first heavy chain variable region;

[0028] 3) a first light chain variable region, a beta-sheet linker, a second heavy chain variable region, a beta-sheet linker, and a first heavy chain variable region; or

[0029] 4) a second heavy chain variable region, a G4S3 linker, a first light chain variable region, a G4S3 linker, and a first heavy chain variable region.

[0030] In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-11.

[0031] In certain embodiments, it further comprises a hinge region, a transmembrane domain, a co-stimulatory domain and / or a signaling domain.

[0032] In certain embodiments, it comprises, from N-terminus to C-terminus, an extracellular antigen-binding domain, a transmembrane domain, a hinge region, a costimulatory domain, and a signaling domain.

[0033] In certain embodiments, the hinge region comprises a hinge region derived from one or more of the following proteins: CD28, IgG1, IgG4, IgD, CD137, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, and LIGHT.

[0034] In certain embodiments, the hinge region comprises a CD8 hinge region.

[0035] In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from one or more of the following proteins: CD8, CD28, CD3e, CD3ε, CD137, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, and SLAM.

[0036] In certain embodiments, the transmembrane domain comprises a CD8 transmembrane domain.

[0037] In certain embodiments, the costimulatory domain comprises a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88.

[0038] In certain embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain.

[0039] In certain embodiments, the signaling domain comprises a signaling domain derived from the following group of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12, and a domain protein comprising at least one ITAM.

[0040] In certain embodiments, the signaling domain comprises a CD3zeta signaling domain.

[0041] In certain embodiments, the CAR comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12-15.

[0042] In another aspect, the present application provides an isolated nucleic acid molecule encoding the CAR described herein.

[0043] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 16-19.

[0044] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.

[0045] In certain embodiments, the vector comprises a viral vector.

[0046] In certain embodiments, the vector comprises a lentiviral vector.

[0047] On the other hand, the present application provides a cell comprising and / or expressing the CAR described in the present application, the isolated nucleic acid molecule described in the present application, and / or the vector described in the present application.

[0048] In certain embodiments, the cells comprise immune effector cells.

[0049] In certain embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, γδ T cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0050] In certain embodiments, the cells comprise T cells.

[0051] In certain embodiments, the cells comprise human cells.

[0052] On the other hand, the present application provides a pharmaceutical composition comprising the CAR described herein, the isolated nucleic acid molecule described herein, the vector described herein, and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0053] On the other hand, the present application provides a CAR described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, and / or the cell described in the present application for preparing a drug, wherein the drug is used to treat a disease or condition associated with the expression of CD19 and / or BCMA.

[0054] On the other hand, the present application provides a CAR described herein, the isolated nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein for use in treating diseases or conditions associated with the expression of CD19 and / or BCMA.

[0055] On the other hand, the present application provides a method for treating a disease or condition associated with the expression of CD19 and / or BCMA, comprising administering to a subject in need thereof an effective amount of the CAR described herein, the isolated nucleic acid molecule described herein, the vector described herein, the immune effector cell described herein and / or the pharmaceutical composition described herein.

[0056] In certain embodiments, the disease or condition comprises a tumor, a hematological disease, and / or an autoimmune disease.

[0057] In certain embodiments, the disease or condition comprises solid tumors and non-solid tumors.

[0058] In certain embodiments, the disease comprises lymphoma and / or leukemia.

[0059] In certain embodiments, the disease comprises multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, follicular lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, megakaryocytic leukemia, Burkitt's lymphoma, anaplastic large cell lymphoma and / or mucosa-associated lymphoid tissue lymphoma.

[0060] In certain embodiments, the disease comprises chronic Guillain-Barré syndrome, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, Sjögren's syndrome, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, demyelinating disease, autoimmune hemolysis, idiopathic thrombocytopenic purpura and / or idiopathic leukopenia.

[0061] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0063] FIG1A-FIG1B show the proliferation of T cells expressing the chimeric antigen receptor described in the present application.

[0064] Figures 2A-2F show the killing effect of T cells expressing the chimeric antigen receptor described in this application on tumor cells.

[0065] Figures 3A-3E show the secretion of cytokines after co-culture of T cells expressing the chimeric antigen receptor described in the present application with tumor cells. DETAILED DESCRIPTION

[0066] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0067] Definition of terms

[0068] In this application, the term "chimeric antigen receptor (CAR)" generally refers to an artificial cell receptor that is engineered to be able to express and specifically bind to an antigen on an immune effector cell. CAR can be used as a therapy for adoptive cell transfer. In this application, CAR may include an extracellular domain, a transmembrane domain, and an intracellular domain. CAR may further include a signal peptide. Among them, the extracellular domain of CAR is generally capable of binding to a target antigen. For example, the extracellular domain of the CAR may include an antigen binding domain and a hinge region, and the intracellular domain of the CAR may include a costimulatory domain and a signaling domain.

[0069] In this application, the term "bispecific" generally refers to a chimeric antigen receptor that can bind to at least two targets and has specificity for two different targets. For example, a bispecific CAR can contain two antigen binding domains, each of which is specific for a different antigenic determinant. For example, the two targets can be located on the same cell surface or on different cell surfaces.

[0070] In this application, the term "extracellular antigen binding domain" or "antigen binding domain" generally refers to a domain capable of binding to a target antigen. The antigen binding domain may comprise a domain capable of specifically binding to an antibody or an antigen-binding fragment thereof. The antigen binding domain may be of natural, synthetic, semi-synthetic, or recombinant origin. For example, the antigen binding domain may comprise a single-chain antibody or an antigen-binding fragment thereof. For example, the antigen binding domain may comprise an scFv. For example, the antigen binding domain may comprise a single-domain antibody or an antigen-binding fragment thereof.

[0071] In this application, the term "antibody" generally refers to an immunoglobulin or its fragment or derivative, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. The term "antibody" may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function. In this application, the term "heavy chain variable region" or "VH" generally refers to the region of the antibody heavy chain structure that includes the heavy chain complementary determining regions CDR1, CDR2, CDR3 and the framework regions FR1, FR2, FR3, and FR4. The heavy chain variable region described herein may include the heavy chain variable region of a VHH antibody. In this application, when describing the heavy chain variable region of a VHH antibody, VHH may be used to refer to it. In this application, the term "light chain variable region" or "VL" generally refers to the region of the antibody light chain structure including the light chain complementary determining regions CDR1, CDR2, CDR3 and the framework regions FR1, FR2, FR3, FR4.

[0072] In the present application, the term "linker" generally refers to a portion or group of portions that join or connect two or more discrete, separate monomer domains. The linker described herein may include a peptide linker of natural and / or synthetic origin, which may be composed of a linear amino acid chain. In the present application, the different domains of CAR may be connected by a linker, and the subregions in a certain domain of CAR may also be connected by a linker. The linker described herein may include a natural linker and an artificially designed linker. In certain embodiments, the linker described herein may be a linker present in a natural protein structure or a functional variant thereof. In certain embodiments, the linker described herein may be a β-stranded linker. In certain embodiments, the linker described herein may be a G4S linker comprising an amino acid sequence (Gly-Gly-Gly-Ser) n, wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In the present application, a G4S3 linker generally refers to a linker comprising the amino acid sequence (Gly-Gly-Gly-Ser)n, n=3; a G4S2 linker generally refers to a linker comprising the amino acid sequence (Gly-Gly-Gly-Ser)n, n=2.

[0073] As used herein, the term "β-stranded linker" generally refers to a structural element that connects two β-sheets. β-sheets are a type of protein secondary structure and refer to parallel or antiparallel chain structures formed by amino acid chains. The β-sheet linker described herein may comprise the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

[0074] In the present application, the term "transmembrane domain" can be used interchangeably with "transmembrane region (abbreviated as TM)", and generally refers to the domain of a peptide, polypeptide or protein that can span the plasma membrane of a cell. In CAR, the hinge region enables the fusion of the extracellular antigen binding domain and the intracellular domain, and enables CAR to be anchored to the plasma membrane of immune effector cells. The transmembrane region in the CAR described herein can be obtained from natural proteins, or can be synthesized, semi-synthesized or recombinantly obtained. The transmembrane domain in the CAR described herein can be derived from a transmembrane domain in a natural transmembrane protein, a functionally active fragment thereof, a truncation thereof and / or a mutant variant thereof. In the present application, the term "CD8 transmembrane domain" generally refers to the transmembrane domain of CD8, a functionally active fragment thereof, a truncation thereof and / or a mutant variant thereof.

[0075] In this application, the term "hinge region" generally refers to a binding domain that plays a role in positioning the antigen binding domain away from the effector cell surface in a chimeric antigen receptor to enable appropriate cell / cell contact, antigen binding and activation. In this application, the hinge region can be located between the binding domain and the transmembrane domain. The hinge region can be derived from natural sources, synthetic sources, semi-synthetic sources or recombinant sources. In this application, the term "CD8 hinge region" generally refers to any polypeptide comprising an amino acid sequence that has sequence identity or similarity to a naturally occurring CD8 hinge region sequence.

[0076] In the present application, the term "intracellular domain" can be used interchangeably with "intracellular domain", generally referring to the intracellular part inside the molecule. For example, the intracellular domain of CAR described in the present application may include a costimulatory domain and a signal transduction domain. In the present application, the term "costimulatory domain" generally refers to a part of CAR, which can provide the second signal required for immune effector cell activation. The costimulatory domain in CAR can be derived from the natural costimulatory molecules on the surface of immune effector cells. In the present application, the term "signaling domain" generally refers to a part of CAR, which participates in the information transduction of effective CAR binding antigen polypeptides into the inside of immune effector cells, to trigger the function of effector cells, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the target cells bound by CAR or other cellular responses triggered by the antigen bound to the extracellular CAR domain.

[0077] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to a viral genome. Some vectors can autonomously replicate (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell they introduce. In the present application, the term "viral vector" generally refers to a non-wild type recombinant viral particle serving as a gene delivery vector and comprising the recombinant viral genome packaged in a viral capsid. The viral species used as a vector can include but is not limited to retroviruses (including slow viruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (for example SV40).

[0078] In this application, the term "immune effector cell" generally refers to cells that participate in an immune response, such as cells that promote an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that are genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0079] In this application, the terms "T lymphocyte" and "T cell" can be used interchangeably and generally refer to thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells can be helper T (Th) cells, such as helper T1 (Th1) cells or helper T2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells) CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells or CD4-CD8- T cells. Alternatively, the T lymphocytes can include naive T cells and memory T cells.

[0080] In this application, the term "killing ability" generally refers to killing of cells by contacting the cells with an effective amount of an antibody, immunoconjugate, bispecific / multispecific molecule or composition. The method can include killing cells that express an antigen, optionally in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0081] In this application, the term "cytokine" generally refers to a protein released by one cell population that acts as an intercellular regulator on another cell. For example, cytokines can include interleukins (IL), interferons (IFN), chemokines, tumor necrosis factors (TNF), and transforming growth factors (TGF).

[0082] In this application, the term "pharmaceutical composition" generally refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is to be administered. In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers.

[0083] In this application, the term "subject" generally refers to an animal, typically a mammal, such as a human, non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects can include fetuses, newborns, infants, adolescents, and adult subjects. Subjects can include animal disease models.

[0084] As used herein, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from the disease.

[0085] In this application, the term "tumor" generally refers to a physiological condition characterized by unregulated cell growth. In this application, a tumor can be benign or malignant. In this application, a tumor can be a solid tumor or a non-solid tumor. In this application, the terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein. In some cases, the term "cancer" is used to refer to a malignant tumor.

[0086] In this application, the term "hematologic disease" generally refers to a pathological condition that primarily or primarily affects the blood and hematopoietic organs. Hematologic diseases may involve abnormal production, function, or destruction of blood cells (including red blood cells, white blood cells, and platelets), as well as disorders of coagulation and bleeding mechanisms.

[0087] As used herein, the term "autoimmune disease" generally refers to a disorder or condition resulting from an autoimmune response mediated by antibodies directed against self-antigens. Autoimmune diseases result in the production of inappropriately produced and / or overproduced autoantibodies directed against self-antigens or autoantigens. Autoimmune diseases are systemic diseases that can occur in almost any part of the body, including the nervous system, gastrointestinal system, endocrine system, skin, skeletal system, and vascular tissue.

[0088] In this application, the term "treat" includes not only treating the disease, but also generally includes slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treated patient.

[0089] In this application, the term "comprises" or "comprising" generally means that the features specifically stated are included, but other elements are not excluded.

[0090] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0091] Detailed Description of the Invention

[0092] On the one hand, the application provides a kind of chimeric antigen receptor (CAR), the CAR has the ability of simultaneously targeting CD19 and BCMA.In certain embodiments, the CAR has the dual specificity to CD19 and BCMA.

[0093] In the present application, the chimeric antigen receptor comprises an extracellular antigen-binding domain, which comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain binds to CD19 and the second antigen-binding domain binds to BCMA.

[0094] In the present application, the first antigen binding domain may be greater than or equal to about 10 5 M -1 (For example, greater than or equal to about 10 5 M -1 , greater than or equal to about 10 6 M -1, greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , greater than or equal to about 10 9 M -1 , greater than or equal to about 10 10 M -1 , greater than or equal to about 10 11 M -1 , greater than or equal to about 10 12 M -1 , greater than or equal to about 10 13 M -1 or greater) of Ka (i.e., the equilibrium association constant for the binding interaction, which is 1 / M); or, -5 M (e.g., less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, less than or equal to about 10 -9 M, less than or equal to about 10 -10 M, less than or equal to about 10 -11 M, less than or equal to about 10 -12 M, less than or equal to about 10 -13 The present invention binds or associates with CD19 with an equilibrium dissociation constant, Kd, ​​of 4 M or less.

[0095] In the present application, the second antigen binding domain may be greater than or equal to about 10 5 M -1 (For example, greater than or equal to about 10 5 M -1 , greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , greater than or equal to about 10 9 M -1 , greater than or equal to about 10 10 M -1 , greater than or equal to about 10 11 M -1 , greater than or equal to about 10 12 M -1 , greater than or equal to about 10 13 M -1 or greater) of Ka (i.e., the equilibrium association constant for the binding interaction, which is 1 / M); or,-5 M (e.g., less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, less than or equal to about 10 -9 M, less than or equal to about 10 -10 M, less than or equal to about 10 -11 M, less than or equal to about 10 -12 M, less than or equal to about 10 -13 The present invention binds or associates with BCMA with an equilibrium dissociation constant, Kd, ​​of 4 M or less.

[0096] In the present application, the affinity of the first antigen-binding domain to CD19, and the affinity of the second antigen-binding domain to BCMA can be determined using routine techniques in the art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assay using a labeled ligand, or using a surface plasmon resonance device (such as Biacore T100, which is available from Biacore, Inc., Piscataway, NJ) or optical biosensor technology.

[0097] In certain embodiments, the first antigen binding domain comprises a first heavy chain variable region (VH) and a first light chain variable region (VL).

[0098] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3 or a functional variant thereof, wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:3; and a protein or polypeptide having more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence shown in SEQ ID NO:3.

[0099] In certain embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:1; and a protein or polypeptide having more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence shown in SEQ ID NO:1.

[0100] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1.

[0101] In certain embodiments, the second antigen-binding domain comprises a second heavy chain variable region (VH). In certain embodiments, the second antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof, wherein the functional variant is selected from the following group: a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 2; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) to the amino acid sequence shown in SEQ ID NO: 2.

[0102] In certain embodiments, the extracellular antigen-binding domain of the chimeric antigen receptor comprises a first antigen-binding domain and a second antigen-binding domain, without limitation to any connection method or connection order. In certain embodiments, the first antigen-binding domain is directly or indirectly connected to the second antigen-binding domain. For example, the first antigen-binding domain is connected to the second antigen-binding domain via a linker.

[0103] In certain embodiments, the extracellular antigen binding domain of the chimeric antigen receptor comprises the first antigen binding domain and the second antigen binding domain, wherein the first antigen binding domain comprises a first heavy chain variable region and a first light chain variable region, wherein the second antigen binding domain comprises a second heavy chain variable region. In certain embodiments, the extracellular antigen binding domain comprises the first heavy chain variable region, the first light chain variable region and the second heavy chain variable region, without being limited to any connection mode and connection order. For example, the first heavy chain variable region, the first light chain variable region and the second heavy chain variable region are connected by a connector. For example, from the N-terminus to the C-terminus in the extracellular antigen-binding domain, the order of the variable regions can be: 1) the first heavy chain variable region, the first light chain variable region, the second heavy chain variable region; 2) the first heavy chain variable region, the second heavy chain variable region, the first light chain variable region; 3) the first light chain variable region, the first heavy chain variable region, the second heavy chain variable region; 4) the first light chain variable region, the second heavy chain variable region, the first heavy chain variable region; 5) the second heavy chain variable region, the first heavy chain variable region, the first light chain variable region; or 6) the second heavy chain variable region, the first light chain variable region, the first heavy chain variable region.

[0104] In certain embodiments, the extracellular antigen binding domain of the chimeric antigen receptor comprises a linker connecting the first heavy chain variable region, the first light chain variable region, and the second heavy chain variable region.

[0105] In certain embodiments, the linker comprises a β-stranded linker and / or a G4S linker. For example, the β-stranded linker may include, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. For example, the G4S linker may comprise an amino acid sequence (Gly-Gly-Gly-Ser)n, wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In certain embodiments, the G4S linker may comprise a G4S2 linker comprising an amino acid sequence (Gly-Gly-Gly-Ser)n, wherein n=2. In certain embodiments, the G4S linker may comprise a G4S3 linker comprising an amino acid sequence (Gly-Gly-Gly-Ser)n, wherein n=3. In certain embodiments, the G4S linker may comprise, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.

[0106] In certain embodiments, the extracellular antigen-binding domain of the chimeric antigen receptor comprises one, two, or more linkers. When the extracellular antigen-binding domain comprises two or more linkers, they may be identical, partially identical, or completely different linkers.

[0107] In certain embodiments, the extracellular antigen binding domain of the chimeric antigen receptor comprises, from N-terminus to C-terminus:

[0108] 1) a first heavy chain variable region, a linker, a first light chain variable region, a linker, and a second heavy chain variable region;

[0109] 2) a first heavy chain variable region, a linker, a second heavy chain variable region, a linker, and a first light chain variable region;

[0110] 3) a second heavy chain variable region, a linker, a first light chain variable region, a linker, and a first heavy chain variable region;

[0111] 4) a second heavy chain variable region, a linker, a first heavy chain variable region, a linker, and a first light chain variable region;

[0112] 5) a first light chain variable region, a linker, a second heavy chain variable region, a linker, and a first heavy chain variable region; or

[0113] 6) a first light chain variable region, a linker, a first heavy chain variable region, a linker, and a second heavy chain variable region;

[0114] The linker can be independently selected from a β-stranded linker, a G4S2 linker and a G4S3 linker.

[0115] In certain embodiments, the extracellular antigen binding domain of the chimeric antigen receptor comprises, from N-terminus to C-terminus:

[0116] 1) a first light chain variable region, a G4S2 linker, a second heavy chain variable region, a G4S2 linker, and a first heavy chain variable region;

[0117] 2) a first light chain variable region, a G4S3 linker, a second heavy chain variable region, a G4S3 linker, and a first heavy chain variable region;

[0118] 3) a first light chain variable region, a beta-sheet linker, a second heavy chain variable region, a beta-sheet linker, and a first heavy chain variable region; or

[0119] 4) a second heavy chain variable region, a G4S3 linker, a first light chain variable region, a G4S3 linker, and a first heavy chain variable region.

[0120] In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-11 or a functional variant thereof, wherein the functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in any one of SEQ ID NOs: 8-11; and a protein or polypeptide having a sequence identity of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) to the amino acid sequence shown in any one of SEQ ID NOs: 8-11.

[0121] In the present application, the chimeric antigen receptor described herein may further comprise an optional signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain and / or a signaling domain. In certain embodiments, a linker may be included between the domains of the chimeric antigen receptor.

[0122] In the present application, the extracellular antigen binding domain of the chimeric antigen receptor can be connected to the transmembrane domain through a hinge region. For example, the chimeric antigen receptor may include one or more hinge regions between the binding domain and the transmembrane domain. For example, the hinge region may include a hinge region derived from one or more of the following histones: CD28, IgG1, IgG4, IgD, CD137, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30 and LIGHT. In certain embodiments, the chimeric antigen receptor comprises a CD8 hinge region, and the CD8 hinge region may comprise the amino acid sequence shown in SEQ ID NO: 21.

[0123] In the present application, the chimeric antigen receptor may comprise one or more transmembrane domains, and the transmembrane domain may be located at the C-terminus of the hinge region. For example, the transmembrane domain may include a transmembrane domain derived from one or more of the following histones: CD8, CD28, CD3e, CD3ε, CD137, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, and SLAM. In certain embodiments, the chimeric antigen receptor comprises a CD8 transmembrane domain, and the CD8 transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO: 22.

[0124] In the present application, the chimeric antigen receptor may include one or more costimulatory domains, and the costimulatory domains may be located at the C-terminus of the transmembrane domain. For example, the costimulatory domain may include a costimulatory domain derived from one or more of the following histones: CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88. In certain embodiments, the chimeric antigen receptor includes a 4-1BB costimulatory domain, and the 4-1BB costimulatory domain may include the amino acid sequence shown in SEQ ID NO: 23.

[0125] In the present application, the chimeric antigen receptor may comprise one or more signaling domains, and the signaling domains may be located at the C-terminus of the costimulatory domain. In the present application, the signaling domain may comprise a signaling domain derived from one or more of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12, and a domain protein comprising at least one ITAM. In certain embodiments, the chimeric antigen receptor comprises a CD3ζ signaling domain, and the CD3ζ signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0126] In the present application, the CAR may further comprise a signal peptide. In the present application, the signal peptide may comprise a signal peptide selected from CD8 and CD45.

[0127] In certain embodiments, the chimeric antigen receptor described herein comprises, from N-terminus to C-terminus, a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain. In certain embodiments, the chimeric antigen receptor described herein may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 12-15 or a functional variant thereof, wherein the functional variant is selected from the following group: a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence as set forth in any one of SEQ ID NOs: 12-15; and a protein or polypeptide having a sequence identity of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) to the amino acid sequence as set forth in any one of SEQ ID NOs: 12-15.

[0128] In another aspect, the present application provides an isolated nucleic acid molecule that encodes the chimeric antigen receptor described herein.

[0129] In certain embodiments, the nucleic acid molecules described herein comprise the nucleotide sequence shown in any one of SEQ ID NOs: 16-19, or a nucleotide sequence having more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to any one of SEQ ID NOs: 16-19.

[0130] In another aspect, the present application provides a vector comprising a nucleic acid molecule capable of encoding the chimeric antigen receptor described herein. In certain embodiments, the vector described herein is capable of expressing the chimeric antigen receptor described herein in a cell.

[0131] In the present application, the vector may further comprise a replication origin, a selection cassette, a promoter, an enhancer, a translation initiation signal (Shine Dalgarno sequence or Kozak sequence), an intron, a polyadenylation sequence, and one or more of 5' and 3' untranslated regions. In the present application, the vector may be selected from a plasmid, a phage, an artificial chromosome (e.g., a yeast artificial chromosome YAC), and a viral vector. The viral vector may include, but is not limited to, a lentiviral vector, an adenoviral vector, and a retroviral vector.

[0132] In another aspect, the present application provides a cell comprising the chimeric antigen receptor described herein, the nucleic acid molecule described herein, and / or the vector described herein. For example, the cell provided herein can be an artificially modified cell.

[0133] In the present application, the cells may include immune effector cells. For example, the immune effector cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, γδT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells. For example, the immune effector cells are T cells. In the present application, the method may include a step of obtaining the immune effector cells from a subject. For example, the T lymphocytes may be obtained from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and a tumor. In addition, the method may further include a step of selecting a specific cell subset from the immune effector cells. For example, a specific T cell subset may be selected by the specific expression of CD3, CD28, CD4, CD8, CD45RA, and CD45RO. In certain embodiments, the cells include human cells. For example, the cells described herein may be modified human cells.

[0134] In another aspect, the present application provides a method for preparing the cell described herein, comprising introducing the chimeric antigen receptor described herein, the nucleic acid molecule described herein and / or the vector described herein into the cell.

[0135] In another aspect, the present application provides a composition comprising the chimeric antigen receptor described herein, the nucleic acid molecule described herein, the vector described herein, and / or the cell described herein. In certain embodiments, the composition is a pharmaceutical composition comprising the chimeric antigen receptor described herein, the nucleic acid molecule described herein, the vector described herein, and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0136] In the present application, the pharmaceutically acceptable carrier can be selected from the following groups: excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants and emulsifiers.

[0137] In certain embodiments, the bioactive ingredient in the pharmaceutical compositions described herein is in an effective amount. The effective amount can be the minimum dose required to achieve a beneficial or desired preventive or therapeutic effect. For example, the effective amount can be affected by factors such as the subject's disease severity, age, weight, and gender.

[0138] On the other hand, the present application provides a use of the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and / or the cell described in the present application in the preparation of a drug.

[0139] In certain embodiments, the medicament is for treating a disease or condition associated with the expression of CD19 and / or BCMA.

[0140] On the other hand, the present application provides a use of the chimeric antigen receptor described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein in treating diseases or conditions related to the expression of CD19 and / or BCMA.

[0141] On the other hand, the present application provides a method for treating a disease or condition associated with the expression of CD19 and / or BCMA, comprising administering to a subject in need thereof an effective amount of the chimeric antigen receptor described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein and / or the pharmaceutical composition described herein.

[0142] In the present application, the diseases or disorders associated with the expression of CD19 and / or BCMA include disorders in which CD19-positive and / or BCMA-positive cells participate or are mediated.

[0143] In certain embodiments, the disease or condition associated with the expression of CD19 and / or BCMA includes a tumor associated with the expression of CD19 and / or BCMA. For example, the tumor includes a solid tumor and a non-solid tumor. In certain embodiments, the disease or condition associated with the expression of CD19 and / or BCMA includes a hematological disease associated with the expression of CD19 and / or BCMA. In certain embodiments, the disease or condition associated with the expression of CD19 and / or BCMA includes an autoimmune system disease associated with the expression of CD19 and / or BCMA.

[0144] For example, the disease or condition associated with the expression of CD19 and / or BCMA may include lymphoma and / or leukemia associated with the expression of CD19 and / or BCMA. For example, the disease or condition associated with the expression of CD19 and / or BCMA may include multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, follicular lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, megakaryocytic leukemia, Burkitt's lymphoma, anaplastic large cell lymphoma and / or mucosa-associated lymphoid tissue lymphoma.

[0145] For example, the disease or condition associated with the expression of CD19 and / or BCMA may include chronic Guillain-Barré syndrome, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, Sjögren's syndrome, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, demyelinating disease, autoimmune hemolysis, idiopathic thrombocytopenic purpura and / or idiopathic leukopenia.

[0146] Without intending to be bound by any theory, the following examples are merely intended to illustrate the chimeric antigen receptor, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0147] Example

[0148] The present application will be further described by the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are based on conventional conditions in this area, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are commercially available.

[0149] The key materials used in the examples of this application are shown in Tables 1 to 3.

[0150] Table 1 Key Materials List (Plasmid)

[0151] Table 2 Key Materials List (Virus)

[0152] Table 3 Key material list (cell)

[0153] Example 1 Construction of CD19-BCMA Dual-Target CAR Vector

[0154] Construction of Pre-Lenti EF1 CAR V2 WPmut Lentiviral Vector:

[0155] (1) The 5'LTR of the shuttle plasmid pRRLSIN.cPPT.PGK-GFP.WPRE (the plasmid map can be found at www.addgene.org / 12252 / ) is the RSV promoter. Changing the 5'LTR promoter from RSV to CMV facilitates efficient transcription of viral genome sequences; the U3 region of the original 3'LTR plasmid has been deleted (3'LTR△

[0156] U3), has been designed for self-inactivation (SIN), and the 3'LTR sequence remains unchanged.

[0157] (2) The hPGK promoter driving the expression of exogenous transgenes was replaced with the EF1 promoter, and the EGFP gene was replaced with a polyclonal sequence;

[0158] (3) The ampicillin resistance gene was replaced with the kanamycin resistance gene from the pUC57-Kan (GenBank: LT671993.1) plasmid;

[0159] (4) The WPRE element of pRRLSIN.cPPT.PGK-GFP.WPRE was modified to prevent the expression of the truncated protein X. Specifically, within the sequence of the wild-type WPRE element, the promoter driving the transcription of the protein X was deleted, and the A in the start codon ATG was changed to T. The new sequence was named WPREmut.

[0160] The above-mentioned modified shuttle plasmid was named Pre-Lenti-EF1a-CAR V2 WPmut (its sequence is shown in SEQ ID NO: 20). On this basis, a plasmid of a CAR that can simultaneously target CD19 and BCMA was constructed. The CAR includes a scFv sequence targeting CD19 and a VHH sequence targeting BCMA, respectively connected with βS linker, (G4S) 2linker or (G4S) 3linker as the antigen binding domain, and the intracellular domain of 4-1BB and CD3ζ molecules as the cytoplasmic region. Different CAR sequences were constructed into the Pre-Lenti-EF1a-CAR V2 WPmut lentiviral vector to obtain a CD19-BCMA dual-target CAR plasmid. Different CARs and their amino acid sequences, antigen binding domain structures and their amino acid sequences and corresponding plasmids are shown in Table 4.

[0161] Table 4 CD19-BCMA dual-target CAR plasmids

[0162] Example 2 Preparation of CD19-BCMA Dual-Target CAR Lentivirus

[0163] In this example, a lentivirus containing a plasmid encoding a CAR that can simultaneously target CD19 and BCMA was prepared.

[0164] The plasmids containing different CARs in Example 1 were mixed with packaging plasmid ZL004: envelope plasmid ZL003: packaging plasmid ZL006 in a ratio of 7:5:3:5 (plasmids ZL004, ZL003 and ZL006 are referenced to the Chinese patent application with publication number CN111979230A). The mixture of the above four plasmids was added to a centrifuge tube containing 293TS basal medium, and PEIpro was added to another centrifuge tube according to the ratio of plasmid: PEIpro = 1:2. After incubating the two tubes at room temperature for 5 minutes, the mixture containing PEIpro was slowly added to the mixture containing the plasmid, the centrifuge tube was gently shaken to mix evenly, and incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to a centrifuge tube with a density of 5.0-8.0×10 6 Cells were suspended in a 293TS cell suspension at 100 cells / ml. Six to eight hours after transfection, the cells were fed with OPM-CHO PFF06 feed medium containing glutamine. After 48 hours, the supernatant was collected to obtain a crude lentiviral solution. The crude lentiviral solution was centrifuged, digested with enzymes, concentrated by ultrafiltration, and sterilized by filtration. The lentiviral solution was then mixed at a ratio of 9:1 virus solution to HSA (20% HSA) to obtain the lentivirus. The prepared lentivirus was aliquoted and stored at -80°C. Flow cytometry was used to determine the transduction titer of the lentivirus. The results are shown in the following table:

[0165] Table 5 Lentiviral transduction titer

[0166] Example 3 Preparation of CD19-BCMA Dual-Target CAR-T Cells

[0167] In this example, T cells expressing the CAR described in this application that can simultaneously target CD19 and BCMA were prepared.

[0168] Peripheral blood was collected from healthy volunteers and CD3-positive T cells were purified using Miltenyi CD3 Regent. The time was recorded as the first day (D1). TexMACS GMP Medium (containing 5% serum replacement) was used as the cell culture medium, and TransAct CD3 / CD28 activation reagent (1:100) and IL-2 (200 IU / ml) were used to activate T cells. After 24 hours of T cell activation, the cells were infected with different CAR lentiviruses prepared in Example 2 (MOI=2). On the 7th day after activation (D7), an appropriate amount of cells was taken from each group to detect the CAR positivity rate; D3 was used to replenish the fluid and replenish the culture medium of the original culture volume. The cell density was adjusted to 5×10 on D5 and D7. 5 cells / mL and continue culturing. On D9, cell killing and cytokine secretion detection experiments were performed.

[0169] During the culture period, the cell expansion times are shown in Figures 1A and 1B. The results indicate that T cells expressing the CAR described in this application that can simultaneously target CD19 and BCMA can proliferate normally. (G4S) n The expansion folds of CAR-T (n=2 or 3) dual-target CAR-T at D5, D7, and D9 of culture were comparable to those of Mock-T and higher than those of βS CAR-T.

[0170] Example 4 CD19-BCMA dual-target CAR-T cell positive rate detection and copy number detection

[0171] CAR positivity rate and CAR copy number per transduced cell are important indicators for evaluating efficacy and safety. Due to their unique structure, dual-target CAR-T cells often have low CAR transduction efficiency or excessively high CAR copy number that does not meet the standard.

[0172] To evaluate the expression of dual-target CAR, this example used flow cytometry to detect the CAR positivity rate on day 7 after activation of T cells from different donors. The test results are shown in Table 6 below. The positivity rate of the dual-target CAR-T group was above 30%, and the transduction efficiency of the CAR containing the G4S linker was higher than that of the CAR containing the βS linker.

[0173] Table 6 CAR positive rate detection values ​​of each group

[0174] The qPCR method is used to detect the copy number of dual-target CAR-T. The brief detection steps are as follows:

[0175] 1. Take the CAR-T cell suspension, count it, and then wash it with PBS 1-5 times;

[0176] 2. Discard the supernatant and add DNase I reaction solution to the cell pellet to digest the exogenous plasmid DNA residue. Use a pipette to mix well and incubate in a 37°C water bath for 15 minutes.

[0177] 3. Place in a dry box incubator at 60°C to completely inactivate DNase I for 5 minutes, and wash the cell pellet once with PBS.

[0178] 4. Extract cell DNA using the instructions of the Biomed Tissue / Cell Genomic DNA Rapid Extraction Kit;

[0179] 5. Turn on the PCR instrument, prepare the PCR reaction system, and set up the PCR reaction program and reaction plate: Set the two-step reaction program: 95℃ pre-denaturation for 2 minutes; 95℃ for 5 seconds, 60℃ for 15 seconds, 40 cycles; reaction volume: 20μl;

[0180] 6. Start running and collect data for analysis;

[0181] 7. Calculation of CAR copy number: Each cell genome contains 2 hALB genes, based on which the CAR copy number of each CART-BCMA cell is calculated. The formula is as follows: CAR copy number = (2×CAR detection value) / hALB detection value

[0182] The copy number can affect the function of CAR-T cells. A copy number that is too low may lead to insufficient persistence of CAR-T cells in the body, affecting long-term efficacy. A copy number that is too high may lead to over-activation of T cells. In addition, the higher the copy number, the higher the risk of secondary tumors caused by activation of oncogenes or inactivation of tumor suppressor genes due to integration. The detection of copy number shows that the copy numbers of the sequences of CD19 and BCMA are different when different linkers are used to link them, (G4S) n The CAR copy number of CAR-T (n=2 or 3) detected at D7 was lower than that of βS CAR-T. The dual-target CAR containing a G4S linker had a structural advantage of lower copy number under similar transduction efficiency.

[0183] Table 7 CAR copy number detection values ​​in each group

[0184] Example 5 CD19-BCMA dual-target CAR-T cell killing assay

[0185] This example detects the killing efficiency of the CD19-BCMA dual-target CAR-T cells described in this application on tumor cells. T cells expressing the CD19-BCMA dual-target CAR described in this application have a killing effect on tumor cells.

[0186] The dual-target CAR-T cells cultured to day 9 were co-cultured with corresponding tumor cell lines (MM.1S: expressing BCMA but not CD19; K562-CD19: expressing CD19 but not BCMA; MM.1S-CD19: expressing BCMA and CD19; Daudi: expressing BCMA and CD19). The killing efficiency was determined by detecting lactate dehydrogenase (LDH). The experiment set different effector-target ratios, with a target cell population of 3×10 4 cells / well, total system 200μL / well in 96-well round bottom plate, 3 replicates per group. Lyse after 16h, centrifuge after 1h and harvest supernatant and add substrate for OD 490nm Detection, calculate the kill percentage based on the detection results.

[0187] As shown in Figure 2, the dual-target CAR-T cells described in this application all showed a killing effect on tumor cells. Figures 2A-2C compare the killing effects of CARs containing different linkers, showing that when the effector-target ratio is 2:1 and 1:1, βS CAR-T has a higher killing effect than (G4S) n CAR-T (n=2 or 3) has a significantly improved killing efficiency against target cells MM.1S, K562-CD19, and MM.1S-CD19. Figures 2D-2F compare the killing effects of T cells expressing dual-target CAR and single-target CAR on different tumor cells. Dual-target CAR showed killing effects on tumor cells expressing BCMA, CD19, and BCMA and CD19. The results showed that the relative position of the sequence targeting CD19 and the sequence targeting BCMA in the antigen binding domain, as well as the choice of linker, will affect the killing effect of dual-target CAR-T cells on tumor cells. Comprehensive comparison shows that (G4S)2CAR-T and (G4S) 3-1 CAR-T has a stronger tumor cell-killing effect.

[0188] Example 6 CD19-BCMA Dual-Target CAR-T Cytokine Secretion Detection

[0189] This example detects that the CD19-BCMA dual-target CAR-T cells described in this application can secrete cytokines when co-cultured with tumor cells.

[0190] As described in Example 5, each group of T cells and each tumor target cell line were co-cultured for 16 hours on the 7th day of culture and then the cell killing test was performed. In this example, the supernatant after 16 hours of co-culture with an effector-target ratio of 1:1 or 0.5:1 was collected to test the secretion of cytokines IFN-γ, TNF-α, IL-2, IL-6, and IL-10. The steps for cytokine secretion detection can be referred to Mouse multi-cytokine detection kit instructions.

[0191] The brief steps are as follows: dilute the standard with deionized water according to the instructions; dilute the supernatant sample appropriately with solution C in the kit according to the estimated cytokine concentration; vortex solution A thoroughly to disperse evenly, calculate the total volume of solution A and solution B required based on the sample volume (each sample requires 25 μL solution A and 25 μL solution B), add 25 μL of cell supernatant sample or 25 μL of serum sample or 25 μL of calibrator of different concentrations to the wells containing the A and B mixtures, and quickly mix gently with a pipette (be careful not to create bubbles), cover the lid, incubate at room temperature in the dark with shaking (200 rpm) for 3 hours, add 900 μL of washing solution to the incubated centrifuge tube, vortex to mix, centrifuge at 2000g for 2 minutes, carefully aspirate the supernatant (the small brown precipitates visible at the bottom are magnetic fluorescent encoded microspheres), add 100 μL of washing solution (solution E), mix well, and prepare for the machine; collect sample data on the flow cytometer and perform data analysis.

[0192] As shown in Figure 3, the dual-target CAR-T cells described in the present application can secrete cytokines when co-cultured with tumor cells. Figures 3A-3D compare the cytokine secretion of CARs containing different linkers. When co-cultured with different target cells, the dual-target CAR-T cells containing different linkers can secrete a certain amount of cytokines; among them, the cytokine secretion of (G4S)n CAR-T (n=2 or 3) is higher than that of βS CAR-T. Figure 3E compares the IFN-γ secretion of T cells expressing dual-target CAR and single-target CAR after co-culture with each tumor target cell line. When co-cultured with MM.1S expressing BCMA, the βS CAR-T group and the single-target BCMACAR-T group had no significant difference in IFN-γ secretion, but compared with (G4S) 3-2 The secretion of IFN-γ in the CAR-T group was higher (P<0.05). When co-cultured with K562-CD19 expressing CD19 protein, there was no significant difference in the secretion of IFN-γ between the βS CAR-T group and the single target CD19 CAR-T group, but it was significantly higher than that in the (G4S) 3-2Compared with the CAR-T group, the secretion of IFN-γ was significantly increased (P<0.0001). When co-cultured with Daudi cells expressing both CD19 and BCMA proteins, the secretion of IFN-γ in the βS CAR-T group was significantly increased compared with the other groups (P<0.0001). The results show that the relative position of the sequence targeting CD19 and the sequence targeting BCMA in the antigen binding domain, as well as the choice of linker, will affect the secretion of cytokines by dual-target CAR-T cells. Comprehensive comparison shows that (G4S)2CAR-T and (G4S) 3-1 CAR-T has significantly stronger cytokine secretion capacity.

Claims

1. A chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain, the extracellular antigen binding domain comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds CD19, the second antigen binding domain binds BCMA, and: 1) the first antigen-binding domain comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3; the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; and 2) The second antigen-binding domain comprises a second heavy chain variable region (VH), and the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

2. The CAR according to claim 1, wherein the extracellular antigen binding domain further comprises a linker, wherein the linker connects the first heavy chain variable region, the first light chain variable region and the second heavy chain variable region.

3. The CAR according to claim 2, wherein the linker comprises a β-stranded linker and / or a G4S linker.

4. The CAR according to claim 3, wherein the G4S linker comprises a G4S3 linker and / or a G4S2 linker.

5. The CAR according to any one of claims 3-4, wherein the β-stranded linker comprises an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO:

7.

6. The CAR according to any one of claims 4-5, wherein the G4S3 linker comprises the amino acid sequence shown in SEQ ID NO:

4.

7. The CAR according to any one of claims 4-6, wherein the G4S2 linker comprises the amino acid sequence shown in SEQ ID NO:

5.

8. The CAR according to any one of claims 1-7, wherein the extracellular antigen binding domain comprises from N-terminus to C-terminus: 1) a first heavy chain variable region, a linker, a first light chain variable region, a linker, and a second heavy chain variable region; 2) a first heavy chain variable region, a linker, a second heavy chain variable region, a linker, and a first light chain variable region; 3) a second heavy chain variable region, a linker, a first light chain variable region, a linker, and a first heavy chain variable region; 4) a second heavy chain variable region, a linker, a first heavy chain variable region, a linker, and a first light chain variable region; 5) a first light chain variable region, a linker, a second heavy chain variable region, a linker, and a first heavy chain variable region; or 6) A first light chain variable region, a linker, a first heavy chain variable region, a linker, and a second heavy chain variable region.

9. The CAR according to any one of claims 1-8, wherein the extracellular antigen binding domain comprises from the N-terminus to the C-terminus: 1) a first light chain variable region, a G4S2 linker, a second heavy chain variable region, a G4S2 linker, and a first heavy chain variable region; 2) a first light chain variable region, a G4S3 linker, a second heavy chain variable region, a G4S3 linker, and a first heavy chain variable region; 3) a first light chain variable region, a β-pleated sheet linker, a second heavy chain variable region, a β-pleated sheet linker, and a first heavy chain variable region; or 4) a second heavy chain variable region, a G4S3 linker, a first light chain variable region, a G4S3 linker and a first heavy chain variable region.

10. The CAR according to any one of claims 1-9, wherein the extracellular antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-11.

11. The CAR according to any one of claims 1-10, further comprising a hinge region, a transmembrane domain, a co-stimulatory domain and / or a signaling domain.

12. The CAR according to any one of claims 1-11, comprising an extracellular antigen binding domain, a transmembrane domain, a hinge region, a co-stimulatory domain and a signaling domain from N-terminus to C-terminus.

13. The CAR according to any one of claims 11-12, wherein the hinge region comprises a hinge region derived from one or more of the following histones: CD28, IgG1, IgG4, IgD, CD137, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30 and LIGHT.

14. The CAR of any one of claims 11-13, wherein the hinge region comprises a CD8 hinge region.

15. The CAR of any one of claims 11-14, wherein the transmembrane domain comprises a transmembrane domain derived from one or more of the following histones: CD8, CD28, CD3e, CD3ε, CD137, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, CD154, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, and SLAM.

16. The CAR of any one of claims 11-15, wherein the transmembrane domain comprises a CD8 transmembrane domain.

17. A CAR according to any one of claims 11-16, wherein the costimulatory domain comprises a costimulatory domain derived from one or more of the following histones: CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

18. The CAR of any one of claims 11-17, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain.

19. A CAR according to any one of claims 11-18, wherein the signaling domain includes a signaling domain derived from the following group of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and a domain protein containing at least one ITAM.

20. The CAR of any one of claims 11-19, wherein the signaling domain comprises a CD3ζ signaling domain.

21. The CAR according to any one of claims 1-20, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 12-15.

22. An isolated nucleic acid molecule encoding the CAR of any one of claims 1-21.

23. The isolated nucleic acid molecule according to claim 22, comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 16-19.

24. A vector comprising the isolated nucleic acid molecule of any one of claims 22-23.

25. The vector of claim 24, which comprises a viral vector.

26. The vector of any one of claims 24-25, which comprises a lentiviral vector.

27. A cell comprising and / or expressing the CAR of any one of claims 1-20, the isolated nucleic acid molecule of any one of claims 22-23, and / or the vector of any one of claims 24-26.

28. The cell of claim 27, comprising an immune effector cell.

29. The cell according to any one of claims 27-28, comprising a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a γδT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte and / or a peripheral blood mononuclear cell.

30. The cell of any one of claims 27-29, wherein the cell comprises a T cell.

31. The cell of any one of claims 27-30, wherein the cell comprises a human cell.

32. A pharmaceutical composition comprising the CAR of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, and / or the cell of any one of claims 27-31, and optionally a pharmaceutically acceptable carrier.

33. Use of the CAR of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, and / or the cell of any one of claims 27-31 in the preparation of a medicament, wherein the medicament is used to treat a disease or condition associated with the expression of CD19 and / or BCMA.

34. Use of the CAR of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the cell of any one of claims 27-31 and / or the pharmaceutical composition of claim 32 in treating a disease or condition associated with the expression of CD19 and / or BCMA.

35. A method for treating a disease or condition associated with the expression of CD19 and / or BCMA, comprising administering to a subject in need thereof an effective amount of the CAR of any one of claims 1-21, the isolated nucleic acid molecule of any one of claims 22-23, the vector of any one of claims 24-26, the immune effector cell of any one of claims 27-31 and / or the pharmaceutical composition of claim 32.

36. The use according to any one of claims 33-34 or the method according to claim 35, wherein the disease or condition comprises a tumor, a blood system disease and / or an autoimmune disease.

37. The use according to any one of claims 33-34 or the method according to claim 35, wherein the disease or disorder comprises solid tumors and non-solid tumors.

38. The use according to any one of claims 33-34 or the method according to claim 35, wherein the disease comprises lymphoma and / or leukemia.

39. The use of any one of claims 33-34 or the method of claim 35, wherein the disease comprises multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, follicular lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, megakaryocytic leukemia, Burkitt's lymphoma, anaplastic large cell lymphoma and / or mucosa-associated lymphoid tissue lymphoma.

40. The use according to any one of claims 33-34 or the method according to claim 35, wherein the disease comprises chronic Guillain-Barré syndrome, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, Sjögren's syndrome, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, demyelinating diseases, autoimmune hemolysis, idiopathic thrombocytopenic purpura and / or idiopathic leukopenia.