BCMA-targeted chimeric antigen receptor, and preparation method and use thereof

Abstract

To provide a BCMA-targeted chimeric antigen receptor, and a preparation method and use thereof.SOLUTION: The present invention provides a BCMA-targeted chimeric antigen receptor which comprises a BCMA-targeted scFv, a hinge region, a transmembrane region, and an intracellular signal domain. The present invention provides a nucleic acid molecule for encoding the chimeric antigen receptor, and a corresponding expression vector, as well as CAR-T cells and use thereof. The chimeric antigen receptor of the present invention targets BCMA-positive cells, and can be used for treating BCMA-positive B-cell lymphoma, multiple myeloma and plasma cell leukemia.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and more specifically, to chimeric antigen receptors targeting BCMA, methods for producing the same, and uses thereof.

Background Art

[0002] BCMA is B cell maturation antigen, also known as CD269 or TNFRSF17, a member of the tumor necrosis factor receptor superfamily, and its ligands are B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). The binding of BCMA to BAFF and APRIL activates NF-kB and induces upregulation of anti-apoptotic Bcl-2 family members such as Bcl-xL or Bcl-2 and Mcl-1. The interaction between BCMA and its ligands maintains a stable balance in the human body environment by regulating humoral immunity, B cell growth, and differentiation in various aspects.

[0003] The expression of BCMA is limited to the B cell lineage, and is expressed in plasmablasts, plasma cells, and some mature B cells, increasing during the differentiation of peripheral B cells. On the other hand, it is not expressed in most B cells such as naive B cells, memory B cells, germinal center B cells, and other organs. The expression of BCMA has been reported to be important for long-lived and resident plasma cells in the bone marrow. Therefore, in BCMA-deficient mice, plasma cells in the bone marrow decrease, but the plasma cell level in the spleen is not affected. Mature B cells differentiate normally into plasma cells in BCMA knockout mice. BCMA knockout mice appear normal and healthy, and the number of B cells is normal, but plasma cells cannot survive for a long time.

[0004] BCMA is highly expressed in malignant plasma cells, and BCMA has been detected, for example, in the HRS cells of patients with multiple myeloma, plasmacytic leukemia, and Hodgkin lymphoma. In the United States, hematological malignancies account for approximately 10% of all malignancies, and myeloma accounts for 15% of all hematological malignancies. In the literature, the expression of BCMA has been reported to be associated with the disease progression of multiple myeloma. The BCMA gene is highly expressed in myeloma specimens, but the expression level is low in chronic lymphocytic leukemia, acute lymphocytic leukemia, and acute T-lymphocytic leukemia. In murine models overexpressing BAFF and APRIL, which are BCMA ligands, B-cell lymphoma grows significantly. It has been demonstrated that ligands binding to BCMA can regulate the growth and survival of multiple myeloma expressing BCMA. Since the binding of BCMA to BAFF and APRIL can keep malignant plasma cells alive, depleting tumor cells expressing BCMA and disrupting the interaction between BCMA ligands and receptors can improve the therapeutic effect on multiple myeloma or other BCMA-positive B-cell malignant lymphomas.

[0005] Multiple myeloma, also known as plasmacytoma or Kahler's disease, is a refractory B-cell malignancy characterized by abnormal proliferation of plasma cells, which are a type of white blood cell responsible for producing antibodies. Data published by the National Cancer Institute of the United States in 2017 showed that myeloma accounted for 1.8% of all tumor cases and had a lethality rate of 2.1%. Statistical results from 2010 to 2014 showed that the annual incidence rate was approximately 6.6 per 100,000 and the mortality rate was approximately 50%. Multiple myeloma belongs to the category of middle-aged and elderly diseases. In Europe and the United States, the median age of onset is 68 years, and there are more men than women. According to statistics in our country, the peak age of onset is 55 - 65 years, and the male-to-female ratio is 2.35:1. In our country, although there is still no reliable epidemiological survey data on multiple myeloma, generally, the incidence rate is close to that of neighboring Southeast Asia and Japan, and it is estimated to be approximately 1 per 100,000. Conventional treatment methods for multiple myeloma include chemotherapy and hematopoietic stem cell transplantation, but these methods have a high recurrence rate. Bortezomib (PS-341) is the first proteasome inhibitor, and in 2003, the FDA approved its use for the treatment of relapsed and refractory multiple myeloma either alone or in combination with existing drugs, and the results were satisfactory. This drug was also commercially available in China in 2005 and is now one of the commonly used options for the treatment of multiple myeloma together with thalidomide, dexamethasone, etc. The treatment methods for multiple myeloma are usually combined, but using multiple drugs simultaneously also has some unfavorable results, such as high costs and cumulative side effects. Clinically, the development of new methods for treating multiple myeloma is still eagerly awaited.

[0006] Recently, immunotherapy, especially adoptive T cell therapy, has shown strong therapeutic effects and bright prospects in clinical trials for treating hematological malignancies. T cells may be genetically modified to express a chimeric antigen receptor (CAR), which includes an antigen recognition portion and a T cell activation region. The CAR can utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and target them without being restricted by MHC. Through such non-MHC-restricted antigen recognition, T cells expressing CAR can recognize antigens without processing them, thus avoiding one of the main mechanisms of tumor escape. In addition, the CAR does not dimerize with the α-chain and β-chain of the endogenous TCR.

[0007] Currently, two chimeric antigen receptor T cell therapy (CAR-T) products targeting CD19 have been commercially available overseas and are used for the treatment of acute lymphoblastic leukemia in children and young patients and for treatment above second-line for relapsed or refractory large B cell lymphoma in adults. However, CD19 is rarely expressed in malignant plasma cells of multiple myeloma. In this field, the development of CAR-T products targeting BCMA for treating multiple myeloma is eagerly desired.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Summary of the Invention The object of the present invention is to provide a chimeric antigen receptor targeting BCMA, a method for producing the same, and uses thereof. Specifically, the object of the present invention is to provide the sequence of a chimeric antigen receptor targeting the BCMA antigen, a method for producing the modified T cells (CART-BCMA) thereof, and the identification of their activity.

Means for Solving the Problems

[0009] The present invention provides a structure of a chimeric antigen receptor for treating BCMA-positive B-cell lymphoma. In a first aspect of the present invention, there is provided a chimeric antigen receptor (CAR) (sequence), wherein the antigen-binding domain of the chimeric antigen receptor is a sequence of a single-chain variable region of an antibody of an extracellular region targeting BCMA. In another preferred example, the antigen-binding domain is a sequence of a single-chain variable region of an antibody targeting the 24th to 41st amino acid residues of the BCMA sequence. In another preferred example, the NCBI accession number of the BCMA sequence is AY684975.1.

[0010] In another preferred example, the structure of the antigen-binding domain is represented by the following formula I. V L -V H (I) (where V H is the variable region of the heavy chain of the antibody, V L is the variable region of the light chain of the antibody, and "-" is a linker peptide or a peptide bond. And the amino acid sequence of V L is represented by SEQ ID NO: 1, the amino acid sequence of V H is represented by SEQ ID NO: 2, or the amino acid sequence of V L is represented by SEQ ID NO: 3, the amino acid sequence of V H is represented by SEQ ID NO: 4, or the amino acid sequence of V L is represented by SEQ ID NO: 5, and the amino acid sequence of V H is represented by SEQ ID NO: 6.)

[0011] In another preferred example, the amino acid sequence of the linker peptide is represented by SEQ ID NO: 10 or SEQ ID NO: 11. In another preferred example, the single-chain variable region of the antibody is a humanized single-chain variable region of an antibody, a murine-derived single-chain variable region of an antibody, or a chimeric single-chain variable region of a human and a murine antibody. In another preferred example, the structure of the chimeric antigen receptor is represented by the following formula II. S-V L -V H-H-TM-C-CD3ζ (II) (where S is an optional signal peptide (i.e., signal peptide), H is a hinge region, TM is a transmembrane domain, C is a co-stimulatory signal molecule, and CD3ζ is an intracellular signaling sequence derived from CD3ζ. V H and V L are as described above.)

[0012] In another preferred example, the above-mentioned S is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof. In another preferred example, the above-mentioned S is a signal peptide derived from CD8. In another preferred example, the amino acid sequence of S is shown in SEQ ID NO: 9.

[0013] In another preferred example, the above-mentioned H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof. In another preferred example, the above-mentioned H is a hinge region derived from CD8. In another preferred example, the amino acid sequence of H is shown in SEQ ID NO: 12.

[0014] In another preferred example, the above-mentioned TM is a transmembrane domain of a protein selected from the group consisting of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof. In another preferred example, the above-mentioned TM is a transmembrane domain derived from CD8. In another preferred example, the sequence of TM is shown in SEQ ID NO: 13.

[0015] In another preferred example, said C is a co-stimulatory signal molecule of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof. In another preferred example, said C is a co-stimulatory signal molecule derived from 4-1BB. In another preferred example, the amino acid sequence of C is shown in SEQ ID NO: 14. In another preferred example, the amino acid sequence of CD3ζ is shown in SEQ ID NO: 15.

[0016] In a second aspect of the present invention, there is provided a nucleic acid molecule encoding the chimeric antigen receptor (CAR) described in the first aspect of the present invention. In another preferred example, said nucleic acid molecule is isolated. In a third aspect of the present invention, there is provided a vector containing the nucleic acid molecule described in the second aspect of the present invention. In another preferred example, said vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof. In another preferred example, said vector is a lentiviral vector.

[0017] In a fourth aspect of the present invention, there is provided a host cell containing the vector described in the third aspect of the present invention, or into which the nucleic acid molecule described in the second aspect of the present invention, which is foreign to the chromosome, is integrated, or which expresses the CAR described in the first aspect of the present invention. In another preferred example, said cell is an isolated cell and / or said cell is a genetically modified cell. In another preferred example, said cell is a mammalian cell. In another preferred example, said cell is a T cell.

[0018] In a fifth aspect of the present invention, there is provided a method for producing CAR-T cells, the method comprising the step of obtaining the CAR-T cells by transducing the nucleic acid molecule according to the second aspect of the present invention or the vector according to the third aspect of the present invention into T cells, wherein the CAR-T cells express the CAR according to the first aspect of the present invention.

[0019] In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising the chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the cell according to the fourth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In another preferred example, the formulation is a liquid formulation. In another preferred example, the dosage form of the formulation is an injection. In another preferred example, in the formulation, the concentration of the CAR-T cells is 1×10 3 ~1×10 8 cells / mL, preferably 1×10 4 ~1×10 7 cells / mL.

[0020] In a seventh aspect of the present invention, there is provided the use of the chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the cell according to the fourth aspect of the present invention, in the manufacture of a medicament or formulation for preventing and / or treating cancer or a tumor. In another preferred example, the tumor is selected from the group consisting of hematological tumors, solid tumors, or combinations thereof. In another preferred example, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0021] In another preferred example, the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or the like. In another preferred example, the tumor is a BCMA-positive tumor, preferably BCMA-positive B cell lymphoma, multiple myeloma, or plasmacytic leukemia.

[0022] In an eighth aspect of the present invention, there is provided a kit for producing the cells described in the fourth aspect of the present invention, the kit comprising a container and a nucleic acid molecule described in the second aspect of the present invention or a vector described in the third aspect of the present invention contained within the container. In a ninth aspect of the present invention, there is provided the use of the cells described in the fourth aspect of the present invention, or the formulation described in the sixth aspect of the present invention, for the prevention and / or treatment of cancer or tumors.

[0023] In a tenth aspect of the present invention, there is provided a method for treating a disease, the method comprising the step of administering an appropriate amount of the cells described in the fourth aspect of the present invention and / or the formulation described in the sixth aspect of the present invention to a subject in need of treatment. In another preferred example, the disease is cancer or a tumor. Of course, within the scope of the present invention, it is understood that each of the above technical features of the present invention and each of the specifically described technical features below (for example, in the examples) can be combined with each other to form new or preferred technical solutions. Due to limited space, they will not be explained one by one here.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

[0025]

Figure 4

Figure 5

[0026]

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0027] Specific Embodiments The inventors have conducted extensive and in-depth research and a large number of screenings, and for the first time obtained a chimeric antigen receptor targeting the BCMA antigen. Specifically, in the present invention, the structure of a chimeric antigen receptor targeting BCMA constructed based on the four monoclonal antibody sequences of BCMA-1, BCMA-20, BCMA-CA8, and BCMA-MO6 was obtained, and the analysis and identification of the expression level, in vitro activation ability, and tumor cell killing ability of these chimeric antigen receptors in primary T cells were completed. In the research, it was revealed that the chimeric antigen receptor of the present invention targets BCMA-positive cells and can be used for the treatment of BCMA-positive B-cell lymphoma, multiple myeloma, plasmacytic leukemia or other diseases.

[0028] Specifically, in the present invention, the relationship between different CAR structures and the expression time and intensity on the cell membrane surface after virus infection was identified, and further, the difference in the difficulty of protein expression of different CAR structures was identified. This finding suggests that the expression level of the CAR protein on the membrane surface and the persistence of the in vivo activity of CART vary depending on the CAR structure under the same infection conditions. Through a large number of screenings, CARs of the structure of the present invention were obtained. The results showed that all the proteins encoded by the CAR structures in the present invention can achieve sufficient expression and membrane localization. In the present invention, the manufacturing process of T cells modified with a CAR structure targeting the BCMA antigen was improved. Mainly, lymphocytes were cultured in vitro in GT-551 serum-free medium supplemented with 1% human serum albumin.

[0029] Terms To facilitate the understanding of the present disclosure, some terms are first defined. As used in this application, unless otherwise clearly defined herein, the following terms all have the following meanings. Throughout the application, other definitions are described. The term "about" refers to a value or composition within an acceptable error range of a specific value or composition determined by those skilled in the art, thereby determining, in part, how the value or composition is measured or measured. The term "administration" refers to introducing the product of the present invention physically into a subject by any one of various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion.

[0030] The term "antibody" (Ab) includes, but is not limited to, globulins and comprises at least two heavy (H) chains and two light (L) chains or antigen-binding portions thereof that specifically bind to an antigen and are linked to each other via disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed among more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.

[0031] Chimeric antigen receptor (CAR) The chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also called an antigen-binding domain). The intracellular domain comprises a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region comprises a part of the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule necessary for an effective response of lymphocytes to an antigen and is not an antigen receptor or its ligand.

[0032] A linker may be introduced between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to the extracellular or intracellular domain of the polypeptide chain. The linker may comprise from 0 to 300 amino acids, preferably from 2 to 100 amino acids, most preferably from 3 to 50 amino acids. In one preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen-binding domain that targets BCMA. When the CAR of the invention is expressed in T cells, it can recognize an antigen based on the specificity of antigen binding. When it binds to the relevant antigen, it affects tumor cells such that the tumor cells do not grow, die, or are affected in other ways, and the patient's tumor burden is reduced or eliminated. The antigen-binding domain is preferably fused to one or more intracellular domains derived from co-stimulatory molecules and the ζ chain. Preferably, the antigen-binding domain is fused to an intracellular domain combined with the 4-1BB signaling domain and the CD3ζ signaling domain.

[0033] As used herein, both "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of an antibody, but has no constant region and is the smallest antibody fragment having all antigen-binding sites. Generally, an Fv antibody further contains a polypeptide linker between the VH and VL domains and can form a structure necessary for antigen binding. The antigen-binding domain is usually an scFv (single-chain variable fragment). The size of an scFv is usually 1 / 6 of a complete antibody. A single-chain antibody preferably has an amino acid sequence encoded by a single nucleotide chain. In a preferred embodiment of the present invention, the scFv includes an antibody that specifically recognizes the extracellular region of BCMA, particularly an antibody that specifically recognizes the 24-41st amino acid residues of the BCMA sequence, and is preferably a single-chain antibody.

[0034] Regarding the hinge region and the transmembrane region (transmembrane domain), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain associated with one of the domains in a native CAR is used. In some examples, by selecting or modifying the transmembrane domain by amino acid substitution to avoid the binding of such a domain to the transmembrane domains of the same or different surface membrane proteins, the interaction with other members of the receptor complex is minimized. The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0035] Preferably, the structure of the CAR of the present invention includes a signal peptide, an antigen recognition sequence (antigen-binding domain), a linker region, a transmembrane region, a co-stimulatory factor signaling region, and a CD3ζ signaling region (ζ-chain portion), and the connection order is as follows. CD8 S-[VL-Linker-VH]-[Hinge-CD8TM]-[4-1BB]-[CD3ζ] Specifically, the sequences used in the present invention are as follows. (1) Signal peptide, the signal peptide sequence derived from CD8: MALPVTALLLPLALLLHAARP (SEQ ID NO: 9) (2) Single-chain variable region of the light chain (VL) sequence derived from the BCMA-1 antibody: DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK (SEQ ID NO: 7) (3) Single-chain variable region of the heavy chain (VH) sequence derived from the BCMA-1 antibody: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS (SEQ ID NO: 8) Here, BCMA-1 is an antibody sequence included in the already published Car-T sequence and is used as a control in the present application.

[0036] (4) Single-chain variable light chain region (VL) sequence derived from the BCMA-20 antibody: DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIK (SEQ ID NO: 1) (5) Single-chain variable heavy chain region (VH) sequence derived from the BCMA-20 antibody: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSS (SEQ ID NO: 2) (6) Single-chain variable light chain region (VL) sequence derived from the BCMA-CA8 antibody: DIQLTQTTSSLSASLGDRVTISCSASTTTSNYLNWYQQKPDGTVELVIYYTSNLHGGGPSRFSGSGSGTDYSLTIGYLEPEDVATYYCQQYRKLPWTFGGGSKLEIKR (SEQ ID NO: 3) (7) Single-chain variable heavy chain region (VH) sequence derived from the BCMA-CA8 antibody: EVQLQQSGAVLARPGASVKMSCKGSGYTFTNYWMHWVKQRPGQGLEWIGATYRGHSDTYYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTRGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 4)

[0037] (8) Single-chain variable light chain (VL) sequence derived from BCMA-MO6 antibody: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 5) (9) Single-chain variable heavy chain (VH) sequence derived from BCMA-MO6 antibody: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 6) (10) Linker sequence between the heavy and light chains of the single-chain variable region of BCMA-1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10) (11) Linker sequence between the heavy and light chains of the single-chain variable regions of BCMA-20, BCMA-CA8, and BCMA-MO6: GGGGSGGGGSGGGGS (SEQ ID NO: 11)

[0038] (12) Sequences of the hinge region and linker region: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 12) (13) Transmembrane sequence of the CD8 (CD8TM) antigen, which is the transmembrane region: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 13) (14) Sequence of the intracellular signaling motif derived from 4-1BB, which is the co-stimulatory factor signal region: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14) (15) Sequence of the immunoreceptor tyrosine-based activation motif (ITAM) of CD3ζ in the TCR complex, which is the CD3ζ signaling region: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 15)

[0039] Chimeric antigen receptor T cells (CAR-T cells) As used herein, the terms "CAR-T cells", "CAR-T", "CART", and "the CAR-T cells of the present invention" all refer to the CAR-T cells described in the second aspect of the present invention. The present invention relates to the construction of the structure of a chimeric antigen receptor targeting BCMA, a method for producing a genetically modified T cell of a chimeric antigen receptor targeting BCMA, and the identification of its activity.

[0040] Vector The nucleic acid sequence encoding the desired molecule can be obtained by recombinant methods known in the art, such as screening a library in cells expressing the gene, obtaining the vector from a vector containing the gene, or directly isolating it from cells and tissues containing the gene by standard techniques. If necessary, the gene of interest can also be produced synthetically. The present invention also provides a vector into which the expression cassette of the present invention is inserted. Retroviruses, such as vectors derived from lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for stable integration and proliferation in their daughter cells over a long period of time. Lentiviruses can be introduced into non-proliferating cells, such as hepatocytes, and thus have advantages over vectors of oncogenic retroviruses, such as murine leukemia virus. They also have the advantage of low immunogenicity.

[0041] Briefly summarized, generally, the expression cassette or nucleic acid sequence of the present invention is operably linked to a promoter and incorporated into an expression vector. The vector is suitable for eukaryotic cell replication and integration. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy by standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0042] The nucleic acid can be cloned into various types of vectors. For example, vectors into which the nucleic acid can be cloned include, but are not limited to, plasmids, phages, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Furthermore, the expression vector can be provided to cells in the form of a viral vector. The technology of viral vectors is known in the art and is 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 that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme cleavage sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0043] Numerous virus-based systems have already been developed and used for introducing genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Genes selected by techniques known in the art can be inserted into vectors and packaged into retroviral particles. The recombinant virus can then be further isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0044] Additional promoter elements, such as enhancers, can regulate the frequency at which transcription is initiated. Usually, these are located in the region 30 - 110 bp upstream of the start point, but recently, it has become clear that many promoters also contain functional elements downstream of the start point. The spacing between promoter elements is often flexible so that the promoter function can be maintained if the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp without a decrease in activity. By the promoter, single elements act jointly or independently to initiate transcription.

[0045] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of highly expressing any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor 1α (EF-1α). However, other constitutive promoter sequences may be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the present invention is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch, thereby allowing the initiation of expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is required, or the termination of expression when expression is not required. Examples of inducible promoters include but are not limited to the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0046] To evaluate the expression of a CAR polypeptide or a part thereof, expression cells can also be identified and selected from a group of cells transduced or infected with a viral vector by making the expression vector introduced into the cells contain either or both of a selectable marker gene and a reporter gene. Also, the selectable marker can be placed on a single DNA fragment and used in the process of co-transfection. Adjacent regions of both the selectable marker gene and the reporter gene may each have appropriate regulatory sequences so that they can be expressed in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0047] Reporter genes are used for the identification of cells that may have been transduced and for the evaluation of the functionality of regulatory sequences. Usually, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue and whose expression can be clearly indicated by a property that is easily detectable, such as enzyme activity. When DNA has already been introduced into recipient cells, the expression of the reporter gene is measured at an appropriate time. Appropriate reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, and green fluorescent protein (for example, Ui-Tei et al., 2000 FEBS Letters 479:79-82). Appropriate expression systems are known and can be produced by known techniques or obtained as commercial products. Usually, a construct having at least five flanking regions that shows the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated to the reporter gene and used for the evaluation of the ability to regulate the promoter of the reagent and activate transcription.

[0048] Methods for introducing genes into cells and methods for expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, and insect cells, by any method known in the art. For example, the expression vectors can be introduced into host cells by physical, chemical, or biological means. Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or foreign nucleic acids are known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0049] Biological methods for introducing polynucleotides of interest into host cells include methods using DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be 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. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as lipid-based systems including macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is liposomes (e.g., artificial membrane vesicles).

[0050] When using a non-viral delivery system, an exemplary delivery carrier is a liposome. It is contemplated to use lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo or in vivo). Also, the nucleic acid may be associated with the lipid. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, adhered to liposomes via a linking molecule that links both liposomes and oligonucleotides, incorporated into liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, formulated with lipids, contained in lipids as a suspension, contained in micelles, complexed with micelles, or bound to lipids in other forms. The lipids, lipid / DNA or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, in a bilayer structure, it may exist as micelles or in a "disrupted" structure. It may simply be dispersed in solution to form aggregates of different sizes and shapes. Lipids are fatty substances and may be natural or synthetic lipids. For example, lipids include lipid droplets that occur naturally in the cytoplasm, as well as compounds such as long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols and aldehydes. In one preferred embodiment of the present invention, the vector is a lentiviral vector.

[0051] Formulations The present invention provides a formulation containing the CAR-T cells described in the first aspect of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, in the formulation, the concentration of the CAR-T cells is 1×10 3 ~1×10 8 cells / mL, more preferably 1×10 4 ~1×10 7cells / mL. In one embodiment, the formulation may include a buffer, such as neutral buffered saline, sulfate buffered saline, carbohydrates such as glucose, mannose, sucrose, dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The formulation of the present invention is preferably prepared for intravenous administration. The formulation may contain an agent. The formulation of the present invention is preferably prepared for intravenous administration.

[0052] Therapeutic use The present invention includes therapeutic use performed on cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced cells target BCMA, a marker of tumor cells, synergistically activate T cells, and can significantly improve the killing efficiency against those tumor cells by inducing an immune response of T cells. Therefore, the present invention also provides a method for stimulating an immune response by T cells against a target cell population or tissue in a mammal, the method comprising the step of administering the CAR-T cells of the present invention to the mammal.

[0053] In one embodiment, the present invention includes a cell therapy in which autologous T cells (or allogeneic donors) of a patient are isolated, activated, genetically modified, and after the generation of CAR-T cells, injected into the body of the same patient. In such a method, the probability of developing graft-versus-host disease is extremely low, and antigens are recognized by T cells without being restricted by MHC. Also, all cancers expressing the antigen can be treated with one CAR-T. Different from antibody therapy, CAR-T cells can replicate in the body and produce long-term persistence in suppressing tumors continuously. In one embodiment, the CAR-T cells of the present invention can persist for an extended time through stable in vivo T cell proliferation. Also, the immune response by CAR may be part of the adoptive immunotherapy process, where the CAR-modified T cells induce an immune response specific to the plateau-binding domain in the CAR. For example, anti-BCMA CAR-T cells elicit a specific immune response resistant to cells expressing BCMA.

[0054] The data disclosed in this specification specifically discloses a lentiviral vector comprising an anti-BCMA scFv, a hinge and transmembrane region, and 4-1BB and CD3ζ signaling domains, but the present invention should be construed to include any number of variations of each component of the construct. Treatable cancers include tumors that are not undergoing angiogenesis or are essentially non-angiogenic, and tumors that have undergone angiogenesis. Cancers may include non-solid tumors (e.g., blood tumors such as leukemia and lymphoma) or solid tumors. The types of cancers treatable with the CAR of the present invention include, but are not limited to, cancers, germ cell tumors and sarcomas, and some leukemias and lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, cancers and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0055] Blood cancers are cancers of the blood or bone marrow. Examples of blood (or hematogenous) cancers include leukemia, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloblastic leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemia (e.g., chronic myeloblastic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (in the case of painless and severe), multiple myeloma, Waldenström's macroglobulinemia, H chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. Solid tumors usually do not include tumors of tissues with cysts or fluid regions. Solid tumors can be either benign or malignant. Different types of solid tumors are named according to the type of cells that form them (e.g., sarcoma, cancer or lymphoma). Examples of solid tumors include, for example, sarcomas and cancers such as fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancy, pancreatic cancer, ovarian cancer.

[0056] The CAR-modified T cells of the present invention are also useful as vaccines for in vitro immunization and / or in vivo therapy against mammals. Preferably, the mammal is a human. For in vitro immunization, at least one of i) cell proliferation, ii) introduction of a nucleic acid encoding the CAR into the cell, and / or iii) cryopreservation of the cell is performed in vitro before administering the cell to the mammal. In vitro protocols are known in the art and will be discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified with a vector expressing the CAR disclosed herein (i.e., in vitro transformation or transduction). The CAR-modified cells can be administered to a mammalian recipient and provide a beneficial effect of treatment. The mammalian recipient may be a human, and the CAR-modified cells may be the recipient's own cells. Optionally, the cells may be allogeneic, syngeneic or xenogeneic to the recipient. In addition to cell-based vaccines for in vitro immunization, the present invention also provides compositions and methods for eliciting an immune response against an antigen in a patient by in vivo immunization.

[0057] The present invention provides a method for treating a tumor, which method comprises the step of administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof. The CAR-modified T cells of the present invention can be administered alone or in combination with a diluent and / or other components, such as IL-2, IL-17 and other cytokines or cell populations, as a pharmaceutical composition. Briefly, the pharmaceutical composition of the present invention contains the target cell population described herein and may be combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, sulfate buffered saline, carbohydrates, such as glucose, mannose, sucrose, dextran, mannitol, proteins, polypeptides or amino acids, such as glycine, antioxidants, chelating agents, such as EDTA and glutathione, adjuvants (such as aluminum hydroxide), and preservatives. The composition of the present invention is preferably prepared for intravenous administration.

[0058] The pharmaceutical composition of the present invention can be administered in a form suitable for a disease that requires treatment (or prevention). The quantity and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage is determined by clinical trials. When described as an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressing effective amount" or "therapeutic amount", the exact amount of the composition of the present invention to be administered takes into account the age, weight, tumor size, degree of infection or metastasis of the patient (subject), and individual differences in the disease condition, and is determined by a physician. Usually, the pharmaceutical composition containing T cells described herein is administered at a dosage of 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 cells / kg body weight (including all integer values within these ranges). The composition of T cells may be administered several times at these dosages. The cells can be administered by infusion techniques known in immunotherapy (e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a specific patient can be adjusted and treated based on monitoring the patient's disease findings, and can be easily determined by a person skilled in the medical field.

[0059] The administration of the composition to the subject may be performed by any convenient means including spraying, injection, oral, infusion, implantation or transplantation. The composition described herein may be administered to the patient subcutaneously, intradermally, intratumorally, intra-articularly, intraspinally, intramuscularly, intravenously (i.v.) or intraperitoneally. In one embodiment, the composition of T cells of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the composition of T cells of the present invention is preferably administered by i.v. injection. The composition of T cells may be injected directly into the tumor, lymph node or site of infection.

[0060] In some embodiments of the invention, cells activated and expanded by the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are used and administered to a patient in combination with any amount of relevant therapeutic means (e.g., before, simultaneously, or after), said therapeutic means including, but not limited to, antiviral therapies, treatment with agents such as cidofovir and interleukin-2, azacitidine (known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine A, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), and cyclophosphamide. For example, in one embodiment, a subject may undergo peripheral blood stem cell transplantation after high-dose chemotherapy. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In another embodiment, the expanded cells are administered before or after surgery.

[0061] The dosage of the above treatments administered to a patient will vary depending on the precise nature of the condition being treated and the recipient being treated. The dosage ratio to be administered to a patient can be carried out in accordance with accepted practice in the art. Typically, for a single treatment or a single treatment cycle, 1 × 10 6 to 1 × 10 10 modified T cells of the invention (e.g., CAR-T-BCMA cells) can be administered to a patient, for example, by means of intravenous infusion.

[0062] The main advantages of the present invention are as follows. (a) The chimeric antigen receptor of the present invention has an extracellular antigen-binding domain that is a specific anti-BCMA scFv, and the CAR formed by binding the specific anti-BCMA scFv to a specific hinge region and an intracellular domain exhibits a very strong killing ability against tumor cells, and moreover has low cytotoxicity and low side effects. (b) The chimeric antigen receptor provided by the present invention can achieve stable expression and membrane localization of the CAR protein after infecting T cells with a lentivirus carrying the CAR gene. (c) The CAR-modified T cells of the present invention have a long survival time in vivo and a strong anti-tumor effect, and the scFv used in the present invention is a humanized antibody or an antibody derived from humans, and the possibility of a specific immune rejection reaction occurring is small.

[0063] The present invention will be further described below with specific examples. It is understood that these examples are only used to explain the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not shown in the following examples are usually according to normal conditions such as those described in, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or according to the recommended conditions of the manufacturer. Unless otherwise specified, % and parts are calculated by weight.

[0064] Example 1 Construction of a lentiviral expression vector The construction of the coding plasmid was achieved by entrusting Shanghai Boyi Biotechnology Co., Ltd. with the synthesis and cloning of the full-length DNA. The pWPT lentiviral vector was used as the cloning vector, and the cloning sites were the BamH I and Sal I sites. The specific sequence is as described above.

[0065] Example 2 Production of CAR-T cells (1) Peripheral blood from a healthy donor was collected, and mononuclear cells were isolated by density gradient centrifugation (PBMCs). (2) On day 0, PBMCs were inoculated into a cell culture flask pre-coated with CD3 monoclonal antibody (OKT3) at a final concentration of 5 μg / mL and Retronectin (purchased from TAKARA) at a final concentration of 10 μg / mL. The medium was GT-T551 cell medium containing 1% human serum albumin, and recombinant human interleukin-2 (IL-2) was added to the medium at a final concentration of 1000 U / mL. The cells were cultured in an incubator at 37°C, saturated humidity, and 5% CO2.

[0066] (3) On day 1, the supernatant for culturing PBMCs was slowly aspirated and discarded. Then, fresh GT-T551 cell medium containing 1% human serum albumin was added, and recombinant human interleukin-2 (IL-2) was added to the medium at a final concentration of 1000 U / mL. The cells were cultured in an incubator at 37°C, saturated humidity, and 5% CO2. (4) On day 3, fresh culture medium, concentrated and purified CAR-BCMA lentivirus solution, protamine sulfate (12 μg / ml), and IL-2 at a final concentration of 1000 U / mL were added. After infecting for 12 hours in an incubator at 37°C and 5% CO2, the culture medium was discarded, fresh medium was added, and the culture was continued in an incubator at 37°C and 5% CO2. (5) From day 6, CART-BCMA cells could be taken for corresponding activity detection tests.

[0067] Example 3 Detection of the integration rate of the CAR gene in the T cell genome and the expression level of the encoded protein on the cell membrane surface. 0.5×10 6Samples of CART-BCMAs cells cultured until the 7th day (Figure 3A), 21st day (Figure 3B), and 29th day (Figure 3C) in Example 2 were taken, stained with the Fc fragment of recombinant human BCMA protein, and then the expression levels of CAR-BCMA protein on the membrane surface of T cells were analyzed by a flow cytometer. As shown in Figure 3, all four CAR structures designed in the present invention were expressed in their corresponding modified T cells, and the localization on the cell membrane surface was completed.

[0068] Example 4 Detection of the in vitro activation ability of CART-BCMAs. The protein CD137 and IFNγ, which are cell activation level indicators, were detected in the CART-BCMAs cells cultured until the 7th day in Example 2. In sequence, 1×10 5 cultured CART-BCMA cells until the 7th day were taken, and they were cultured at a ratio of 1:1 in 200 μl of GT-551 medium with the BCMA-positive K562-BCMA+E7 tumor cell line, the BCMA-negative K562 tumor cell line, or without adding tumor cells for 18 h. Then, the expression level of CD137 on the membrane surface of T cells was detected by flow cytometry, and the secretion level of IFNγ in the culture supernatant was detected by the ELISA method. As shown in Figures 4A and 4B, the expression of CD137 was detected on the surface of all four CART cells, and the expression of IFNγ was detected in all the culture supernatants. Among them, CAR-BCMA-20 had the best CD137 activation level and IFNγ release level. CART-BCMA-MO6 constructed based on the humanized MO6 antibody sequence had a weaker CD137 activation level compared to CART-BCMA-CA8 constructed based on the murine-derived antibody sequence, but had a high IFNγ release level.

[0069] Example 5 Detection of the induction activity of terminal apoptosis of tumor cells by CART-BCMAs cells. (1) At ratios of 1:1, 2.5:1, 5:1, 10:1, and 20:1 (the ratios shown in Figure 5), 1×10 4Individual CFSE-labeled BCMA-negative cells (NH929) or BCMA-positive constructed cells NH929-BCMA overexpressing tumor cell lines were mixed and co-cultured in 100 μl of GT-551 medium for 4 h. After that, they were stained with 100 μl of 25% PI dye for 15 min, and the ratio of PI-positive cells in CFSE-positive cells was analyzed by a flow cytometer. (2) At ratios of 1:1, 5:1, 10:1, 20:1, 40:1 (the ratios shown in Fig. 6B), the CART-BCMAs cells cultured until day 22 in Example 2 were each 1×10 4 Individual CFSE-labeled BCMA-negative cells (NH929), BCMA-positive constructed cells NH929-BCMA overexpressing tumor cell lines, or the MM.1S cell line expressing natural BCMA were mixed and co-cultured in 100 μl of GT-551 medium for 4 h. After that, they were stained with 100 μl of 25% PI dye for 15 min, and the ratio of PI-positive cells in CFSE-positive cells was analyzed by a flow cytometer. As shown in Figs. 5 and 6, all four CART cells were able to preferably induce apoptosis of BCMA-positive tumor cells. Among them, CART-BCMA-20 preferably induced late apoptosis of BCMA-positive tumor cells more than CART-BCMA-1, and CART-BCMA-MO6 and CART-BCMA-CA8 had similar abilities to induce late apoptosis of BCMA-positive tumor cells.

[0070] Example 6 Inhibitory effect of CART-BCMAs on the RPMI-8226 multiple myeloma xenograft model RPMI-8226 cells in the logarithmic growth phase were collected, and 4.0×10 6 tumor cells were subcutaneously inoculated into the right dorsal part of 6-8-week-old B-NDG mice. When the tumor volume reached about 120 mm 3 , the animals were randomly divided into 4 groups according to tumor volume such that the difference in tumor volume between each group was less than 10% of the average value. Then, from the tail vein, solvent control, 7.5×10 6 individuals of NT and 7.5×10 6Individual CART-BCMAs cells were injected. As shown in Figure 7, compared with the control group, single intravenous injection of CART-BCMA-1 and CART-BCMA-20 effectively inhibited the growth of human myeloma RPMI-8226 cells (relative tumor growth rate %T / CRTV ≤ 40%, P < 0.05), and significantly extended the survival time of human myeloma-bearing mice (the survival period in the control group was 23 days, and the median survival period in the CART-BCMAs treatment group > 33 days). Among them, there was no significant difference in the relative tumor growth rate and the median survival period between the mice treated with CART-BCMA-1 and CART-BCMA-20.

[0071] Comparative Example During the screening process of the chimeric antigen receptor of the present application, the inventors tested a large number of candidate sequences, and the following will be described with examples. The screened antibodies are BCMA-1, BCMA-2, BCMA-69, BCMA-72, BCMA-2A1, BCMA-1E1, BCMA-J22.9, BCMA-20, BCMA-CA8, BCMA-MO6. It is the structure of a chimeric antigen receptor targeting BCMA constructed based on the above antibodies. Here, BCMA-1 and BCMA-2 are already published Car-T sequences. As a positive control for screening, the method for manufacturing CAR-T cells is the same as in Example 2, and the detection method is the same as in Examples 3 and 4. As shown in Figure 1, the implementation results of two lots of CAR-T cells are respectively shown. When detecting the expression of Car-T with BCMA-Fc fusion protein, high expression was observed in primary T cells. Refer to Figure 1A. In Figure 1B, it can be seen that BCMA-1, BCMA-20, BCMA-1E1, BCMA-CA8, BCMA-MO6, and BCMA-J22.9 were activated by the BCMA antigen. In Figure 1C, it was shown that the Car-T of the activated BCMA-1, BCMA-20, BCMA-1E1, BCMA-CA8, BCMA-MO6, and BCMA-J22.9 generated relatively high IFN-γ. Summarizing these results, it was shown that the CAR-T obtained with BCMA-1, BCMA-20, CA8, and MO6 had similar functions. Therefore, BCMA-20, CA8, and MO6 CAR-T were further analyzed and studied.

[0072] All documents related to the present invention are cited for reference in this application so that each document is cited independently. After reading the above content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and it should be understood that those equivalent forms are included in the scope of the claims of the present invention.

Claims

1. A chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor is a sequence of an antibody single-chain variable region of an extracellular domain that targets BCMA.

2. The chimeric antigen receptor according to claim 1, wherein the antigen-binding domain is a sequence of an antibody single-chain variable region targeting the 24th to 41st amino acid residues of the BCMA sequence.

3. The chimeric antigen receptor according to claim 1, wherein the structure of the antigen-binding domain is represented by the following formula I: Ｌ -V Ｈ (I) (However, V Ｈ is the heavy chain variable region of an antibody, and V Ｌ is the light chain variable region of an antibody, and "-" is a connecting peptide or peptide bond. Ｌ The amino acid sequence of V is shown in SEQ ID NO:

1. Ｈ The amino acid sequence of V is shown in SEQ ID NO:2; Ｌ The amino acid sequence of V is shown in SEQ ID NO:

3. Ｈ The amino acid sequence of V is shown in SEQ ID NO:4, Ｌ The amino acid sequence of V is shown in SEQ ID NO:

5. Ｈ The amino acid sequence is shown in SEQ ID NO:

6.

4. The chimeric antigen receptor according to claim 1, wherein the structure of the chimeric antigen receptor is represented by the following formula II: S-V Ｌ -V Ｈ -H-TM-C-CD3ζ (II) (wherein S is any signal peptide; H is a hinge region; TM is a transmembrane domain; C is a costimulatory signal molecule; and CD3ζ is an intracellular signaling sequence derived from CD3ζ. Ｈ and V Ｌ are as above.)

5. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the vector of claim 6, or chromosomally incorporating an exogenous nucleic acid molecule of claim 5, or expressing the chimeric antigen receptor of claim 1.

8. A method for producing a CAR-T cell, the CAR-T cell expressing the CAR according to claim 1, the method comprising: obtaining the CAR-T cell by transducing a nucleic acid molecule according to claim 5 or a vector according to claim 6 into a T cell.

9. A formulation comprising the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 5, the vector of claim 6, or the host cell of claim 7, together with a pharma- ceutically acceptable carrier, diluent or excipient.

10. Use of the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 5, the vector of claim 6, or the host cell of claim 7, characterized in that it is used for the manufacture of a drug or formulation for preventing and / or treating cancer or tumors.

11. The use according to claim 10, characterized in that the tumor is a BCMA-positive tumor.

12. The use according to claim 10, characterized in that the tumor is a BCMA-positive B-cell lymphoma, multiple myeloma, or plasma cell leukemia.

Images