CN116854825A

CN116854825A - Humanized chimeric antigen receptor targeting BCMA and uses thereof

Info

Publication number: CN116854825A
Application number: CN202310780655.4A
Authority: CN
Inventors: 刘丹; 王璞; 施明; 王旭; 赵璇; 郑骏年
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2023-06-29
Filing date: 2023-06-29
Publication date: 2023-10-10
Anticipated expiration: 2043-06-29
Also published as: CN116854825B

Abstract

The invention discloses a humanized chimeric antigen receptor targeting BCMA and application thereof. The present invention provides a humanized chimeric antigen receptor targeting BCMA, as well as nucleic acid molecules encoding the chimeric antigen receptor, engineered cells and methods for preparing said cells, and the use of said cells for preparing a product for diagnosing or treating a disease associated with BCMA expression, in particular for treating multiple myeloma or acute lymphoblastic leukemia. The chimeric antigen receptor of the targeted BCMA can effectively avoid target escape and prevent tumor recurrence.

Description

Humanized chimeric antigen receptor targeting BCMA and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a humanized chimeric antigen receptor targeting BCMA and application thereof.

Background

B cell maturation antigen (B-cel 1maturation antigen, BCMA, also known as CD269, TNFRSF 17) is a member of the Tumor Necrosis Family Receptor (TNFR) expressed by cells of the B cell lineage. BCMA expression is highest on terminally differentiated B cells. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. Recently, BCMA expression has been associated with a number of cancers, autoimmune diseases and infectious diseases. Cancers with increased BCMA expression include some hematological cancers such as myeloma, hodgkin's lymphoma and non-hodgkin's lymphoma, various leukemias, or glioblastomas.

Multiple Myeloma (MM) is a blood system tumor originating from malignant plasma cells in bone marrow, and frequently accompanied symptoms are Multiple osteolytic pathological changes, anemia, kidney injury, hypercalcemia, and the like. Since malignant tumors are a clonal disease, many subclones form within tumor cells over time, genetic and phenotypic heterogeneity of tumor cells often occurs in the same patient. In the past half century, the treatment of MM has made remarkable progress in clinical applications through proteasome inhibitors, immunomodulators, and monoclonal antibody therapies, making the treatment of MM into the age of targeted immunotherapy. However, MM remains largely incurable, and recurrence and intractability remain major problems of treatment. Acute Leukemia (AL) is a malignant clonal disease that originates from hematopoietic stem/progenitor cells, is a clinically common malignant hematological disease, with mortality leading to malignancy in adults under 35 years of age, and severely jeopardizes human health. Refractory/Relapsed (R/R) is the major factor responsible for low long-term survival of AL. The current treatment means of AL is mainly chemotherapy and hematopoietic stem cell transplantation conventional treatment means, and along with the development of medical technology, the diagnosis and treatment technology of AL is continuously improved, but the traditional treatment is mainly. AL mainly includes acute lymphoblastic leukemia (Acute Lymphocytic Leukemia, ALL) and acute myelogenous leukemia (Acute Myeloid Leukemia, AML).

Chimeric antigen receptor T cell (Chimeric Antigen Receptor T cell, CAR-T) therapy is a novel treatment method with definite therapeutic effects on certain hematological tumors. Chimeric Antigen Receptor (CAR) structures are genetically engineered molecules that direct T cells to specifically attack tumor cells by antigen-antibody interactions rather than by Major Histocompatibility Complex (MHC) -dependent means. Among the various types of cell therapies, CAR-T is the most predominant and effective antitumor drug in the treatment of cancer, particularly hematological malignancy. Thus, it is of great importance to study CAR-T therapy for the treatment of multiple myeloma and acute leukemia. However, CAR-T still faces many difficulties in the field of tumor application at present, such as insufficient sensitivity of CAR-T to tumor cells, low killing ability, large clinical dosage, and long in vitro preparation time. How to enhance the sensitivity and killing ability of the CAR-T to tumor cells, improve the treatment effect of the CAR-T, reduce the clinical dosage of the CAR-T, improve the in vitro proliferation ability of the CAR-T and shorten the preparation time is a problem to be solved by the CAR-T therapy.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a humanized chimeric antigen receptor (BCMA-CAR mutant or BCMA-CAR) targeting BCMA ^mut ) BCMA-CAR prepared by the method ^mut -CAR-T to enhance TCR signaling activity, enhancing the killing ability of CAR-T against tumors.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first aspect of the present invention provides a humanized chimeric antigen receptor targeting BCMA, said chimeric antigen receptor comprising an antibody fragment scFv that specifically binds BCMA; the antibody fragment scFv is obtained by substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1; the substituted site in the amino acid sequence is selected from any one or more of the following sites in SEQ ID NO. 1: y102, N103, N172, N175, T192 or S193.

Further, the site of substitution in the amino acid sequence is selected from the following sites in SEQ ID No. 1: y102, N103, N172, N175, T192, and S193.

Further, the amino acid substitutions of Y102, N103, N172, N175, T192, and S193 comprise Y102H, N103G, N G, N175G, T a and S193G.

Further, the amino acid sequence of the antibody fragment scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 3.

Further, the amino acid sequence of the antibody fragment scFv is shown as SEQ ID NO. 3.

Further, the chimeric antigen receptor further comprises a transmembrane domain.

Further, the transmembrane domain comprises a transmembrane domain of: igG1, igG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3 epsilon, or variants thereof.

Further, the transmembrane domain is a CD8 transmembrane domain.

Further, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO. 7.

Further, the chimeric antigen receptor further comprises a hinge region.

Further, the hinge region comprises a hinge region of: igG1, igG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3 epsilon, or variants thereof.

Further, the hinge region is a CD8 hinge region.

Further, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO. 5.

Further, the chimeric antigen receptor further comprises an intracellular signaling domain.

Further, the intracellular signaling domain comprises an intracellular signaling domain of: fcγ R, fc ε R, fc α R, fcRn, CD3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3b, C3dg, C3d, zap70, or variants thereof.

Further, the intracellular signaling domain is a cd3ζ intracellular signaling domain.

Further, the amino acid sequence of the CD3 zeta intracellular signaling domain is shown as SEQ ID NO. 11.

Further, the chimeric antigen receptor further comprises a costimulatory signaling domain.

Further, the costimulatory signaling domain comprises the costimulatory signaling domain of: CD19, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, ICAM, LFA-1, lck, CD2, CD4, CD5, CD7, CD226, CD8 alpha, CD8 beta, LIGHT, CD83, DAP10, DAP12, GITR, DR3, NKG2C, HVEM, B7-H3 or variants thereof.

Further, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

Further, the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO. 9.

Further, the chimeric antigen receptor further comprises a signal peptide.

Further, the signal peptide comprises a signal peptide of: the alpha chain, beta chain, CD3 ζ, CD3 epsilon, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, igG, or variants thereof of the T cell receptor.

Further, the signal peptide is a CD8 signal peptide.

Further, the amino acid sequence of the CD8 signal peptide is shown as SEQ ID NO. 13.

Further, the chimeric antigen receptor contains a signal peptide, an scFv, a hinge region, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain, which are sequentially linked.

Further, the chimeric antigen receptor contains a CD8 signal peptide, scFv, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signal domain, cd3ζ intracellular signaling domain, connected in sequence.

Further, the amino acid sequence of the chimeric antigen receptor is an amino acid sequence formed by sequentially connecting SEQ ID NO.13, 3, 5, 7, 9 and 11.

In a second aspect, the invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor according to the first aspect of the invention.

Further, the coding sequence of the chimeric antigen receptor comprises a coding sequence of a signal peptide, a coding sequence of an scFv, a coding sequence of a hinge region, a coding sequence of a transmembrane domain, a coding sequence of a co-stimulatory signaling domain, a coding sequence of an intracellular signaling domain, which are sequentially linked.

Further, the coding sequence of the chimeric antigen receptor comprises a coding sequence of a CD8 signal peptide, a coding sequence of scFv, a coding sequence of a CD8 hinge region, a coding sequence of a CD8 transmembrane domain, a coding sequence of a 4-1BB costimulatory signal domain, and a coding sequence of a CD3 zeta intracellular signaling domain, which are sequentially connected.

Further, the coding sequence of the CD8 signal peptide is shown as SEQ ID NO. 14.

Further, the coding sequence of the scFv is shown as SEQ ID NO. 4.

Further, the coding sequence of the CD8 hinge region is shown as SEQ ID NO. 6.

Further, the coding sequence of the CD8 transmembrane domain is shown as SEQ ID NO. 8.

Further, the coding sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO. 10.

Further, the coding sequence of the CD3 zeta intracellular signaling domain is shown as SEQ ID NO. 12.

In a third aspect, the invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

Further, the vector comprises a cloning vector and an expression vector.

Further, the vector comprises a DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector, or viral vector.

Further, the viral vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, a herpesviral vector, or a retroviral vector.

Further, the viral vector is a lentiviral vector.

In a fourth aspect, the invention provides an engineered cell comprising or expressing the chimeric antigen receptor according to the first aspect, the nucleic acid molecule according to the second aspect or the vector according to the third aspect of the invention.

Further, the cells comprise immune cells.

Further, the immune cells comprise T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof.

Further, the immune cells are T cells.

Further, the T cells comprise primary T cells or Jurkat cells.

In a fifth aspect, the invention provides a method of preparing a cell according to the fourth aspect of the invention, the method comprising introducing into the cell a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention.

In a sixth aspect the present invention provides a derivative comprising a detectably labelled chimeric antigen receptor according to the first aspect of the invention and/or a nucleic acid molecule according to the second aspect of the invention, a chimeric antigen receptor according to the first aspect of the invention and/or a nucleic acid molecule according to the second aspect of the invention which confers antibiotic resistance, a chimeric antigen receptor according to the first aspect of the invention and/or a nucleic acid molecule according to the second aspect of the invention which is conjugated or coupled to a therapeutic agent.

Further, the detectable label comprises a radionuclide, fluorophore, chemiluminescent agent, microparticle, enzyme, colorimetric label, magnetic label, hapten, molecular beacon, or aptamer beacon.

Further, the therapeutic agent comprises a radionuclide, cytokine, gold nanoparticle, viral particle, liposome, nanomagnetic particle, prodrug activating enzyme, or chemotherapeutic agent.

The seventh aspect of the invention provides a pharmaceutical composition or kit comprising a chimeric antigen receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a cell according to the fourth aspect of the invention.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

An eighth aspect of the invention provides any one of the following applications:

1) Use of a chimeric antigen receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a cell according to the fourth aspect of the invention, a derivative according to the sixth aspect of the invention or a pharmaceutical composition or kit according to the seventh aspect of the invention for the preparation of a product for the diagnosis or treatment of a disease associated with BCMA expression;

2) Use of a chimeric antigen receptor according to the first aspect of the invention for the preparation of a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a cell according to the fourth aspect of the invention, a derivative according to the sixth aspect of the invention or a pharmaceutical composition or kit according to the seventh aspect of the invention;

3) Use of a nucleic acid molecule according to the second aspect of the invention for the preparation of a vector according to the third aspect of the invention, a cell according to the fourth aspect of the invention, a derivative according to the sixth aspect of the invention or a pharmaceutical composition or kit according to the seventh aspect of the invention;

4) Use of a vector according to the third aspect of the invention for the preparation of a cell according to the fourth aspect of the invention, a derivative according to the sixth aspect of the invention or a pharmaceutical composition or kit according to the seventh aspect of the invention;

5) Use of a cell according to the fourth aspect of the invention for the preparation of a derivative according to the sixth aspect of the invention or a pharmaceutical composition or kit according to the seventh aspect of the invention;

6) The use of a derivative according to the sixth aspect of the invention for the preparation of a pharmaceutical composition or kit according to the seventh aspect of the invention.

Further, the disease associated with BCMA expression comprises cancer, a precancerous condition, or a non-cancer related indication.

Further, the disease associated with BCMA expression is cancer.

Further, the cancer comprises myeloma, myelodysplastic and myelodysplastic syndrome, acute lymphoblastic leukemia, chronic leukemia, blast plasmacytoid dendritic cell tumor, burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, plasmablastoid lymphoma, plasmacytoid dendritic cell tumor, glioblastoma, waldenstrom's macroglobulinemia, hodgkin's lymphoma or non-hodgkin's lymphoma.

Further, the cancer is myeloma or acute lymphoblastic leukemia.

Further, the myeloma is multiple myeloma.

The invention has the advantages and beneficial effects that:

BCMA-CAR constructed according to the present invention ^mut The TCR signal activity is stronger after contacting with target cells, and BCMA-CAR is obviously improved ^mut Killing efficiency of T, reduced BCMA-CAR ^mut Clinical dose of-T, shortening BCMA-CAR ^mut The in vitro preparation time of T is safe and effective, and can be used for immune cell treatment of blood tumor, solid tumor and autoimmune disease.

Drawings

FIG. 1 is a BCMA-CAR and a BCMA-CAR ^mut Wherein 1A is a construction pattern diagram of a BCMA-CAR and 1B is a construction pattern diagram of a BCMA-CAR ^mut Is a construction pattern diagram of (1);

FIG. 2 is a schematic diagram of the construction of Jurkat-Reporter stable cell lines;

FIG. 3 is a flow cytometric detection of BCMA-CAR-Jurkat-Reporter, BCMA-CAR ^mut BCMA-CAR and BCMA-CAR of Jurkat-Reporter cells ^mut Positive expression rate and average fluorescence intensity;

FIG. 4 is a flow cytometric assay of BCMA-CAR-Jurkat-Reporter, BCMA-CAR after receiving chemical stimulus ^mut -effect plot of GFP positive expression rate and average fluorescence intensity of Jurkat-Reporter cells, wherein 4A is effect plot of GFP positive expression rate and 4B is effect plot of average fluorescence intensity;

FIG. 5 is a graph showing the effect of flow cytometry on detecting the positive expression rate of BCMA on the surface of U266 target cells and Nalm6-BCMA-GFP, wherein 5A is a graph showing the effect of positive expression rate of BCMA on the surface of Nalm6-BCMA-GFP, and 5B is a graph showing the effect of positive expression rate of BCMA on the surface of U266 cell;

FIG. 6 is a flow cytometric detection of BCMA-CAR-Jurkat-Reporter, BCMA-CAR ^mut -graph of TCR signal intensity after Jurkat-Reporter cells received biological stimulation, wherein 6A is graph of TCR signal intensity after U266 cells, and 6B is graph of TCR signal intensity after Nalm6-BCMA-GFP cells;

FIG. 7 is a flow cytometric assay of patient-derived T-cell-produced BCMA-CAR-T cells and BCMA-CAR ^mut BCMA-CAR and BCMA-CAR for T cells ^mut Positive expression rate and average fluorescence intensity;

FIG. 8 is a graph showing the effect of flow cytometry on detecting the positive expression of BCMA on the surface of CHO cells;

FIG. 9 is an RTCA evaluation of patient-derived T cell-produced BCMA-CAR-T and BCMA-CAR ^mut -a T-killing function effect graph, wherein 9A is a normalized cell index effect graph with RTCA real-time monitoring effect target ratio of 1:1, and 9B is a normalized cell index effect graph with RTCA real-time monitoring effect target ratio of 1:5;

FIG. 10 is a flow cytometric assay of normal human-derived T-cell-prepared BCMA-CAR-T cells and BCMA-CAR ^mut BCMA-CAR and BCMA-CAR for T cells ^mut Positive expression rate and averageEffect graph of fluorescence intensity;

FIG. 11 is a graph of the effect of different CAR-T cell killing functions of normal human-derived T cell preparations, wherein 11A is the flow cytometric detection of BCMA-CAR-T cells and BCMA-CAR ^mut -ratio effect profile of T cells on residual tumor cells at different effective target ratios, 11B is a statistical plot of residual tumor cell ratio.

Detailed Description

The invention develops a BCMA-CAR mutant (BCMA-CAR) through extensive and intensive research ^mut ) And construct BCMA-CAR ^mut -T。BCMA-CAR ^mut T after stimulation with target antigen, the activity intensity of TCR signal is significantly higher than BCMA-CAR-T, indicating that BCMA-CAR is contained ^mut Has stronger killing function to target cells.

In the present invention, the term "BCMA" generally refers to B cell maturation antigen protein (also known as TNFRSF17, BCM or CD 269), i.e. a Tumor Necrosis Factor Receptor (TNFR) family member, expressed on plasma cells and mature B cells. For example, human BCMA is a 184 amino acid long protein encoded by a 994 nucleotide long primary mRNA transcript (nm_ 001192.2). The amino acid sequence of human BCMA is indicated by UniPretKB accession number Q02223. In the present invention, the term "BCMA" may comprise mutated proteins, for example proteins which may comprise point mutations, fragments, insertions, deletions and splice variants of full-length wild-type BCMA. In the present invention, the term "BCMA" may also include a portion of an intact BCMA protein, as long as the relevant biological activity is retained.

In the present invention, the term "targeting" when referring to protein expression is intended to include, but is not limited to, directing a protein or polypeptide to an appropriate destination within or outside of a cell. Targeting is typically achieved by a signal peptide or targeting peptide, which is a stretch of amino acid residues in the polypeptide chain. These signal peptides may be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. The polypeptides may also be engineered to have a signal peptide at the C-terminus. The signal peptide may direct extracellular cleavage of the polypeptide, localization to plasma membranes, golgi apparatus, endosomes, endoplasmic reticulum and other cellular compartments. As used herein, the term "targeting" when referring to targeting of a tumor refers to the ability of a cell to recognize and kill a tumor cell (i.e., a target cell). The term "targeting" in this context refers to the ability of, for example, a CAR expressed by a cell to recognize and bind to a cell surface antigen expressed by a tumor.

In the present invention, the terms "chimeric antigen receptor" and "CAR" are used interchangeably and refer to a chimeric polypeptide comprising a plurality of functional domains, which are arranged in sequence from amino-to carboxy-terminus: (a) An extracellular domain comprising an antigen binding domain (e.g., comprising an antibody fragment scFv) and a hinge region; (b) a transmembrane domain; and (c) an intracellular region (comprising a costimulatory signaling domain, one or more intracellular signaling domains), wherein the aforementioned domains may optionally be linked by one or more spacer domains. The CAR may further comprise a signal peptide sequence, which is typically removed during post-translational processing, and presents the CAR on the surface of a cell transformed with an expression vector comprising a nucleic acid sequence encoding the CAR. CARs may be prepared according to principles well known in the art.

The term "extracellular domain" as used in the present invention is used in the same sense as "Antigen Binding Domain (ABD)". The term "extracellular domain" or "antigen binding domain" refers to a portion of an antigen binding molecule that has the ability to non-covalently, reversibly, and specifically bind to an antigen. As used herein, the term "antigen binding domain" encompasses antibody fragments that retain the ability to non-covalently, reversibly, and specifically bind to an antigen. In a specific embodiment of the invention, the extracellular domain or antigen-binding domain refers to an antibody or antibody fragment scFv that specifically binds BCMA. The antigen binding domain may be derived from natural sources, synthetic sources, semisynthetic sources, or recombinant sources.

In the present invention, the term "transmembrane domain" is used interchangeably with "transmembrane region (TM)" and refers to a portion of the CAR that fuses the extracellular domain and the intracellular region and anchors the CAR to the plasma membrane of immune effector cells. The transmembrane domain may be obtained from a natural protein, or may be obtained synthetically, semisynthetically or recombinantly. The chimeric antigen receptor of the present invention further comprises a hinge region, and is a sequence that promotes binding of the chimeric antigen receptor to an antigen via a mutant antibody and enhances signal transduction to cells. The hinge region may be disposed between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The hinge region also means any oligopeptide or polypeptide that functions by linking the transmembrane domain to the extracellular domain, and/or the transmembrane domain to the intracellular region. The hinge region comprises up to 300 amino acids, for example about 10 to 100 amino acids, or about 25 to 50 amino acids.

The transmembrane domains and hinge regions include, but are not limited to, the transmembrane domains and hinge regions of: the T cell receptor alpha, beta or zeta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD34, CD37, CD45, CD64, CD80, CD86, OX40, CD137, ICOS, CD152, CD154, igG1, igG4, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, GITR, or variants thereof. In a specific embodiment of the invention, the transmembrane domain and hinge region is a CD8 transmembrane domain and hinge region.

In the present invention, the term "intracellular signaling domain" is generally part of a CAR that is involved in transducing information of an effective anti-BCMA CAR binding human BCMA polypeptide into the interior of immune effector cells to elicit effector cell functions such as activation, cytokine production, proliferation and cytotoxic activity, including release of cytotoxic factors to the CAR-bound target cells or other cellular responses elicited by antigens bound to the extracellular domain of the CAR. In the present invention, the intracellular signaling domain may comprise, but is not limited to, the intracellular signaling domain of: fcγ R, fc ε R, fc α R, fcRn, CD3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3b, C3dg, C3d, zap70, or variants thereof. In a specific embodiment of the invention, the intracellular signaling domain is a cd3ζ intracellular signaling domain.

In the present invention, the term "costimulatory signaling domain" generally refers to the intracellular signaling domain of a costimulatory molecule. For example, the co-stimulatory molecule may be a cell surface molecule other than an antigen receptor or Fc receptor that, upon antigen binding, may provide a second signal required for efficient activation and function of T lymphocytes. For example, the costimulatory signaling domain may be selected from the group consisting of: CARD11, CD2, CD4, CD5, CD7, CD8 a, CD8 β, CD19, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX 40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LFA-1, lck, LAT, LIGHT, DAP, DAP12, NKD2CSLP76, GITR, DR3, NKG2C, TRIM, B7-H3, ZAP70, or variants thereof, or a co-stimulatory signaling domain derived from a killer immunoglobulin-like receptor (KIR). In a specific embodiment of the invention, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

In the present invention, "CAR" includes, but is not limited to, a first generation CAR, a second generation CAR, a third generation CAR, or a fourth generation CAR. The term "first generation CAR" refers to a CAR in which the intracellular region comprises only a single conductive signal, e.g., only a cd3ζ single signal is contained in the intracellular region of the CAR. The second generation CARs include a costimulatory signaling domain, including but not limited to CD28, 4-1BB, in addition to a single signaling domain such as the cd3ζ signaling domain, which enhances the persistence, cytokine secretion, and anti-tumor activity of CAR-T. Third generation CARs include two costimulatory signal domains. The fourth generation of CARs, also known as "armored vehicles", are based on the third generation of CARs, further adding structures that can improve CAR-T cell function, such as receptor structures for cytokines or chemokines.

Exemplary cellular regions that can be incorporated into the CARs of the invention comprise (amino to carboxyl): CD3 ζ; CD28-CD3 zeta; CD28-OX40-CD3 ζ; CD28-41BB-CD3 zeta; 41BB-CD28-CD3 zeta and 41BB-CD3 zeta. In a specific embodiment of the invention, the intracellular comprises a 41BB-CD3 zeta signal.

The term "specific binding" as used herein refers to the property of complementary binding with high affinity, determined by the spatial conformation of the antigenic determinant and the variable region of the antibody molecule. This high affinity determines that the antibody molecule, once bound to the antigen, can exert its corresponding physiological function,

the term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid vhh domains, multispecific antibodies formed from antibody fragments (e.g., bivalent fragments comprising two Fab fragments linked at the hinge region by disulfide bonds), and isolated CDRs or other epitope-binding fragments of antibodies.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

In the present invention, the CAR portion comprising the antibody fragment may exist in a variety of forms, wherein the antigen binding domain is expressed as part of a continuous polypeptide chain comprising, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) humanized antibody, or a bispecific antibody. In one aspect, the antigen binding domain of the CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment scFv.

"amino acid substitution" or "substitution" as used herein refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence in a polypeptide with a different amino acid. Amino acid substitutions may be made using genetic or chemical methods well known in the art. For example, single amino acid substitutions or multiple amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions) may be made in a naturally occurring sequence (e.g., in a portion of the polypeptide outside of the domain that forms intermolecular contacts).

The term "conservative amino acid substitution" is the substitution of one amino acid residue with another amino acid residue having a side chain of similar chemical properties. Families of amino acid residues with similar side chains have generally been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of phenylalanine with tyrosine is a conservative substitution. In general, conservative substitutions in the sequence of a polypeptide, soluble protein, and/or antibody of the present disclosure do not eliminate binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence to the target binding site. Methods for identifying amino acid conservative substitutions that do not eliminate binding are well known in the art.

The term "non-conservative amino acid substitution" refers to the substitution of a member from one of these classes for a member from another class. In making such changes, according to various embodiments, the hydropathic index of amino acids may be considered (hydropathic index). Each amino acid has been assigned a hydropathic index based on the hydrophobicity and charge characteristics of the amino acid. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

The art understands the importance of the hydrophilic amino acid index in conferring biological functions of protein interactions. It is known that certain amino acids may be substituted with other amino acids having similar hydrophilicity indices or scores and still retain similar biological activity.

An antibody fragment scFv (or "BCMA binding domain") that specifically binds to BCMA as used in the present invention has a different binding moiety comprising one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions compared to the amino acid sequence shown in SEQ ID No.1, the BCMA binding domain retaining at least an active moiety that specifically binds to BCMA. Specifically, the BCMA binding domain used in the present invention can be obtained by substituting a part of amino acids in the amino acid sequence shown in SEQ ID No. 1. One embodiment of the BCMA binding domain used in the present invention obtained by amino acid substitution can be obtained by substituting at least one of amino acids 102, 103, 172, 175, 192 or 193 in the amino acid sequence shown as SEQ ID No. 1. In a specific embodiment of the invention, the BCMA binding domain comprises amino acid substitutions of amino acid residue positions Y102, N103, N172, N175, T192 and S193 of SEQ ID No. 1; in a specific embodiment of the invention, the amino acid substitutions are Y102H, N103G, N172G, N175G, T192A and S193G; further, the amino acid sequence of the BCMA binding domain used in the present invention is shown as SEQ ID NO. 3.

As used herein, the term "% identity" when used anywhere in the context of two or more nucleic acid or protein/polypeptide sequences means that the two or more sequences or subsequences are the same or have (or at least have) a specified percentage of amino acid residues or nucleotides that are the same, as measured using a sequence comparison algorithm or by manual alignment and visual inspection (see, e.g., NCBI website) (i.e., have or at least about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% identity in a specified region (preferably over its full length sequence) when compared and aligned to obtain maximum correspondence over a comparison window or specified region).

In the present invention, the term "isolated" or "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

The term "isolated nucleic acid molecule" as used herein generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof, isolated from the natural environment or synthesized.

The term "encoding" as used herein refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, in a biological process as a template for the synthesis of other polymers and macromolecules having defined nucleotide sequences (e.g., rRNA, tRNA and mRNA) or defined amino acid sequences and biological properties derived therefrom. Thus, a gene, cDNA or RNA encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is normally provided in the sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be referred to as a protein or other product encoding the gene or cDNA.

In the present invention, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell.

The term "cloning vector" as used in the present invention is defined as a substance capable of carrying and replicating a DNA fragment into a host cell. In the present invention, the cloning vector may further comprise a polyadenylation signal, a transcription termination sequence and a plurality of cloning sites. In this case, the plurality of cloning sites comprises at least one endonuclease restriction site. In addition, the cloning vector may further include a promoter. Furthermore, the term "expression vector" as used in the present invention is defined as a DNA sequence required for transcription and translation of cloned DNA in a suitable host. Further, the term "expression vector" as used herein refers to a genetic construct comprising the necessary regulatory elements operably linked to an insertion sequence such that the insertion sequence is expressed if it is present in a cell of an individual. Expression vectors can be generated and purified using standard recombinant DNA techniques. The kind of the expression vector is not particularly limited as long as it has a function of expressing a desired gene and producing a desired protein in various host cells such as prokaryotic and eukaryotic cells. However, it is preferably a vector capable of producing a large amount of foreign proteins in a form similar to the natural state, while having a promoter exhibiting strong activity and strong expression ability. The expression vector preferably comprises at least a promoter, an initiation codon, a gene encoding the desired protein, and a termination codon. In addition, it may suitably include a DNA encoding a signal peptide, additional expression control sequences, untranslated regions at the 5 'and 3' ends of the desired gene, selectable marker regions, replication units, and the like.

For example, the carrier comprises: a DNA vector; an RNA vector; a plasmid; a transposon vector; CRISPR/Cas9 vector; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papilloma viruses, papilloma-polyomaviruses (e.g., SV 40). It will be apparent that the viral vectors according to the present disclosure need not be limited to components of a particular virus, that the viral vectors may comprise components derived from two or more different viruses, and may also comprise synthetic components. In a specific embodiment of the invention, the vector is a lentiviral vector. A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Lentiviruses are unique among retroviruses, which are capable of infecting non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, and therefore they are one of the most effective methods of gene delivery vectors. Lentiviruses include, but are not limited to: human Immunodeficiency Virus (HIV), including HIV type and type 2 HIV; weissner-Meidi virus (VMV) virus; goat arthritis-encephalitis virus (CAEV); equine Infectious Anemia Virus (EIAV); feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); simian Immunodeficiency Virus (SIV). Lentiviral-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

In the present invention, the term "expression" refers to biosynthesis of a gene product, preferably to transcription and/or translation of a nucleotide sequence (e.g., an endogenous gene or a heterologous gene) in a cell. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA, and optionally subsequent translation of the mRNA into one or more polypeptides.

In the present invention, the term "immune cell" refers to a cell that can elicit an immune response, and "immune cell" and grammatical variations thereof can refer to any source of immune cells. The term "immune cell" may also be human or non-human. "immune cells" include, but are not limited to, leukocytes, T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof. Further, the immune cells are T cells; still further, examples of the T cells include, but are not limited to, primary T cells ("lymphocyte progenitor cells"), central memory T cells, effector memory T cells, T memory stem cells (Tscm), iPSC-derived T cells, synthetic T cells, jurkat cells, or combinations thereof.

In the present invention, the term "T cell" or "T lymphocyte" is a term understood by those skilled in the art and means a lymphocyte type that develops in the thymus and plays a central role in immune response. T cells can be distinguished from other lymphocytes by the presence of T Cell Receptors (TCRs) on the cell surface. The term "primary T cells" is meant to include T cells obtained from an individual as opposed to T cells that are maintained in culture for an extended period of time. Thus, primary T cells are in particular peripheral blood T cells obtained from a subject. The population of primary T cells may consist essentially of a subset of T cells. Alternatively, the population of primary T cells may consist of different subsets of T cells. The term "Jurkat cell" or "Jurkat" means an immortalized cell line of human T lymphocytes and is widely used for the study of acute T cell leukemia, T cell signaling and functional luciferase.

As used herein, the term "introducing" refers to a method of delivering a vector comprising a polynucleotide encoding an antibody into a host cell. Such introduction may be performed by different methods known in the art, including but not limited to the use of lipofection, DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, and protoplast fusion. In addition, transfection refers to the use of viral particles to deliver desired material into cells by infection. Alternatively, the vector may be introduced into the host cell by gene bombardment.

In the present invention, the term "derivative" may be a nucleic acid molecule, as a DNA molecule, encoding a polynucleotide as defined above, or a nucleic acid molecule comprising a polynucleotide as defined above, or a polynucleotide of complementary sequence. In the context of the present invention, the term "derivative" also refers to longer or shorter polynucleotides and/or polypeptides having, for example, a percentage of identity of at least 41%, 50%, 60%, 65%, 70% or 75%, more preferably at least 85%, such as at least 90%, even more preferably at least 95% or 100%, with the mentioned sequence or its complement or its DNA or RNA counterpart. The term "derivative" also includes modified synthetic oligonucleotides. The term "derivative" may also include nucleotide analogs, i.e., naturally occurring ribonucleotides or deoxyribonucleotides substituted with non-naturally occurring nucleotides. The term "derivative" also includes nucleic acids or polypeptides that can be produced by mutating one or more nucleotides or amino acids in its sequence, equivalent or precursor sequence. The term "derivative" also includes at least one functional fragment of a polynucleotide.

In the present invention, the term "detectable label" is used to indicate that an entity may be visualized or otherwise detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. The detectable label may be selected such that it produces a signal that can be measured and whose intensity is proportional to the amount of bound entity. A wide variety of systems for labeling and/or detecting proteins and peptides are known in the art. The label may be directly detectable (i.e. it does not require any further reaction or manipulation to achieve detection, e.g. the fluorophore is directly detectable), or it may be indirectly detectable (i.e. it is detectable by reaction or binding with another entity that is detectable, e.g. the hapten is detectable by immunostaining after reaction with an appropriate antibody comprising a reporter such as a fluorophore). Detectable labels include, but are not limited to, radionuclides, fluorophores, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, or aptamer beacons.

In the present invention, the term "therapeutic agent" refers to a compound that when present in an effective amount produces a desired therapeutic effect in a subject in need thereof. Therapeutic agents include, but are not limited to, radionuclides, cytokines, gold nanoparticles, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes, chemotherapeutic agents.

In the present invention, a composition refers to any mixture of two or more products, substances or compounds (including cells). It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. The term "pharmaceutical composition" as used in the present invention refers to a formulation in a form that is effective for the biological activity of one or more active ingredients, and that is free of other components that are unacceptably toxic to the subject to which the formulation is administered. Thus, it is a composition suitable for medical use in mammalian subjects (typically humans). Pharmaceutical compositions generally comprise an effective amount of an active agent and a carrier, excipient or diluent. The carrier, excipient or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively. Such formulations may be sterile.

The term "pharmaceutically acceptable carrier, excipient or diluent" as used herein includes any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. The use of any conventional carrier medium is also considered to be within the scope of the present invention unless it is incompatible with the compounds of the present invention, e.g., due to any undesirable biological effects that may occur or otherwise interact in a deleterious manner with one or more of any of the other components of the pharmaceutical composition.

As used herein, the term "kit" refers to any delivery system for delivering a substance, including but not limited to kits for research and clinical applications. Such delivery systems include systems that permit storage, transport, or delivery of reactive reagents (e.g., oligonucleotides, enzymes, etc., in a suitable container) and/or supporting substances (e.g., buffers, written instructions for performing an assay, etc.) from one location to another.

The prepared product containing the humanized chimeric antigen receptor targeting the BCMA can diagnose or treat diseases related to BCMA expression. Diseases associated with BCMA expression include, but are not limited to: 1) Diseases associated with cells expressing BCMA (e.g., wild-type or mutant BCMA) include, for example, proliferative diseases (e.g., cancer or malignancy) or pre-cancerous conditions (e.g., myelodysplastic syndrome, or pre-leukemia); or 2) non-cancer related indications associated with cells expressing BCMA (e.g., wild-type or mutant BCMA).

For the avoidance of doubt, diseases associated with BCMA expression may include conditions associated with cells that do not currently express BCMA but once express BCMA. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is hematologic cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a malignancy of differentiated plasma B cells. In one aspect, cancers associated with BCMA (e.g., wild-type or mutant BCMA) expression include cancers and malignancies, including, but not limited to: such as one or more acute lymphoblastic leukemias including, but not limited to, e.g., B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("tal"), acute Lymphoblastic Leukemia (ALL); one or more chronic leukemias, including, but not limited to, chronic Myelogenous Leukemia (CML), chronic Lymphocytic Leukemia (CLL), for example. Other cancers or hematological disorders associated with BCMA (e.g., wild-type or mutant BCMA) expression include, but are not limited to, e.g., B-cell prolymphocytic leukemia, lymphoblastic plasmacytoid dendritic cell tumor, burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, myeloma (multiple myeloma), myelodysplastic and myelodysplastic syndrome, hodgkin's lymphoma, non-hodgkin's lymphoma, plasmablastoid lymphoma, plasmacytoid dendritic cell tumor, glioblastoma, waldenstrom macroglobulinemia, and "pre-leukemia" (which is a diverse collection of hematological disorders associated with ineffective production (or dysplasia) by myeloid blood cells). In the present invention, the tumor is myeloma or acute lymphoblastic leukemia; preferably, the myeloma is multiple myeloma.

Other diseases associated with BCMA expression (e.g., wild-type or mutant BCMA) expression include, but are not limited to, e.g., atypical and/or non-classical cancers, malignant tumors, pre-cancerous conditions or proliferative diseases associated with BCMA expression (e.g., wild-type or mutant BCMA), such as prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, or lung cancer.

Non-cancer related conditions associated with BCMA (e.g., wild-type or mutant BCMA) include viral infections, e.g., HIV, fungal infections, e.g., cryptococcus neoformans; autoimmune diseases; such as rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris and Sjogren's syndrome; inflammatory bowel disease, ulcerative colitis; transplantation-related allo-specific immune disorders associated with mucosal immunity; and unwanted immune responses to biological agents (such as factor VIII), where humoral immunity is important. In some embodiments, non-cancer related indications associated with BCMA expression include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the cell expressing the tumor antigen expresses or expresses mRNA encoding the tumor antigen at any time. In one embodiment, the tumor antigen expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In one embodiment, the cell expressing the tumor antigen produces a detectable level of tumor antigen protein at a time point and then produces substantially no detectable tumor antigen protein.

The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. Simple modifications of the invention in accordance with the essence of the invention are all within the scope of the invention as claimed.

EXAMPLE 1 construction of BCMA-CAR or BCMA-CAR expression ^mut Jurkat-Reporter cell 1, construction of 3X NFAT Response element-minIL-2P-EGFP Reporter System

1) Nucleic acid sequence of the 3×nfat transcription factor response element (3× NFAT Response element):

GGAGGAAAAACTGTTTCATACAGAAGGCGTACGCCTTCTGTATGAAACAGTTTTTCCTCCACGCCTTCTGTATGAAACAGTTTTTCCTCCTCGAGG；

2) Nucleic acid sequence of the minimal IL-2 promoter (minIL-2P):

ACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGCCCAAGCTTGGCATTCCGGTACTGTTGGTAAA；

3) Nucleic acid sequence of reporter gene EGFP:

GCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA。

the nucleic acid sequence of the 3X NFAT Response element-minIL-2P-EGFP Reporter system comprises a nucleic acid sequence of a 3X NFAT transcription factor response element, a minimal IL-2 promoter and a Reporter EGFP linked in sequence. The nucleotide sequence of the 3X NFAT Response element-minIL-2P-EGFP Reporter system is cloned to pHAGE-EF1 alpha vector, and a lentiviral shuttle plasmid carrying the Reporter system is constructed.

2、BCMA-CAR、BCMA-CAR ^mut Amino acid sequence and nucleic acid sequence of (a)

1) The scFv amino acid sequence of BCMA-CAR (SEQ ID No. 1):

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR；

2) scFv nucleic acid sequence of BCMA-CAR (SEQ ID No. 2):

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAAGCTTCTGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTCCGACAGGCCCCTGGACAAGGACTTGAATGGATGGGCGCCACCTACAGAGGCCACAGCGACACCTACTACAACCAGAAATTCAAGGGCCGCGTGACCATCACCGCCGACAAGTCTACAAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGCCATCTACAACGGCTACGACGTGCTGGATAATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTAGTGGTGGTGGCGGATCTGATATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACCATTACCTGTAGCGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCAACCTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCAGAGGACTTCGCCACCTATTACTGCCAGCAGTACCGGAAGCTGCCCTGGACATTTGGACAGGGCACCAAGCTGGAAATCAAGCGG；

3)BCMA-CAR ^mut scFv amino acid sequence of (SEQ ID No. 3):

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIHGGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISGYLGWYQQKPGKAPKLLIYYAGNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR；

4)BCMA-CAR ^mut scFv nucleic acid sequence (SEQ ID No. 4):

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAAGCTTCTGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTCCGACAGGCCCCTGGACAAGGACTTGAATGGATGGGCGCCACCTACAGAGGCCACAGCGACACCTACTACAACCAGAAATTCAAGGGCCGCGTGACCATCACCGCCGACAAGTCTACAAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGCCATCCATGGTGGCTACGACGTGCTGGATAATTGGGGCCAGGGCACCCTGGTCACAGTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTAGTGGTGGTGGCGGATCTGATATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACCATTACCTGTAGCGCCAGCCAGGACATCTCCGGTTACCTGGGTTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTCCTGATCTACTACGCCGGTAACCTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCAGAGGACTTCGCCACCTATTACTGCCAGCAGTACCGGAAGCTGCCCTGGACATTTGGACAGGGCACCAAGCTGGAAATCAAGCGG；

5) CD8 hinge region amino acid sequence (SEQ ID NO. 5):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD；

6) CD8 hinge region nucleic acid sequence (SEQ ID NO. 6):

ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGAT；

7) CD8 transmembrane domain amino acid sequence (SEQ ID NO. 7):

IYIWAPLAGTCGVLLLSLVITLYC；

8) CD8 transmembrane domain nucleic acid sequence (SEQ ID NO. 8):

ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGT；

9) 4-1BB costimulatory signal domain amino acid sequence (SEQ ID NO. 9):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

10 4-1BB costimulatory signal domain nucleic acid sequence (SEQ ID NO. 10):

AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG；

11 CD3 zeta intracellular signaling domain amino acid sequence (SEQ ID No. 11):

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRZ；

12 CD3 zeta intracellular signaling domain nucleic acid sequence (SEQ ID No. 12):

CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAA；

13 CD8 signal peptide (CD 8 SP) amino acid sequence (SEQ ID No. 13): MALPVTALLLPLALLLHAARP;

14 CD8 signal peptide nucleic acid sequence (SEQ ID NO. 14):

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC。

mut represents mutant, 5'LTR represents 5' terminal promoter, and 3'LTR represents 3' terminal promoter.

The amino acid sequence of the BCMA-CAR is formed by sequentially connecting SEQ ID NO.13, 1, 5, 7, 9 and 11; the nucleic acid sequence of BCMA-CAR is formed by sequentially connecting SEQ ID NO.14, 2, 6, 8, 10 and 12; BCMA-CAR ^mut The amino acid sequence of (2) is formed by sequentially connecting SEQ ID NO.13, 3, 5, 7, 9 and 11; BCMA-CAR ^mut The nucleic acid sequence of (2) is formed by sequentially connecting SEQ ID NO.14, 4, 6, 8, 10 and 12.

TABLE 1 alignment of mutation sites on scFv sequences

Mutation site (aa)	Pre-mutation codons	Post-mutation codons
			102	TAC(Y)	CAT(H)
103	AAC(N)	GGT(G)
			172	AAC(N)	GGT(G)
175	AAC(N)	GGT(G)
			192	ACC(T)	GCC(A)
193	AGC(S)	GGT(G)

2. Construction of Jurkat-Reporter stable cell line

And infecting Jurkat cells with lentivirus carrying a Reporter system, and finally obtaining Jurkat-Reporter cells through monoclonal separation. The cells are stimulated by external stimuli to trigger Ca ²⁺ Influx, which causes NFAT to activate, core, bind to NFAT responsive elements, and initiate EGFP expression. By means of a fluorescence microscope and a flow cytometer, the change of the signal activity of the TCR can be visually monitored in real time, and the intensity of the TCR can be quantitatively analyzed.

3. Construction of BCMA-CAR or BCMA-CAR ^mut Overexpressed Jurkat-Reporter cells

Will load BCMA-CAR or BCMA-CAR ^mut Is infected with Jurkat-Reporter cells to obtain BCMA-CAR-Jurkat-Reporter cells and BCMA-CAR ^mut Jurkat-Reporter cells, detection of BCMA-CAR or BCMA-CAR by flow cytometry using APC-Protein-L staining ^mut Positive expression rate and average fluorescence intensity. The Control group (Control) was Jurkat-Reporter cells.

4. Experimental results

The results are shown in FIG. 3 for BCMA-CAR or BCMA-CAR ^mut High level expression on Jurkat-Reporter cells, BCMA-CAR or BCMA-CAR ^mut The positive expression rate of (2) is higher than 94%, and the average fluorescence intensities are 3395 and 4344 respectively. Indicating BCMA-CAR or BCMA-CAR ^mut All can be used for subsequent functional activity evaluation of the CAR, and interference of the CAR expression level difference on experimental results is avoided.

Example 2 comparison of BCMA-CAR loading or BCMA-CAR ^mut Reactivity of the Reporter cells of (E) to chemical and biological stimuli

1. Intensity of TCR signal after receiving chemical stimulus

Phorbol ester (PMA) and ionophore Ionomycin (Ionomycin) combined to treat BCMA-CAR-Jurkat-Reporter cells and BCMA-CAR respectively ^mut Jurkat-Reporter cells, after 12h flow cells examined GFP positive expression rate and average fluorescence intensity. The Control group (Control) was Jurkat-Reporter cells.

2. Screening of BCMA-positive expressing cells

A strain of exogenous BCMA-overexpressed lymphoblastic leukemia cells of Nalm6-BCMA-GFP, which is obtained by infecting Nalm6 cells with BCMA-GFP-loaded lentivirus, and screening under pressure; u266 cell is a myeloma cell which endogenously expresses BCMA. Flow cells were used to detect target cell surface BCMA expression levels using PE-CD269 staining.

3. Intensity of TCR signal after receiving biological stimulus

BCMA-CAR-Jurkat-Reporter cells and BCMA-CAR ^mut Jurkat-Reporter cells were incubated with U266 cells or Nalm6-BCMA-GFP cells, respectively, at an effective target ratio of 1:2.5, and GFP expression levels were detected by flow cytometry after 24 h.

4. Experimental results

The results of the reactivity and intensity of the chemical stimulus are shown in FIG. 4, and the cells of the control groupBCMA-CAR-Jurkat-Reporter cells and BCMA-CAR ^mut GFP positive expression rates of Jurkat-Reporter cells were 64.13%, 60.34%, 60.86%, respectively, and average fluorescence intensities were 1150, 1002, 1007, respectively. The PMA combined with the stimulation of the Ionomycin can activate a Reporter system, and the GFP expression level of 3 cells is obviously up-regulated. And GFP expression has similar variation trend and variation intensity in 3 Reporter cells, suggesting to load BCMA-CAR or BCMA-CAR ^mut Without significantly affecting the reactivity of the Reporter system. Thus 2 CAR-overexpressing Reporter cells can be used as tool cells for subsequent comparison of CAR function, activity.

The result of screening BCMA positive expression cells is shown in FIG. 5, and the BCMA positive expression rate in Nalm6-BCMA-GFP cells is 39.51%, and the average fluorescence intensity is up-regulated by 15 times, so that the Nalm6-BCMA-GFP cells are successfully constructed; about 91.94% of U266 cells were strongly positive for BCMA expression with an average fluorescence intensity as high as 1660, well above 457 of Nalm6-BCMA-GFP cells. The above results indicate that both Nalm6-BCMA-GFP cells and U266 cells can be used as target cells for the detection of CAR functional activity.

The results of TCR signal intensity after biological stimulation are shown in figure 6, where co-incubation with U266 cells up-regulates GFP expression levels in 2 Reporter cells, particularly BCMA-CAR ^mut the-Jurkat-Reporter cells are more obvious, and the GFP positive expression rate is as high as 59.70 percent, which is 2.5 times that of BCMA-CAR-Jurkat-Reporter cells; co-incubation with Nalm6-BCMA-GFP cells also up-regulates GFP expression levels in 2 Reporter cells, where BCMA-CAR ^mut GFP positive expression rate of the Jurkat-Reporter cells was 23.25% and 2.3 times that of BCMA-CAR-Jurkat-Reporter cells. Indicating that the mutated BCMA-CAR elicits a stronger TCR signal upon stimulation with the target antigen. Preliminary prompt, BCMA-CAR ^mut T may have a stronger killing activity.

EXAMPLE 3 construction of BCMA-CAR-T and BCMA-CAR ^mut T cells

1. Experimental method

Isolation and purification of T cells in PBMC of multiple myeloma patients, respectively infected with BCMA-CAR and BCMA-CAR ^mut Successful preparation of BCMA-CAR-T and BCMA-CAR ^mut -T cells. Detection of BCMA-CAR and BCMA-CAR by flow cytometry Using APC-Protein-L staining ^mut Positive expression rate and average fluorescence intensity.

2. Experimental results

The results are shown in FIG. 7 for BCMA-CAR and BCMA-CAR ^mut The positive expression rates of (a) were 43.73% and 43.19%, respectively, and the average fluorescence intensities 611 and 562. The expression level of the 2 CARs on T cells is similar, so that the interference of differential expression of the CARs on the subsequent functional experimental results can be reduced.

Example 4 RTCA evaluation of BCMA-CAR-T and BCMA-CAR ^mut -T killing function

1. Experimental method

BCMA-loaded lentivirus was used to infect CHO cells and pressure-screened to obtain a cell that stably expressed BCMA (CHO-BCMA). Positive expression rates of BCMA were detected by flow cytometry using PE-CD269 staining.

BCMA-CAR-T, BCMA-CAR using RTCA technique ^mut -the killing function of T is detected. First, CHO-BCMA cells were treated according to 10 ⁴ The wells were inoculated into cell culture plates. The next day, the Mock-T, BCMA-CAR-T, BCMA-CAR was again used ^mut T cells are added to the corresponding wells in an effective target ratio of 1:1 or 1:5. The killing activity of the CAR-T cells on target cells is indirectly reflected by monitoring the change of the signals of the adherent cells in real time and continuously.

2. Experimental results

The flow cell assay results are shown in FIG. 8, wherein about 62.16% of CHO cells express BCMA, indicating successful construction of CHO-BCMA cells;

the real-time RTCA monitoring results are shown in FIG. 9, and BCMA-CAR is achieved whether the effective target ratio is 1:1 or 1:5 ^mut The killing of target cells by T cells is significantly stronger than BCMA-CAR-T cells.

Example 5 flow cytometric assessment of BCMA-CAR-T and BCMA-CAR ^mut -T killing function

1. Experimental method

Isolation and purification of normal human-derived T cells, respectively infected with BCMA-CAR and BCMA-CAR-carrying ^mut Successful preparation of BCMA-CAR-T and BCMA-CAR ^mut -T cells. Flow cytometry using FITC-CD34 stainingMeasurement of BCMA-CAR and BCMA-CAR ^mut Positive expression rate and average fluorescence intensity.

Mock-T, BCMA-CAR-T and BCMA-CAR ^mut T cells co-incubated with U266 cells at an effective target ratio of 2:1, 1:1, 1:3, respectively. After 48h the flow cell detects the ratio of CAR-T cells to target cells (residual tumor cells) in the remaining cells.

3. Experimental results

Flow cytometric detection of BCMA-CAR and BCMA-CAR ^mut The positive expression rate and the average fluorescence intensity are shown in FIG. 10, and the results of the BCMA-CAR and the BCMA-CAR are shown in the following Table ^mut The positive expression rate on the T cells is 66.57% and 64.43%, and the average fluorescence intensity of the 2 cells is similar, so that the cell can be used as an effector cell for a detection experiment of the CAR-T killing function.

The difference of activity of different CAR-T killer target cells U266 detected by flow cytometry is shown in FIG. 11, and the ratio of BCMA-CAR-T group residual tumor cells is obviously higher than that of BCMA-CAR under 3 effective target ratios ^mut Group T, as well as the statistical analysis results. Indicating that mutating specific sites on the BCMA scFv sequence can increase the antitumor activity of BCMA-CAR-T. This set of data also further corroborates the experimental results of RTCA.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. A humanized chimeric antigen receptor targeting BCMA, wherein said chimeric antigen receptor comprises an antibody fragment scFv that specifically binds BCMA; the antibody fragment scFv is obtained by substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1; the substituted site in the amino acid sequence is selected from any one or more of the following sites in SEQ ID NO. 1: y102, N103, N172, N175, T192, or S193;

preferably, the site of substitution in the amino acid sequence is selected from the following sites in SEQ ID No. 1: y102, N103, N172, N175, T192, and S193;

preferably, the amino acid substitutions of Y102, N103, N172, N175, T192 and S193 comprise Y102H, N103G, N172G, N175G, T a and S193G;

preferably, the amino acid sequence of the antibody fragment scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 3;

preferably, the amino acid sequence of the antibody fragment scFv is shown in SEQ ID NO. 3.

2. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further comprises a transmembrane domain;

Preferably, the transmembrane domain comprises the transmembrane domain of: igG1, igG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3 epsilon, or variants thereof;

preferably, the transmembrane domain is a CD8 transmembrane domain;

preferably, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO. 7;

preferably, the chimeric antigen receptor further comprises a hinge region;

preferably, the hinge region comprises a hinge region of: igG1, igG4, CD8, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD34, OX40, CD3 epsilon, or variants thereof;

preferably, the hinge region is a CD8 hinge region;

preferably, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO. 5;

preferably, the chimeric antigen receptor further comprises an intracellular signaling domain;

preferably, the intracellular signaling domain comprises an intracellular signaling domain of: fcγ R, fc ε R, fc α R, fcRn, CD3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3b, C3dg, C3d, zap70, or variants thereof;

Preferably, the intracellular signaling domain is a cd3ζ intracellular signaling domain;

preferably, the amino acid sequence of the cd3ζ intracellular signaling domain is shown in SEQ ID No. 11;

preferably, the chimeric antigen receptor further comprises a costimulatory signaling domain;

preferably, the costimulatory signaling domain comprises the costimulatory signaling domain of: CD19, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, ICAM, LFA-1, lck, CD2, CD4, CD5, CD7, CD226, CD8 alpha, CD8 beta, LIGHT, CD83, DAP10, DAP12, GITR, DR3, NKG2C, HVEM, B7-H3 or variants thereof;

preferably, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain;

preferably, the amino acid sequence of the 4-1BB costimulatory signal domain is shown as SEQ ID NO. 9;

preferably, the chimeric antigen receptor further comprises a signal peptide;

preferably, the signal peptide comprises a signal peptide of: the alpha chain, beta chain, CD3 ζ, CD3 epsilon, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, igG, or variants thereof of the T cell receptor;

preferably, the signal peptide is a CD8 signal peptide;

Preferably, the amino acid sequence of the CD8 signal peptide is shown as SEQ ID NO. 13;

preferably, the chimeric antigen receptor comprises a signal peptide, scFv, hinge region, transmembrane domain, costimulatory signaling domain, intracellular signaling domain, connected in sequence;

preferably, the chimeric antigen receptor comprises a CD8 signal peptide, scFv, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signaling domain, cd3ζ intracellular signaling domain, connected in sequence;

preferably, the amino acid sequence of the chimeric antigen receptor is an amino acid sequence formed by sequentially connecting SEQ ID NO.13, 3, 5, 7, 9 and 11.

3. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 1 or 2;

preferably, the coding sequence of the chimeric antigen receptor comprises a coding sequence of a signal peptide, a coding sequence of an scFv, a coding sequence of a hinge region, a coding sequence of a transmembrane domain, a coding sequence of a costimulatory signal domain, a coding sequence of an intracellular signaling domain, which are linked in sequence;

preferably, the coding sequence of the chimeric antigen receptor comprises the coding sequence of a CD8 signal peptide, the coding sequence of an scFv, the coding sequence of a CD8 hinge region, the coding sequence of a CD8 transmembrane domain, the coding sequence of a 4-1BB costimulatory signal domain, the coding sequence of a CD3 zeta intracellular signaling domain, which are linked in sequence;

Preferably, the coding sequence of the CD8 signal peptide is shown as SEQ ID NO. 14;

preferably, the coding sequence of the scFv is shown as SEQ ID NO. 4;

preferably, the coding sequence of the CD8 hinge region is shown in SEQ ID NO. 6;

preferably, the coding sequence of the CD8 transmembrane domain is shown in SEQ ID NO. 8;

preferably, the coding sequence of the 4-1BB costimulatory signal domain is shown in SEQ ID NO. 10;

preferably, the coding sequence of the CD3 zeta intracellular signaling domain is shown in SEQ ID NO. 12.

4. A vector comprising the nucleic acid molecule of claim 3;

preferably, the vector comprises a cloning vector, an expression vector;

preferably, the vector comprises a DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector, or viral vector;

preferably, the viral vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, a herpesviral vector or a retroviral vector;

preferably, the viral vector is a lentiviral vector.

5. An engineered cell comprising or expressing the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, or the vector of claim 4;

Preferably, the cells comprise immune cells;

preferably, the immune cells comprise T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof;

preferably, the immune cells are T cells;

preferably, the T cells comprise primary T cells or Jurkat cells.

6. A method of making the cell of claim 5, comprising introducing the nucleic acid molecule of claim 3 or the vector of claim 4 into a cell.

7. A derivative comprising a detectably labeled chimeric antigen receptor of claim 1 or 2 and/or a nucleic acid molecule of claim 3, a chimeric antigen receptor of claim 1 or 2 and/or a nucleic acid molecule of claim 3 that confers antibiotic resistance, a chimeric antigen receptor of claim 1 or 2 that is conjugated or coupled to a therapeutic agent and/or a nucleic acid molecule of claim 3;

preferably, the detectable label comprises a radionuclide, fluorophore, chemiluminescent agent, microparticle, enzyme, colorimetric label, magnetic label, hapten, molecular beacon, or aptamer beacon;

Preferably, the therapeutic agent comprises a radionuclide, cytokine, gold nanoparticle, viral particle, liposome, nanomagnetic particle, prodrug activating enzyme, or chemotherapeutic agent.

8. A pharmaceutical composition or kit comprising the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4 or the cell of claim 5;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

9. Any of the following applications:

1) Use of the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4, the cell of claim 5, the derivative of claim 7 or the pharmaceutical composition or kit of claim 8 for the preparation of a product for diagnosing or treating a disease associated with BCMA expression;

2) Use of the chimeric antigen receptor of claim 1 or 2 in the preparation of the nucleic acid molecule of claim 3, the vector of claim 4, the cell of claim 5, the derivative of claim 7 or the pharmaceutical composition or kit of claim 8;

3) Use of the nucleic acid molecule of claim 3 in the preparation of the vector of claim 4, the cell of claim 5, the derivative of claim 7 or the pharmaceutical composition or kit of claim 8;

4) Use of the vector of claim 4 for the preparation of the cell of claim 5, the derivative of claim 7 or the pharmaceutical composition or kit of claim 8;

5) Use of the cell of claim 5 for the preparation of the derivative of claim 7 or the pharmaceutical composition or kit of claim 8;

6) Use of a derivative according to claim 7 for the preparation of a pharmaceutical composition or kit according to claim 8.

10. The use according to claim 9, wherein the disease associated with BCMA expression comprises cancer, precancerous lesions or non-cancer related indications;

preferably, the disease associated with BCMA expression is cancer;

preferably, the cancer comprises myeloma, myelodysplastic and myelodysplastic syndrome, acute lymphoblastic leukemia, chronic leukemia, blast plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, plasmablastoid lymphoma, plasmacytoid dendritic cell tumor, glioblastoma, waldenstrom's macroglobulinemia, hodgkin's lymphoma or non-hodgkin's lymphoma;

Preferably, the cancer is myeloma or acute lymphoblastic leukemia;

preferably, the myeloma is multiple myeloma.