A CAR-T cell that simultaneously targets BCMA and CD19 and its application

Abstract

The present invention discloses a CAR-T cell that simultaneously targets BCMA and CD19 and its application. The CAR that simultaneously targets BCMA and CD19 provided by the present invention has advantages such as safety and effectiveness. The constructed CAR-T cell has a significant therapeutic effect on tumors and autoimmune diseases expressing BCMA and / or CD19. Compared with ordinary single-target CARs, the dual-target CAR has a stronger ability to recognize target cells, which is beneficial to preventing the occurrence of antigen escape phenomenon, and has broad clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a CAR-T cell that simultaneously targets BCMA and CD19 and its application. Background Art

[0002] Multiple myeloma (MM) is the second most common hematological malignancy, which is prone to occur in middle-aged and elderly people. Its characteristic is the clonal proliferation of plasma cells in the bone marrow or the production of monoclonal immunoglobulins at extramedullary sites. With the continuous aggravation of the global population aging, the incidence of MM shows an obvious upward trend year by year. Although significant progress has been made in the treatment of MM in the past decade, improving the survival rate of patients, MM is still considered an incurable disease, and almost all MM patients will eventually experience recurrence. Effective and tolerable treatment regimens for relapsed and refractory (R / R) MM patients remain an unmet need. The median overall survival (OS) of stage III refractory patients is 9.3 months, and the median OS of stage V refractory patients is 5.6 months.

[0003] Chimeric antigen receptor (CAR) T cells targeting B cell maturation antigen (BCMA) have shown good efficacy in the treatment of relapsed and refractory MM patients, with an overall response rate of 48%-100% and a complete response rate of 6%-76%. However, there are still some patients who are unresponsive to BCMA CAR-T cell therapy. Among the patients who are effective in BCMA CAR-T cell therapy, 27%-64% of the patients experience disease recurrence. It has been found in studies that BCMA downregulation or loss, that is, antigen escape, is observed in 4%-33% of the patients who progress after CAR-T treatment, which may be one of the reasons for tumor recurrence. To solve the problem of antigen escape, some researchers have developed dual-target CAR-T cells that target different targets on the surface of MM cells, such as BCMA / CD38 dual-target CAR-T cells for the treatment of MM.

[0004] Another important reason for the recurrence of patients after receiving BCMACAR-T therapy is that tumor stem cells cannot be effectively eliminated. Previous studies have shown that a small subset of MM cells express CD19, and these cells are considered to be less differentiated MM cells or myeloma-like stem cells, which are associated with drug resistance and low survival rates. Recent studies have confirmed that CD19 is expressed at ultra-low density on a subset of myeloma cells (10.3%-80%) in most patients, and myeloma cells with low CD19 expression can be eliminated by anti-CD19 CAR-T cells. Based on these observations, the inventors of the present invention conducted a prospective study and demonstrated that the combined injection of humanized anti-CD19 CAR-T and anti-BCMACAR-T cells is feasible in R / R MM patients. Based on these preliminary data, the inventors of the present invention further designed a second-generation bispecific BC19 CAR, which contains an anti-BCMA single-chain variable fragment (scFv) and a humanized anti-CD19 scFv. And the efficacy of the BC19 CAR-T cells in R / R MM patients receiving this CAR-T cell therapy was demonstrated in preclinical and phase I / II clinical trials. Summary of the Invention

[0005] In order to overcome the technical problems existing in the prior art, the object of the present invention is to provide a CAR-T cell that simultaneously targets BCMA and CD19 and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a chimeric antigen receptor that targets BCMA and CD19.

[0008] Furthermore, the chimeric antigen receptor comprises a bispecific antibody that targets BCMA and CD19;

[0009] The bispecific antibody is obtained by sequentially connecting in series an antibody that targets BCMA, a Linker, and an antibody that targets CD19;

[0010] The amino acid sequence of the antibody that targets BCMA is as shown in SEQ ID NO:2 or an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:2;

[0011] The amino acid sequence of the antibody that targets CD19 is as shown in SEQ ID NO:4 or an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:4;

[0012] The amino acid sequence of the Linker is as shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 3;

[0013] Preferably, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region, a co-stimulatory signal domain, and an intracellular signal transduction domain;

[0014] More preferably, the signal peptide includes the signal peptides of the following molecules: CD8, CD4, CD5, CD9, CD28, CD16, CD3ζ, CD3ε, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6; More preferably, the hinge region and transmembrane region include the hinge regions and transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor; More preferably, the co-stimulatory signal domain includes the co-stimulatory signal domains of the following molecules: 4-1BB, CD27, CD19, CD4, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3; More preferably, the intracellular signal transduction domain includes the intracellular signal transduction domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d;

[0015] Most preferably, the signal peptide is the CD8 signal peptide; Most preferably, the hinge region and transmembrane region are the CD8 hinge region and transmembrane region; Most preferably, the co-stimulatory signal domain is the 4-1BB co-stimulatory signal domain; Most preferably, the intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain;

[0016] Most preferably, the amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO:1 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:1; most preferably, the amino acid sequence of the CD8 hinge region and transmembrane region is as shown in SEQ ID NO:5 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:5; most preferably, the amino acid sequence of the 4-1BB co-stimulatory signal domain is as shown in SEQ ID NO:6 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:6; most preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:7 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:7;

[0017] Most preferably, the chimeric antigen receptor is obtained by sequentially connecting in series a CD8 signal peptide, an antibody targeting BCMA, a Linker, an antibody targeting CD19, a CD8 hinge region and transmembrane region, a 4-1BB co-stimulatory signal domain, and a CD3ζ intracellular signaling domain; most preferably, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:8 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:8; most preferably, the nucleotide sequence of the chimeric antigen receptor is as shown in SEQ ID NO:9 or is a nucleotide sequence having at least 90% homology with the nucleotide sequence corresponding to SEQ ID NO:9.

[0018] In a specific embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is as shown in SEQ ID NO:8, and the nucleotide sequence is as shown in SEQ ID NO:9. It is first discovered through experimental verification in the present invention that in the CAR, the connection order of the antibody targeting BCMA and the antibody targeting CD19 will have a great impact on the effect of the CAR. Based on the connection order where the antibody targeting BCMA is in the front and the antibody targeting CD19 is in the back, the therapeutic effect of the CAR-T cells prepared is significantly better, that is, the chimeric antigen receptor of the present invention has achieved unexpected technical effects.

[0019] The second aspect of the present invention provides an optimized chimeric antigen receptor targeting BCMA and CD19.

[0020] Furthermore, the chimeric antigen receptor comprises a bispecific antibody targeting BCMA and CD19;

[0021] The bispecific antibody is obtained by sequentially connecting in series an antibody targeting BCMA, a Linker, and an antibody FMC63 targeting CD19;

[0022] The amino acid sequence of the antibody targeting BCMA is as shown in SEQ ID NO: 2 or an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 2;

[0023] The amino acid sequence of the antibody FMC63 targeting CD19 is as shown in SEQ ID NO: 12 or an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 12;

[0024] The amino acid sequence of the Linker is as shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 3;

[0025] Preferably, the chimeric antigen receptor further comprises a signal peptide, a hinge region and a transmembrane region, a co-stimulatory signal domain, and an intracellular signal transduction domain;

[0026] More preferably, the signal peptide includes the signal peptides of the following molecules: CD8, CD4, CD5, CD9, CD28, CD16, CD3ζ, CD3ε, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6; more preferably, the hinge region and transmembrane region include the hinge regions and transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor; more preferably, the co-stimulatory signal domain includes the co-stimulatory signal domains of the following molecules: 4-1BB, CD27, CD19, CD4, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3; more preferably, the intracellular signal transduction domain includes the intracellular signal transduction domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d;

[0027] Most preferably, the signal peptide is the CD8 signal peptide; most preferably, the hinge region and transmembrane region are the CD8 hinge region and transmembrane region; most preferably, the co-stimulatory signal domain is the 4-1BB co-stimulatory signal domain; most preferably, the intracellular signaling domain is the CD3ζ intracellular signaling domain;

[0028] Most preferably, the amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO:1 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:1; most preferably, the amino acid sequence of the CD8 hinge region and transmembrane region is as shown in SEQ ID NO:5 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:5; most preferably, the amino acid sequence of the 4-1BB co-stimulatory signal domain is as shown in SEQ ID NO:6 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:6; most preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:13 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:13;

[0029] Most preferably, the chimeric antigen receptor is obtained by sequentially connecting in series the CD8 signal peptide, an antibody targeting BCMA, a Linker, an antibody FMC63 targeting CD19, the CD8 hinge region and transmembrane region, the 4-1BB co-stimulatory signal domain, and the CD3ζ intracellular signaling domain; most preferably, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:14 or is an amino acid sequence having at least 90% homology with the amino acid sequence corresponding to SEQ ID NO:14; most preferably, the nucleotide sequence of the chimeric antigen receptor is as shown in SEQ ID NO:15 or is a nucleotide sequence having at least 90% homology with the nucleotide sequence corresponding to SEQ ID NO:15.

[0030] In a specific embodiment of the present invention, the amino acid sequence of the optimized chimeric antigen receptor is as shown in SEQ ID NO:14, and the nucleotide sequence is as shown in SEQ ID NO:15. It is first discovered through experimental verification in the present invention that the optimized CAR obtains the optimal expression efficiency in T cells, significantly superior to other codon optimization methods, and can be stably and highly expressed in T cells from different patients, that is, the optimized chimeric antigen receptor of the present invention achieves unexpected technical effects.

[0031] The third aspect of the present invention provides a nucleic acid molecule.

[0032] Furthermore, the nucleic acid molecule encodes the chimeric antigen receptor described in the first aspect of the present invention or the chimeric antigen receptor described in the second aspect of the present invention;

[0033] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor described in the first aspect of the present invention is as shown in SEQ ID NO: 9 or a nucleotide sequence having at least 90% homology with the nucleotide sequence corresponding to SEQ ID NO: 9;

[0034] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor described in the second aspect of the present invention is as shown in SEQ ID NO: 15 or a nucleotide sequence having at least 90% homology with the nucleotide sequence corresponding to SEQ ID NO: 15.

[0035] In some embodiments, the nucleic acid molecule may comprise natural, non-natural or altered nucleotides; and it may comprise natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester present between the nucleotides of an unmodified oligonucleotide. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, in some cases it may be appropriate for the nucleic acid to comprise one or more insertions, deletions, inversions and / or substitutions, and thus nucleic acids formed by insertions, deletions, inversions and / or substitutions based on the nucleic acid molecules provided by the present invention are also included within the scope of protection of the present invention.

[0036] The fourth aspect of the present invention provides an expression vector.

[0037] Furthermore, the expression vector comprises the nucleic acid molecule described in the third aspect of the present invention;

[0038] Preferably, the vector includes a DNA vector, an RNA vector, a transposon vector, a CRISPR / Cas9 vector; more preferably, the DNA vector includes a plasmid; more preferably, the RNA vector includes a virus-derived vector; most preferably, the virus-derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, a herpesviral vector.

[0039] In some embodiments, examples of the vectors that can be used in the present invention include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, vectors of viral origin. Various vectors known in the art can be selected, for example, commercially available vectors can be selected, and then the nucleotide sequence encoding the chimeric antigen receptor described in the first aspect of the present invention or the chimeric antigen receptor described in the second aspect of the present invention is operably linked to an expression regulatory sequence to form an expression vector. In some embodiments, the vectors of viral origin include, but are not limited to: lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors, herpesviral vectors, baculoviral vectors, papillomaviral vectors, polyomaviral vectors.

[0040] The fifth aspect of the present invention provides an engineered host cell.

[0041] Furthermore, the engineered host cell contains the nucleic acid molecule described in the third aspect of the present invention and / or the expression vector described in the fourth aspect of the present invention;

[0042] Preferably, the host cell includes mammalian cells; more preferably, the host cell is an immune cell; most preferably, the immune cell includes T cells, B cells, NK cells, iNKT cells, γδT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils or any combination thereof; most preferably, the immune cell is a T cell; most preferably, the T cell is of human origin; most preferably, the engineered host cell is a T cell modified with the chimeric antigen receptor described in the first aspect of the present invention or a T cell modified with the chimeric antigen receptor described in the second aspect of the present invention.

[0043] In some embodiments, the engineered host cell is an autologous cell or an allogeneic cell. In some embodiments, the engineered immune cell is obtained from a subject suffering from BCMA- and / or CD19-related diseases. In some embodiments, the engineered immune cell is obtained from a healthy donor.

[0044] The sixth aspect of the present invention provides a derivative.

[0045] Furthermore, the derivatives include the chimeric antigen receptor described in the first aspect of the present invention with a detectable label and / or the chimeric antigen receptor described in the second aspect of the present invention and / or the nucleic acid molecule described in the third aspect of the present invention and / or the engineered host cell described in the fifth aspect of the present invention, the chimeric antigen receptor described in the first aspect of the present invention with antibiotic resistance and / or the chimeric antigen receptor described in the second aspect of the present invention and / or the nucleic acid molecule described in the third aspect of the present invention and / or the engineered host cell described in the fifth aspect of the present invention, the chimeric antigen receptor described in the first aspect of the present invention conjugated or coupled with a therapeutic agent and / or the chimeric antigen receptor described in the second aspect of the present invention and / or the nucleic acid molecule described in the third aspect of the present invention and / or the engineered host cell described in the fifth aspect of the present invention;

[0046] Preferably, the detectable label includes fluorescent dyes, colloidal gold, chemiluminescent markers, chemiluminescent catalysts; more preferably, the chemiluminescent markers include luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, adamantane, rare earth elements, ruthenium bipyridyl complexes; more preferably, the chemiluminescent catalysts include horseradish peroxidase, alkaline phosphatase;

[0047] Preferably, the antibiotic resistance genes include penicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, kanamycin resistance gene;

[0048] Preferably, the therapeutic agents include radionuclides, cytokines, gold nanoparticles, virus particles, liposomes, magnetic nanoparticles, prodrug activating enzymes, chemotherapeutic agents; more preferably, the radionuclides include 67 Ga, 68 Ga, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y, 90 Y, 89 Zr, 120 I, 123 I, 13 N, 15 O, 186 Re, 110 In, 111In; more preferably, the cytokine includes IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, M-CSF; more preferably, the nano-magnetic particles include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II); more preferably, the chemotherapeutic agent includes cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxalate platinum, and leucovorin.

[0049] The seventh aspect of the present invention provides a pharmaceutical composition or a biological preparation.

[0050] Furthermore, the pharmaceutical composition contains the chimeric antigen receptor described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, and / or the derivative described in the sixth aspect of the present invention;

[0051] The biological preparation contains the chimeric antigen receptor described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention, and / or the pharmaceutical composition described above.

[0052] In some embodiments, the pharmaceutical composition or the biological preparation may further contain other second therapeutic agents that can be used for treating and / or preventing BCMA- and / or CD19-related diseases. There is no particular limitation on the second therapeutic agent, as long as the reagent or drug that can cooperate with the pharmaceutical composition or the biological preparation described in the present invention to achieve the expected effect is within the protection scope of the present invention.

[0053] In some embodiments, the pharmaceutical composition or the biological preparation may further contain a pharmaceutically acceptable carrier and / or excipient. Suitable pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used as needed to help the stability of the formulation or to improve the activity or bioavailability of the active substance. In some embodiments, when using the pharmaceutical composition or the biological preparation, a safe and effective amount of the pharmaceutical composition or the biological preparation described in the present invention is administered to a human.

[0054] In some embodiments, the administration forms of suitable pharmaceutical compositions include forms suitable for parenteral administration, such as by injection or infusion, e.g., by bolus injection or continuous infusion, intravenously, inhalably or subcutaneously. In the case where the product is for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulating agents such as suspending agents, preservatives, stabilizers and / or dispersing agents.

[0055] In some embodiments, the pharmaceutical composition can be made into various dosage forms as needed, and the physician can determine the dose beneficial to the patient according to factors such as the type of patient, age, weight and general disease condition, and the mode of administration. A skilled physician can usually easily determine the prescription and the dosage and mode of administration of the prescription effective for the desired treatment and / or prevention.

[0056] The eighth aspect of the present invention provides a kit.

[0057] Furthermore, the kit comprises the chimeric antigen receptor described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention and / or the derivative described in the sixth aspect of the present invention.

[0058] The ninth aspect of the present invention provides any one of the following methods, which methods comprise:

[0059] (1) A method for preparing the engineered host cell described in the fifth aspect of the present invention, which method comprises the following steps: introducing the nucleic acid molecule described in the third aspect of the present invention or the expression vector described in the fourth aspect of the present invention into a host cell;

[0060] Preferably, the introducing method includes lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxviral vector, herpes simplex virus vector, adenoviral vector, adeno-associated viral vector;

[0061] (2) A method for stimulating an immune response in a target cell population or tissue in a mammal, which method comprises the following steps: administering to the mammal an effective amount of the engineered host cell described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention, the pharmaceutical composition described in the seventh aspect of the present invention and / or the biological agent described in the seventh aspect of the present invention;

[0062] (3) A method for inhibiting the activity of BCMA and / or CD19 protein in vitro, the method comprising the following steps: contacting the engineered host cell described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention, the pharmaceutical composition described in the seventh aspect of the present invention, and / or the biological preparation described in the seventh aspect of the present invention with somatic cells of an organism.

[0063] In addition, the present invention also provides a method for treating and / or preventing BCMA- and / or CD19-related diseases, the method comprising administering an effective amount of the engineered host cell described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention, the pharmaceutical composition and / or biological preparation described in the seventh aspect of the present invention to a subject in need thereof.

[0064] In a specific embodiment of the present invention, the chimeric antigen receptor immune cell (CAR-T) is administered by infusion, and the administered dose is 1×10 6 cells / kg (body weight). It should be noted that the protection scope of the present invention is not limited by the specific administration mode and administered dose. As long as the CAR-T cells, pharmaceutical composition or biological preparation described in the present invention produce the expected therapeutic and / or preventive effect in a subject, it falls within the protection scope of the present invention.

[0065] The tenth aspect of the present invention provides any one of the following applications, and the application comprises:

[0066] (1) The application of the chimeric antigen receptor described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention, the pharmaceutical composition described in the seventh aspect of the present invention, the biological preparation described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention in the preparation of a drug for treating and / or preventing BCMA- and / or CD19-related diseases;

[0067] (2) The application of the chimeric antigen receptor described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, and the derivative described in the sixth aspect of the present invention in the preparation of a kit for preparing chimeric antigen receptor immune cells for treating and / or preventing BCMA- and / or CD19-related diseases;

[0068] (3) Use of the chimeric antigen receptor described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention, the pharmaceutical composition described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention in the preparation of a biological agent for the treatment and / or prevention of BCMA- and / or CD19-related diseases;

[0069] (4) Use of the pharmaceutical composition described in the seventh aspect of the present invention and the biological agent described in the seventh aspect of the present invention in the treatment and / or prevention of BCMA- and / or CD19-related diseases;

[0070] (5) Use of the kit described in the eighth aspect of the present invention in the preparation of chimeric antigen receptor immune cells for the treatment and / or prevention of BCMA- and / or CD19-related diseases;

[0071] (6) Use of the chimeric antigen receptor described in the first aspect of the present invention and the chimeric antigen receptor described in the second aspect of the present invention in the preparation of nucleic acid molecules, expression vectors, engineered host cells, and derivatives;

[0072] (7) Use of the nucleic acid molecule described in the third aspect of the present invention in the preparation of expression vectors, engineered host cells, and derivatives;

[0073] (8) Use of the expression vector described in the fourth aspect of the present invention in the preparation of engineered host cells and derivatives;

[0074] (9) Use of the engineered host cell described in the fifth aspect of the present invention in the preparation of derivatives;

[0075] (10) Use of the bispecific antibody described in the first aspect of the present invention and the bispecific antibody described in the second aspect of the present invention in the preparation of chimeric antigen receptor immune cells for the treatment and / or prevention of BCMA- and / or CD19-related diseases;

[0076] Preferably, the BCMA- and / or CD19-related diseases include tumors and autoimmune diseases;

[0077] More preferably, the tumor is a tumor expressing BCMA and / or CD19;

[0078] More preferably, the autoimmune disease is an autoimmune disease expressing BCMA and / or CD19;

[0079] Most preferably, the tumors include multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, small lymphocyte lymphoma, follicular lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, megakaryocytic leukemia, Burkitt lymphoma, anaplastic large cell lymphoma, mucosa-associated lymphoid tissue lymphoma;

[0080] Most preferably, the autoimmune diseases include chronic Guillain-Barré syndrome, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, demyelinating lesions, autoimmune hemolysis, idiopathic thrombocytopenic purpura, idiopathic leukopenia.

[0081] In the present invention, the BCMA and / or CD19-related diseases are not limited to the specific diseases listed in the present invention, and any diseases related to the expression of BCMA and / or CD19 are within the protection scope of the present invention, including but not limited to: tumors expressing BCMA and / or CD19, autoimmune diseases expressing BCMA and / or CD19. In a specific embodiment of the present invention, the BCMA and / or CD19-related diseases particularly refer to multiple myeloma and chronic Guillain-Barré syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It is a result diagram for the construction and in vitro functional verification of BCMA / CD19 bispecific CAR-T cells. Among them, Figure A: Schematic diagrams of the structures of 4 kinds of CARs; Figure B: Detection of the expression levels of CARs in 4 kinds of CAR-T cells by PE-Protein L staining and flow cytometry analysis; Figure C: Detection results of the differentiation phenotypes of 4 kinds of CAR-T cell preparations, and the differentiation phenotypes include naive (CD45RA+CD62L+), central memory (CM) (CD45RA-CD62L+), effector memory (EM) (CD45RA-CD62L-), and effector (CD45RA+CD62L-) cells; Figure D: Draw the amplification curves of 4 kinds of CAR-T cells during in vitro culture; Figure E: Quantitative analysis of the cytotoxicity of 4 kinds of CAR-T cells by RTCA technology. Target cells: SKOV3 (left), SKOV3-BCMA (middle), and SKOV3-CD19 (right); E: T ratio is 3:1 (upper figure) or 1:3 (lower figure); Figure F: Evaluation of the anti-tumor activity of 4 kinds of CAR-T cells by flow cytometry. Target cells: U266 (left) and Nalm6 (right), E: T ratio is 1:1; Figure G: Detection of the secretion level of interferon γ (IFN-γ) in the culture supernatant by ELISA.

[0083] Figure 2For the detection of the binding specificity of BC19 CAR to BCMA or CD19. Among them, Figure A: Schematic diagram of the BC19 fusion protein with Fc tag (upper figure) and His tag (lower figure); Figure B: Western Blot was used to detect the relative molecular mass and purity of the two BC19 fusion proteins; Figure C: Flow cytometry was used to evaluate the binding ability of the two BC19 fusion proteins to Nalm6 or U266 cells; Figure D: The binding specificity of the two BC19 fusion proteins was detected using SKOV3, SKOV3-BCMA, and SKOV3-CD19 cells.

[0084] Figure 3 To evaluate the in vivo anti-tumor activity of BC19 CAR T cells using a multiple myeloma model. Among them, Figure A: Flow chart of the animal experiment. On the 7th day, 2×10 6 U266 cells were injected into the tail vein of NCG mice. The mice were orally administered the γ-secretase inhibitor LY3039478 at 1 mg / kg, three times a week, to stabilize the expression of BCMA on the surface of U266 cells. The mice were randomly divided into 4 groups (n = 3 or 4). On the 0th day, 2×10 6 BCMACAR T, CD19 CAR T, BC19 CAR T, or Mock T cells were injected into the tail vein; Figure B: Bioluminescence imaging was used to monitor the growth of transplanted tumors in vivo on the 0th, 14th, 21st, 28th, 35th, and 42nd days; Figure C: The luciferase activity of different groups was quantified and ANOVA analysis and Dunnett's test were performed; Figure D: The overall survival rate of the mice was represented by the Kaplan-Meier curve and analyzed using the log-rank test.

[0085] Figure 4 To construct a Nalm6 xenograft tumor model to investigate the efficacy of BC19 CAR-T cells in killing CD19+ target cells in vivo. Among them, Figure A: Flow chart of the animal experiment. On the 7th day, 1×10 6 NALM-6 cells were injected into each NCG mouse. On the 0th day, the mice were treated with 2×10 6 BCMACAR T, CD19 CAR T, BC19 CAR T, or mock T cells respectively; Figure B: Bioluminescence imaging was used to monitor the growth of transplanted tumors in vivo on the -7th, 0th, 7th, 14th, 21st, 28th, and 35th days; Figure C: The luciferase activity of different groups was quantified and ANOVA analysis and Dunnett's test were performed; Figure D: The overall survival rate of the mice was represented by the Kaplan-Meier curve and analyzed using the log-rank test.

[0086] Figure 5 Flow chart of the enrollment and exclusion of 64 R / R MM patients.

[0087] Figure 6 CT imaging results of extramedullary lesions in 3 MM patients before and after BC19 CAR-T treatment. In the upper figure, before CAR-T treatment; in the lower figure, after CAR-T treatment.

[0088] Figure 7 Results of the correlation analysis between disease characteristics and treatment response in 50 patients with evaluable efficacy.

[0089] Figure 8 Results of the statistical analysis of the treatment effects at different time points after 50 patients received BC19 CAR-T infusion.

[0090] Figure 9 Kaplan-Meier analysis of overall survival (OS), progression-free survival (PFS) and duration of response (DOR). In figure A: Kaplan-Meier curve of OS for 50 patients; in figure B: Kaplan-Meier curve of PFS for 50 patients; in figure C: Kaplan-Meier curve of DOR for 46 patients who achieved partial remission or better. The tick marks indicate the data censoring time at the last follow-up.

[0091] Figure 10 Results of the conformational feature analysis of FMC63 and its variants S1 and S2. In figure A: results of humanization analysis; in figure B: results of 3D structure simulation and superposition mode analysis.

[0092] Figure 11 Positive expression rates of 3 BC19 CAR mutants detected by APC-Protein-L staining combined with flow cytometry.

[0093] Figure 12 Quantitative analysis of the mean fluorescence intensity of 3 BC19 CAR mutants by flow cytometry.

[0094] Figure 13 Basic characteristics of 7 relapsed / refractory (R / R) multiple myeloma patients who received BC19 CAR mutant T cell therapy.

[0095] Figure 14Efficacy evaluation of 3 CIDP patients after receiving BC19 CAR mutant T cell therapy. Among them, Figure A: Typical pictures of the motor function of both lower extremities of the patient before and after CAR-T therapy. The left picture was taken before CAR-T therapy, showing the loss of motor function of both lower extremities of the patient. The right picture was taken after therapy, showing partial recovery of the motor function of both lower extremities of the patient. Figure B: Evaluation of the B cell clearance potential of CAR T cells by flow cytometry. Figures C-D: Results of MRC3 scores and INCAT disability scores before CAR-T therapy and on days 21, 26, 28, and 199 after therapy. Among them, Figure C shows the MRC3 score, and Figure D shows the INCAT disability score. Detailed implementation mode

[0096] The present invention will be further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and should not be construed as a limitation of the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0097] Example 1 Construction of CAR-T cells targeting BCMA and CD19 simultaneously and verification of their therapeutic effects

[0098] I. Experimental methods

[0099] 1. Construction of viral vectors

[0100] Bispecific C19B CAR (CD19 in front and BCMA behind) and BC19 CAR (BCMA in front and CD19 behind) contain anti-CD19 single-chain antibody and anti-BCMA single-chain antibody, which are connected by (G4S)4. Monospecific CD19 CAR and BCMA CAR contain the above-mentioned anti-CD19 single-chain antibody and anti-BCMA single-chain antibody respectively. The above scFv sequences are respectively concatenated with the hinge region and transmembrane region of CD8 molecule, the cytoplasmic part of 4-1BB, and the cytoplasmic part of CD3ζ molecule, and cloned into the lentiviral shuttle plasmid. Figure 1 Figure A in this example provides a schematic diagram of the structures of the 4 CARs constructed. The packaging and titer detection of CAR lentivirus were completed by GeneChem (Shanghai, China).

[0101] BC19 CAR is obtained by sequentially concatenating the CD8 leader sequence, BCMA (scfv), Linker, CD19 (scfv), CD8 hinge region and transmembrane region, CD137 (4-1BB) co-stimulatory signal domain, and CD3ζ intracellular signal transduction domain.

[0102] The C19B CAR is obtained by successively connecting in series a CD8 leader sequence, CD19 (scfv), a Linker, BCMA (scfv), a CD8 hinge region and transmembrane region, a CD137 (4-1BB) co-stimulatory signal domain, and a CD3ζ intracellular signaling domain.

[0103] The CD19 CAR is obtained by successively connecting in series a CD8 leader sequence, CD19 (scfv), a CD8 hinge region and transmembrane region, a CD137 (4-1BB) co-stimulatory signal domain, and a CD3ζ intracellular signaling domain.

[0104] The BCMACAR is obtained by successively connecting in series a CD8 leader sequence, BCMA (scfv), a CD8 hinge region and transmembrane region, a CD137 (4-1BB) co-stimulatory signal domain, and a CD3ζ intracellular signaling domain.

[0105] Among them, the amino acid sequences of the CD8 leader sequence, BCMA (scfv), Linker, CD19 (scfv), CD8 hinge region and transmembrane region, CD137 (4-1BB) co-stimulatory signal domain, and CD3ζ intracellular signaling domain are respectively as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0106] Among them, the amino acid sequence of the BC19 CAR is as shown in SEQ ID NO:8, and the nucleotide sequence is as shown in SEQ ID NO:9. The amino acid sequence of the C19B CAR is as shown in SEQ ID NO:10, and the nucleotide sequence is as shown in SEQ ID NO:11.

[0107] 2. CAR-T cell production

[0108] After obtaining informed consent, primary mononuclear cells (PBMCs) are isolated from the peripheral blood of normal individuals, diffuse large B-cell lymphoma, or B-cell acute lymphoblastic leukemia (B-ALL) patients using Ficoll density centrifugation (Sigma-Aldrich, Germany). The EasySep TM Human T Cell Isolation Kit (STEMCELL, #17951) is used to purify T lymphocytes. Human T-Expander CD3 / CD28 (Thermo, CAT#11141D) activates and expands T cells. 48 - 72 hours after T cell activation, the T cells are infected with lentivirus carrying CAR. Then, they are expanded in vitro for 10 - 12 days in an X-VIVO 15 culture system containing 200 U / mL IL-2, 5 ng / mL IL-15, and 5 ng / mL IL-7. If the cells meet the predetermined release criteria, including a transduction efficiency ≥ 10%, cell viability ≥ 70%, negative for mycoplasma, and negative for bacterial and fungal cultures, the cells are injected into the patient.

[0109] 3. T cell proliferation assay

[0110] On day 0, T cells are seeded in 24-well plates at a density of 5×10 5 / well, infected with lentivirus carrying CAR, and at the same time, the cells are harvested and stained with 0.4% trypan blue, and the trypan blue-negative cells are counted on a hematology analyzer. Subsequently, trypan blue staining and live cell counting are performed again on days 2, 4, 7, 9, 11, 14, and 17 after infection, and a cell growth curve is plotted.

[0111] 4. Flow cytometry

[0112] Biotinylated Protein L (405203; BioLegend, USA) is used to detect the expression of CD19, BCMA, C19B, and BC19 CAR, and then all samples are stained with streptavidin-APC (405203; BioLegend, USA). Anti-CD45RA-PE (561811; BD, USA) and anti-CD62L-PECy7 (564419; BD) are used to detect the differentiation phenotypes of CAR-T cell products, including naive (CD45RA+CD62L+), central memory (CM) (CD45RA-CD62L+), effector memory (EM) (CD45RA-CD62L-), and effector (CD45RA+CD62L-) cells.

[0113] Flow cytometry is used to evaluate the killing activity of CAR-T cells. CAR-T cells labeled with CellTracker (0.25 μM, 25 min) are co-incubated with U266 cells at an effector-to-target ratio of 1:1 for 24 hours, and the proportions of CAR-T and U266 among live cells after killing are analyzed.

[0114] BC19 scFv affinity detection. The expression of target antigens on the cell surface of Nalm6 (CD19+BCMA-) and U266 (CD19-BCMA+) cells was detected using BC19 Bs scFv-FC or BC19 Bs scFv-His recombinant proteins (Sino Biological, China) at 0, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL. The expression of target antigens on the cell surface of SKOV3, SKOV3-BCMA, and SKOV3-CD19 cells was detected using 0.5 μg / mL BC19 Bs scFv-FC or BC19 Bs scFv-His recombinant proteins. Finally, all samples were stained with APC anti-human IgG Fc Antibody (410712; BioLegend, USA) or APC anti-His Tag Antibody (362605, BioLegend, USA).

[0115] Data were collected using a CytoFLEX S flow cytometer from Beckman Coulter, USA, and analyzed using NovoExpress software from ACEA Biosciences.

[0116] 5. Cytotoxicity of tumor-specific CAR-T cells (RTCA)

[0117] Adherent target cells (SKOV3, SKOV3-BCMA, SKOV3-CD19) were seeded into 96-well E-Plates (ACEA Biosciences, USA) at a density of 1×10 4 / well and monitored overnight in an impedance-based xCELLigence real-time cell analyzer (RTCA) system (ACEA Biosciences, USA). The next day, 4 types of CAR-T or Mock-T cells were added to the corresponding wells at an effector-to-target ratio of 3:1 or 1:3, and the RTCA system continuously monitored the cell impedance in the E-Plates for 96 hours and plotted a real-time impedance change curve.

[0118] 6. Enzyme-linked immunosorbent assay (ELISA) for detecting interferon-γ

[0119] 1×10 4Adherent target cells (SKOV3, SKOV3-BCMA, SKOV3-CD19) and suspension target cells (U266, Nalm6) of the pores were cultured with 4 kinds of CAR-T or Mock-T cells at an effector-to-target ratio of 1:1 (each cell) in a U-bottom 96-well plate, and 200 μL of X-Vivo 15 serum-free medium (04-418Q, Lonza, USA) containing 10% fetal bovine serum was added to each well. After 24 h, 150 μL of the culture medium supernatant was transferred to a V-bottom 96-well plate and centrifuged at 300 g for 5 min to remove cell debris. The supernatant was transferred to a new 96-well plate, and the human interferon-γ secretion level was detected using an ELISA kit (430104, BioLegend, USA) according to the manufacturer's instructions. The experiment was independently conducted three times.

[0120] 7. Establishment of multiple myeloma xenograft tumor model

[0121] Male NCG mice aged 6 - 8 weeks (Jiangsu Jicui Yakang Co., Ltd., China) were inoculated with 2×10 6 / U266 / luc cells per mouse via the tail vein on day -7 to establish a multiple myeloma model. On day -1, in vivo tumor growth was evaluated by luciferase real-time imaging (Berthold Technologies, Germany), and the mice were randomly divided into 4 groups according to the fluorescence intensity, namely Mock T group (n = 3 mice), CD19 CAR-T group (n = 3 mice), BCMACAR-T group (n = 4 mice), and CD19-BCMACAR-T group (n = 4 mice). On day 0, the corresponding CAR-T cells were administered at 2×10 6 / per mouse via the tail vein for treatment, and then luciferase real-time imaging was performed every 7 days for 42 consecutive days to evaluate tumor progression. The mortality of the mice within 60 days after modeling was observed, and the Kaplan-Myer survival curve was plotted. γ-secretase inhibitor LY3039478 (HY-12449, MCE, USA) was administered by gavage at 1 mg / kg on day -1 and three times a week after injection of CAR-T for 42 days.

[0122] Male NCG mice aged 6 - 8 weeks (Jiangsu Jicui Yakang Co., Ltd., China) were inoculated with 1×10 6 / Nalm6 / luc cells per mouse via the tail vein on day -7 to establish a multiple myeloma model. On day -1, the mice were randomly divided into 4 groups with 5 mice in each group according to the results of luciferase real-time imaging. On day 0, 2×10 6 / Only CAR-T cell therapy was performed, and then luciferase real-time imaging was carried out every 7 days for 35 consecutive days to evaluate tumor progression. At the same time, the mouse mortality rate was recorded to plot the Kaplan-Myer survival curve.

[0123] 8. Clinical trial design

[0124] The inventors conducted an open-label, single-arm, multicenter phase I / II study of BC19 CAR-T cells in patients with R / R MM. Eligible patients for this study must be: 1) less than 70 years old; 2) meet the diagnostic criteria for R / R MM defined by the International Myeloma Working Group (IMWG); 3) the patient has relapsed or at least 2 previous treatments have been ineffective, including proteasome inhibitors and immunomodulatory drugs, etc.; 4) the patient has measurable lesions and good organ function, and the ECOG score ≤ 2. Regardless of the expression of CD19, the positive expression of BCMA on MM cells needs to be confirmed by flow cytometry, but no pre-specified expression level is required. Female patients must be negative for human chorionic gonadotropin and have no plans to become pregnant within 6 months after treatment. Patients with mental or psychological diseases, severe allergies or a history of severe allergies (especially patients allergic to interleukin [IL]-2) are excluded.

[0125] This study was initiated and designed by the Department of Hematology, Affiliated Hospital of Xuzhou Medical University, and approved by the institutional review boards of each participating institution. This project has been registered on Chictr.org.cn with the number ChiCTR2000033567. All patients participating in this trial and receiving treatment signed a written informed consent form before participation. All clinical investigations were conducted in accordance with the Declaration of Helsinki Principles.

[0126] 9. Procedures

[0127] On days 11 - 15, peripheral blood mononuclear cells (PBMCs) of the patient were obtained by leukapheresis for the preparation of BC19 CAR-T cells. Day 0 was the first day of BC19 CAR-T cell infusion. After immunocytoreductive chemotherapy with cyclophosphamide and fludarabine (750 mg / m 2 / day, 30 mg / m 2 / day, from day 5 to day 2), the patient received a single dose of autologous BC19 CAR-T cell infusion on day 0 at a dose of 1 × 10 6 cells / kg (body weight).

[0128] Response assessment was performed on day 14 and day 28 after infusion and at each follow-up stage. Patients were followed up monthly for the first 6 months, then every 3 months for 2 years, or until death or withdrawal from the study. Response evaluation included the number of bone marrow plasma cells (BM), immunofixation electrophoresis, serum free light chain levels, serum paraprotein, serum immunoglobulin concentration, and 24-hour urine protein analysis. Minimal residual disease (MRD) was monitored at specified times after CAR-T cell infusion and defined as the absence of plasma cells in BM (<0.01%) according to the EuroFlow protocol. For patients with extramedullary disease, the evaluation included imaging techniques (MRI, CT, or PET-CT) and physical examination. Serum cytokine levels were measured using a cytokine bead array (CBA) according to the manufacturer's instructions. Cytogenetic and genomic aberrations were identified by karyotyping and fluorescence in situ hybridization. As previously described, real-time quantitative PCR (QPCR) was used to evaluate CAR-DNA copy number. Cytokine release syndrome (CRS) was graded according to the criteria of Lee and colleagues (Lee, D.W. et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 124, 188-195 (2014)). Neurological events and other events were graded according to the Common Terminology Criteria for Adverse Events, version 4.03. Hypoplasia of B cells detected by multiparameter flow cytometry (MPFC) was defined as <1% blood CD19+ with a recovery rate ≥3%.

[0129] 10. Endpoints and Assessments

[0130] The primary endpoint was safety. Safety mainly referred to the severity, frequency, and duration of adverse events. The key secondary endpoint was the overall response rate (ORR). ORR was defined as the proportion of patients who achieved stringent complete remission (sCR), CR, very good partial remission (VGPR), or PR at any time after infusion according to the IMWG criteria. Other secondary endpoints included duration of response (DOR), progression-free survival (PFS), and OS. DOR was defined as the time from the first evidence of at least a PR to disease relapse or progression. PFS was defined as the time from CAR-T cell infusion to disease progression or death from any cause. OS was defined as the time from injection to death. The cutoff date was June 30, 2022.

[0131] 11. Statistical Analysis

[0132] The statistical tests used in the preclinical study were described in the corresponding legends. All 50 patients who received CAR-T cell infusion were included in the analysis.

[0133] Descriptive statistics included the mean of the standard deviation of continuous variables or the median of the minimum and maximum values of continuous variables, as well as the count and percentage of categorical variables. Missing data were not entered unless otherwise stated. For categorical variables, the exact method (Clopper-Pearson 95% confidence interval) and Fisher's exact test were used. For two groups of continuous variables, the Mann-Whitney U test was used, and for multiple groups, the Kruskal-Wallis test was used. Forest plots included the confidence intervals for individual groups and differences. The DOR, PFS, and OS of patients were determined using the Kaplan-Meier method and compared using the log-rank test. All analyses were performed using SPSS 26 or GraphPad Prism 8. A P value less than 0.05 (two-tailed) was considered significant.

[0134] II. Experimental Results

[0135] 1. Construction and in vitro functional verification of BCMA / CD19 bispecific CAR-T cells

[0136] The inventors separately linked anti-CD19 scFv or anti-BCMA scFv in series with a classical second-generation CAR structure (including the CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory domain, CD3ζ) to construct CD19 CAR and BCMA CAR. On this basis, the inventors linked anti-CD19 scFv and anti-BCMA scFv through (G4S)4 to construct a bispecific CAR targeting CD19 and BCMA. Among them, the one with anti-CD19 scFv in front was named C19B CAR; the one with anti-BCMA scFv in front was named BC19 CAR( Figure 1 A). Lentiviruses carrying CD19 CAR, BCMA CAR, C19B CAR, and BC19 CAR were transduced into T cells from healthy donors. After detection, all four CARs could be stably expressed on the cells. The positive rates of single-target CARs (CD19 66.1%, BCMA 65.3%) were slightly higher than those of bispecific CARs (C19B 58.4%, BC19 60.2%)( Figure 1 B). The results of differentiation phenotype detection showed that the BC19 CAR-T cell preparation and the C19B CAR-T cell preparation were mainly composed of naive T cells (35.7%, 31.3%) and central memory T cells (46.6%, 46.1%), which were similar to the phenotypes of single-target CD19 or BCMA CAR-T cell preparations( Figure 1 C). The in vitro expansion ability of C19B CAR-T cells was slightly higher than that of the other three groups of CAR-T and NT cells, but there was no statistical difference, and no obvious cell death was observed in each group( Figure 1 D).

[0137] To explore the killing activities of 4 types of CAR-T cells against CD19-positive or BCMA-positive tumor cells through real-time cytotoxicity assay (RTCA), the inventors used SKOV3 cells (CD19- / BCMA-), SKOV3 / BCMA cells (CD19- / BCMA+), and SKOV3 / CD19 cells (CD19+ / BCMA-) as target cells, and used CD19, BCMA, C19B, and BC19 CAR-T cells as effector cells, with effector-to-target ratios of 3:1 and 1:3 respectively. Compared with single-target and C19B CAR-T, BC19 CAR-T showed stronger cytotoxicity and higher interferon-γ release when co-incubated with SKOV3-CD19 cells. When BC19 CAR-T was co-incubated with SKOV3-BCMA cells, the cytotoxicity was significantly higher than that of single-target and C19B CAR-T cells, but there was no significant difference in the secretion level of interferon-γ among the three. After co-incubation with SKOV3 cells, none of the 4 types of CAR-T cells showed obvious reaction( Figure 1 E and Figure 1 G).

[0138] The inventors used the hematological tumor cell lines U266 cells (CD19- / BCMA+) and Nalm6 cells (CD19+ / BCMA-) as target cells to compare the killing activities of the 4 types of CAR-T cells again. After co-incubation with Nalm6 cells, the performance of BC19 CAR-T was better than that of CD19 CAR-T or C19B CAR-T cells. When co-incubated with U266 cells, the killing activities of BC19 CAR-T and BCMACAR-T were comparable and better than that of C19B CAR-T cells. In summary, both exogenous and endogenous expression of target antigens can induce BC19CAR-T cells to produce highly efficient and specific anti-tumor responses( Figure 1 F). Given that BC19 CAR-T cells showed more excellent activities in in vitro killing experiments, among the two bispecific CAR-Ts, we selected BC19 CAR-T cells for subsequent experiments.

[0139] 2. BC19 scFv fusion protein can specifically recognize and bind to membrane-expressed target antigen

[0140] To detect the specificity of the binding of BC19 CAR to the membrane antigen BCMA or CD19, the inventors constructed and expressed and purified 2 types of fusion proteins with Fc or His tags, namely BCMA scFv-Linker-CD19 scFv-Fc and BCMA scFv-Linker-CD19 scFv-His( Figure 2A). The molecular weight (MW) of BCMA scFv-Linker-CD19 scFv-Fc was 75 kDa as analyzed by Western Blot, and that of BCMA scFv-Linker-CD19 scFv-His was 57 kDa, which was in line with expectations. Figure 2 B). Flow cytometry analysis found that both BC19 scFvs could recognize and bind CD19 on the surface of Nalm6 cells and BCMA on the surface of U266 cells, and the binding of BC19 scFv to the target antigen was dose-dependent. Figure 2 C). Subsequently, the inventors incubated the two fusion proteins with SKOV3, SKOV3 / BCMA, and SKOV3 / CD19 cells respectively. The experimental results showed that neither of the two fusion proteins could bind to SKOV3 cells (CD19− / BCMA−), but could bind to SKOV3 / BCMA or SKOV3 / CD19 cells. Figure 2 D). The above data indicated that BCMA scFv-Linker-CD19 scFv could specifically recognize and bind to the target antigen expressed endogenously or exogenously, and thus was suitable for the construction of bispecific CAR.

[0141] 3. BC19 CAR inhibits the growth of U266 multiple myeloma xenograft tumors in vivo and prolongs the survival time of mice

[0142] To detect the therapeutic effect of bispecific BC19 CAR-T cells in multiple myeloma, NCG mice were injected with 2×10 6 U266 / luc cells per mouse via the tail vein. Meanwhile, γ-secretase inhibitors LY3039478 were used to stabilize the expression of BCMA on the cell membrane surface. On the seventh day after tumor inoculation, Mock T, CD19 CAR-T, BCMACAR-T, and BC19CAR-T cells were administered respectively. Figure 3 A). Bioluminescence imaging and survival analysis results showed that no light signal from the tumor was detected in the BCMACAR-T and BC19 CAR-T groups on the 14th day after CAR-T infusion, indicating good anti-tumor activity. However, tumor recurrence occurred in the BCMACAR-T group on the 21st day, and the prognosis was worse than that of the BC19 CAR-T treatment group. Figure 3 B - 3D). The above results indicated that bispecific BC19 CAR-T cells had efficient and persistent anti-tumor effects in a multiple myeloma model.

[0143] 4. BC19 CAR-T has the effect of inhibiting the growth of CD19-positive tumors in vivo

[0144] The inventors established a Nalm6 xenograft tumor model to investigate the efficacy of BC19 CAR-T cells in killing CD19+ target cells in vivo. The inventors inoculated NCG mice via the tail vein with 1×10 6 / mouse NALM-6 / luc cells, and 7 days later administered 2×10 6 / mouse CAR-T cells for treatment, with the grouping the same as above ( Figure 4 A). The results showed that both CD19 CAR-T and BC19 CAR-T cells exhibited good anti-tumor activity after CAR-T infusion, but tumor recurrence occurred in the CD19 CAR-T group 35 days later ( Figure 4 B-4D). The above results indicate that bispecific BC19 CAR-T cells can effectively eliminate CD19+ target cells in a mouse model.

[0145] 5. Patient characteristics

[0146] During the period from June 5, 2020 to February 28, 2022, 64 patients with R / R MM underwent eligibility screening, and 54 patients were initially enrolled and leukapheresis was performed. The production of BC19 CAR-T cells for the patients who underwent leukapheresis was all successful. 4 patients stopped treatment due to rapid disease progression before infusion ( Figure 5 ). Finally, 50 patients received injections of BC19 CAR-T cells, and the baseline characteristics of the patients are listed in Table 1.

[0147] The median age of the patients was 57 years (range 31 to 70 years), and the median time from MM diagnosis to CAR-T cell infusion was 29.5 months (range 4 to 162 years). A total of 46 patients (96%) had stage II or III disease, 21 patients (42%) had extramedullary disease, 34 patients (68%) had high-risk cytogenetic features, defined as del(17p), t(4;14), or t(14;16). The median number of prior treatments for the patients before enrollment was 4 lines (range 2 to 11). Among them, 20 patients (40%) had previously received auto-HSCT treatment, and 5 patients (10%) had previously received BCMA, CD19, or GPRC5D-targeted CAR-T cell treatment.

[0148] The proportions of patients exposed to or refractory to proteasome inhibitors, immunomodulatory drugs, and CD38 antibodies are shown in Table 1. 2 patients (4%) started auto-HSCT for consolidation 2.2 months and 3.7 months after CAR-T cell treatment, respectively. The other patients did not receive further consolidation treatment.

[0149] Table 1 Patient characteristics

[0150]

[0151] 6. Efficacy

[0152] Among 50 evaluable patients for efficacy, 46 (92%; 95% CI, 81 to 98) had an overall response (PR or better) to BC19 CAR-T cells, including 19 (38%) sCR, 12 (24%) CR, 8 (16%) VGPR, and 7 (14%) PRs (Table 2). Four patients (8%) had stable disease as the best response. The median time to first PR or better was 23.5 days (range, 14 - 30), and the median time to best response was 1.9 months (range, 0.5 - 6.1).

[0153] Seventeen of 21 patients with extramedullary disease (81%; 95% CI, 58 to 95) had an overall response ( Figure 6 ), and among 5 patients previously treated with CAR-T cells, 2 achieved sCR, 1 achieved PR, and the other 2 had SD.

[0154] Among all patients, 41 patients had MRD testing. Thirty-four patients (83%; 95% CI, 68 to 93) achieved MRD negativity, including 22 with CR or sCR (Table 2). The median time to MRD positivity after injection of CAR-T cells was 0.5 months (range, 0.5 - 2.1). The time to achieve MRD negativity was not related to the depth of response.

[0155] Univariate analysis showed that the overall response rate ORR was consistent across key covariates, including disease stage, high-risk cytogenetic profile, baseline tumor burden, BCMA expression, CD19 detection, complex karyotype, number of prior treatment lines, and time since diagnosis. Patients with extramedullary disease or previously treated with CAR-T cells had a lower ORR ( Figure 7 ).

[0156] At a median follow-up of 11.4 months (range 2.5 to 24), 27 of 46 patients with PR or better (59%; 95% CI, 43 to 73) had a durable response. Fifteen of 46 patients (33%; 95% CI, 20 - 48) had relapse or progression during follow-up, including 8 of 31 patients with CR or better and 7 of 15 patients with VGPR or PR ( Figure 8 ). Among 15 patients who relapsed or progressed with BCMA and CD19 detection, 1 (7%) relapsed with BCMA-negative myeloma cells and 14 (93%) relapsed with CD19-negative myeloma cells.

[0157] The median OS and PFS for all 50 patients were 19.7 months (95% CI, 5.0 - 34.4 months) and 19.7 months (95% CI, 7.7 - 31.7 months), respectively. Figure 9 A - B). The median DOR for 46 patients with PR or better was not reached. Figure 9 C). The 1 - year PFS, OS, and DOR rates were 54% (95% CI, 38 - 70), 85% (95% CI, 75 - 95), and 60% (95% CI, 44 - 76), respectively. The 12 - month PFS and OS rates for patients with CR or better were 64% (95% CI, 46 - 82) and 86% (95% CI, 74 - 99), respectively.

[0158] Table 2 Therapeutic effects of BC19 CAR - T on patients

[0159]

[0160] Example 2 Construction of BCMA / CD19 bispecific CAR mutants, CAR - T cells expressing the CAR mutants, and verification of their therapeutic effects

[0161] I. Experimental methods

[0162] 1. Construction of BC19 CAR mutants

[0163] In this example, the fully human anti - CD19 scFv sequence in the BC19 CAR described in Example 1 was replaced with the FMC63 sequence (a murine monoclonal antibody sequence targeting CD19). Using the method described in Example 1, a BC19 CAR mutant was constructed. The BC19 CAR mutant was obtained by sequentially connecting the CD8 leader sequence, BCMA (scfv), Linker, CD19 (scfv, FMC63), CD8 hinge region and transmembrane region, CD137 (4 - 1BB) co - stimulatory signal domain, and CD3ζ intracellular signal transduction domain in series.

[0164] Among them, the amino acid sequences of the CD8 leader sequence, BCMA (scfv), Linker, CD19 (scfv, FMC63), CD8 hinge region and transmembrane region, CD137 (4-1BB) co-stimulatory signal domain, and CD3ζ intracellular signal transduction domain in the BC19 CAR mutant are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:13 respectively. The amino acid sequence of the BC19 CAR mutant is shown in SEQ ID NO:14, and the nucleotide sequence is shown in SEQ ID NO:15.

[0165] 2. Sequence alignment of FMC63 and the fully human anti-CD19 scFv described in Example 1

[0166] In this example, the fully human anti-CD19 scFv and FMC63 used in the construction process of BC19 CAR in Example 1 were subjected to sequence alignment, and the main differences were found to be: (1) The 11th and 13th amino acids of the CDR-H2 of the heavy chain variable region of the fully human anti-CD19 scFv are both S, while the 11th amino acid of the CDR-H2 region of FMC63 is N and the 13th is A; (2) The framework region of FMC63 was humanized by the fully human anti-CD19 scFv.

[0167] In order to investigate the influence of the above differences on the conformational characteristics of FMC63, two mutants of FMC63 (mutant S1, mutant S2) were further constructed in this example. Among them, mutant S1 only humanized the framework region of FMC63; mutant S2 is the CD19 scFv sequence used in BC19 CAR described in Example 1. Humanization analysis and 3D structure modeling were performed on FMC63 (abbreviation F), mutant S1, and mutant S2 respectively.

[0168] 3. Codon optimization of BC19 CAR mutant

[0169] To further improve the expression efficiency of the BC19 CAR mutant in human T cells, in this example, the amino acid sequence of the BC19 CAR mutant was codon-optimized on the GenScript website (Human T), the GenScript website (Human), and GeneChem Co., Ltd. respectively. The optimized sequences were named Codon 1#, Codon 2#, and Codon 3# respectively. Among them, the amino acid sequences corresponding to Codon 1#, Codon 2#, and Codon 3# are all shown as SEQ ID NO:14, and the nucleotide sequences corresponding to Codon 1#, Codon 2#, and Codon 3# are shown as SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:15 respectively.

[0170] 4. Construction of BC19 CAR-T cells

[0171] The above three sequences (Codon 1#, Codon 2#, and Codon 3#) were respectively cloned into the pHAGE vector, and subsequent lentiviral packaging and titer detection were carried out. To avoid interference of individual differences on the experimental results, in this example, PBMC cells derived from 3 multiple myeloma patients were randomly selected for isolation and purification of T cells. Each purified T cell was infected with lentiviruses carrying Codon 1#, Codon 2#, and Codon 3# respectively at MOI = 10, and CAR-T cells expressing 3 BC19 CAR mutants (Codon 1#, Codon 2#, and Codon 3#) were successfully prepared. Finally, through APC-Protein-L staining and flow cytometry analysis, it was determined which codon optimization method among the 3 codon optimization methods could achieve the optimal expression efficiency of the BC19 CAR mutant in T cells.

[0172] 5. Verification of the therapeutic effect of BC19 CAR-T cells

[0173] During the period from September 2020 to April 2022, a total of 7 patients with relapsed / refractory (R / R) multiple myeloma received injections of BC19 CAR mutant T cells. The administration method was as described in Example 1, and the administration dose was 1×10 6cells / kg (body weight). The median number of prior treatments for the enrolled patients was 4 (range 2 to 5), 4 patients (68%) had extramedullary disease (EMD), and 4 patients (68%) had high-risk cytogenetic features (HR). Among them, 3 patients (40%) had previously received auto-HSCT treatment, and 1 patient (10%) had previously received CAR-T cell treatment. Moreover, 6 patients (86%) had experienced 3 or more disease relapses. The BC19 CAR mutant was the Codon 3# mutant optimized by GeneChem as described above.

[0174] Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare peripheral nerve disease characterized by progressive symmetric motor and sensory loss, and weakness associated with loss of deep tendon reflexes, which is caused by damage to the nerve surface layer called myelin sheath. During the period from 2022 to 2023, a total of 3 patients diagnosed with CIDP received BC19 CAR mutant T cell treatment. The administration method of the BC19 CAR mutant T cells was as described in Example 1, and the administration dose was 1×10 6 cells / kg (body weight). Among them, 3 patients were evaluable for efficacy. The BC19 CAR mutant was the Codon 3# mutant optimized by GeneChem as described above.

[0175] II. Experimental Results

[0176] Results of humanization analysis and 3D structure modeling showed that compared with FMC63, S1 and S2 had higher humanization degree and lower immunogenicity ( Figure 10 A), meeting our expectations. It can be seen from the structural simulation and superposition mode that except for the structural variation near the Linker, the structures of the CDR regions were well superimposed, and the orientation and conformation of the backbone carbon atoms did not change much. That is to say, the modes of FMC63, S1 and S2 recognizing CD19 should be quite the same, and antigenic epitope drift would not occur ( Figure 10 B).

[0177] Results of the influence of different codon optimization methods on the expression efficiency of BC19 CAR mutants in T cells showed that the positive expression rate and mean fluorescence intensity of Codon 3# were significantly higher than those of the other two BC19 CAR mutants (Codon 1#, Codon 2#), and Codon 3# could be stably and highly expressed in T cells from different patients. Figure 11 and Figure 12) Therefore, all BC19 CAR mutants used in subsequent experiments were Codon 3# mutants optimized by GeneChem.

[0178] The results of treatment effect verification showed that all 7 relapsed / refractory multiple myeloma patients (100%) who received BC19 CAR mutant T cell therapy achieved very good partial remission (VGPR or better), and 2 patients (29%) achieved sCR / CR ( Figure 13 ). The most common acute adverse events after CAR-T cell infusion were CRS and immune effector cell-associated neurotoxicity syndrome (ICANS). Among the 7 patients, except for 1 patient (8%) who died of grade 5 CRS, no patient had grade 3 or higher CRS (0%), and no patient had grade 3 or higher CRS accompanied by ICANS. The above results indicate that the BC19 CAR mutant T cells constructed in this example can be used for the safe and effective treatment of relapsed / refractory multiple myeloma.

[0179] The results of treatment effect verification showed that the Medical Research Council Score (MRC Score) of all 3 patients with chronic Guillain-Barré syndrome who received BC19 CAR mutant T cell therapy was significantly improved, indicating that the muscle strength of the patients was improved. Among them, the MRC values of 2 patients reached 60 after treatment, and the muscle strength returned to normal. In addition, the INCAT disability scores of the 3 patients were also significantly reduced, and the INCAT value of 1 patient dropped to 0, indicating that the patient recovered normal sensation. Moreover, B cell depletion kinetics detection showed that 7-10 days after CAR-T infusion, the B cells in the blood of all 3 patients dropped to 0%, indicating that the antibody-producing B cells were completely depleted. Among them, on the day of CAR-T administration, the B cells completely disappeared from the peripheral blood of 1 patient, and B cell reconstitution occurred 10 days later, but no recurrence of CIDP was observed during the follow-up period, and no drugs related to CIDP were received during this period ( Figure 14 ). No patient had grade 3 or higher CRS (0%), and no patient had grade 3 or higher CRS accompanied by ICANS. This study provides new hope for CIDP patients to achieve remission without treatment and no longer live with the disease.

Claims

1. A chimeric antigen receptor targeting BCMA and CD19, characterized in that, The chimeric antigen receptor comprises a bispecific antibody targeting BCMA and CD19; The bispecific antibody is obtained by sequentially connecting in series an antibody targeting BCMA, a Linker, and an antibody targeting CD19; The amino acid sequence of the antibody targeting BCMA is as shown in SEQ ID NO:2; The amino acid sequence of the antibody targeting CD19 is as shown in SEQ ID NO:4; The amino acid sequence of the Linker is as shown in SEQ ID NO:3; The antibody targeting BCMA is in the front and the antibody targeting CD19 is at the back.

2. The chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor further comprises a signal peptide, a hinge region and a transmembrane region, a co-stimulatory signal domain or an intracellular signal transduction domain.

3. The chimeric antigen receptor according to claim 2, characterized in that, The signal peptide is selected from the signal peptides of the following molecules: CD8, CD4, CD5, CD9, CD28, CD16, CD3ζ, CD3ε, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS or IgG6.

4. The chimeric antigen receptor according to claim 2, characterized in that, The hinge region and transmembrane region are selected from the hinge regions and transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor or IL-11 receptor.

5. The chimeric antigen receptor according to claim 2, characterized in that, The co-stimulatory signal domain is selected from the co-stimulatory signal domains of the following molecules: 4-1BB, CD27, CD19, CD4, CD28, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1 or B7-H3.

6. The chimeric antigen receptor according to claim 2, characterized in that, The intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12 or CD66d.

7. The chimeric antigen receptor according to claim 2, characterized in that, The signal peptide is the CD8 signal peptide.

8. The chimeric antigen receptor according to claim 2, characterized in that, The hinge region and transmembrane region are the CD8 hinge region and transmembrane region.

9. The chimeric antigen receptor according to claim 2, characterized in that, The co-stimulatory signal domain is the 4-1BB co-stimulatory signal domain.

10. The chimeric antigen receptor according to claim 2, characterized in that, The intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

11. The chimeric antigen receptor according to claim 7, characterized in that, The amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO:1; 12. The chimeric antigen receptor according to claim 8, characterized in that, The amino acid sequence of the CD8 hinge region and transmembrane region is as shown in SEQ ID NO:5; 13. The chimeric antigen receptor according to claim 9, characterized in that, The amino acid sequence of the 4-1BB co-stimulatory signal domain is as shown in SEQ ID NO:6; 14. The chimeric antigen receptor according to claim 10, characterized in that, The amino acid sequence of the CD3ζ intracellular signal transduction domain is as shown in SEQ ID NO:7; 15. The chimeric antigen receptor according to claim 2, characterized in that, The chimeric antigen receptor is obtained by sequentially connecting in series the CD8 signal peptide, the antibody targeting BCMA, the Linker, the antibody targeting CD19, the CD8 hinge region and transmembrane region, the 4-1BB co-stimulatory signal domain, and the CD3ζ intracellular signal transduction domain.

16. The chimeric antigen receptor according to claim 15, characterized in that, The amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

8.

17. The chimeric antigen receptor according to claim 16, characterized in that, The nucleotide sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

9.

18. An optimized chimeric antigen receptor targeting BCMA and CD19, characterized in that, The chimeric antigen receptor comprises a bispecific antibody targeting BCMA and CD19; The bispecific antibody is obtained by sequentially concatenating an antibody targeting BCMA, a Linker, and a mutant of the antibody FMC63 targeting CD19; The amino acid sequence of the antibody targeting BCMA is as shown in SEQ ID NO:2; The amino acid sequence of the mutant of the antibody FMC63 targeting CD19 is as shown in SEQ ID NO:12; The amino acid sequence of the Linker is as shown in SEQ ID NO:3; The antibody targeting BCMA is in the front, and the mutant of the antibody FMC63 targeting CD19 is in the back.

19. The chimeric antigen receptor according to claim 18, characterized in that, The chimeric antigen receptor further comprises a signal peptide, a hinge region and a transmembrane region, a co-stimulatory signal domain or an intracellular signal transduction domain.

20. The chimeric antigen receptor according to claim 19, characterized in that, The signal peptide is selected from the signal peptides of the following molecules: CD8, CD4, CD5, CD9, CD28, CD16, CD3ζ, CD3ε, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS or IgG6.

21. The chimeric antigen receptor according to claim 19, characterized in that, The hinge region and transmembrane region are selected from the hinge regions and transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor or IL-11 receptor.

22. The chimeric antigen receptor according to claim 19, characterized in that, The co-stimulatory signal domain is selected from the co-stimulatory signal domains of the following molecules: 4-1BB, CD27, CD19, CD4, CD28, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1 or B7-H3.

23. The chimeric antigen receptor according to claim 19, characterized in that, The intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12 or CD66d.

24. The chimeric antigen receptor according to claim 19, characterized in that, The signal peptide is the CD8 signal peptide.

25. The chimeric antigen receptor according to claim 19, characterized in that, The hinge region and transmembrane region are the CD8 hinge region and transmembrane region.

26. The chimeric antigen receptor according to claim 19, characterized in that, The co-stimulatory signal domain is the 4-1BB co-stimulatory signal domain.

27. The chimeric antigen receptor according to claim 19, characterized in that, The intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

28. The chimeric antigen receptor according to claim 24, characterized in that, The amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO:

1.

29. The chimeric antigen receptor according to claim 25, characterized in that, The amino acid sequence of the CD8 hinge region and transmembrane region is as shown in SEQ ID NO:

5.

30. The chimeric antigen receptor according to claim 26, characterized in that, The amino acid sequence of the 4-1BB co-stimulatory signal domain is as shown in SEQ ID NO:

6.

31. The chimeric antigen receptor according to claim 27, characterized in that, The amino acid sequence of the CD3ζ intracellular signal transduction domain is as shown in SEQ ID NO:

13.

32. The chimeric antigen receptor according to claim 19, characterized in that, The chimeric antigen receptor is obtained by sequentially concatenating a CD8 signal peptide, an antibody targeting BCMA, a Linker, a mutant of the antibody FMC63 targeting CD19, a CD8 hinge region and transmembrane region, a 4-1BB co-stimulatory signal domain, and a CD3ζ intracellular signaling domain.

33. The chimeric antigen receptor according to claim 32, characterized in that, The amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

14.

34. The chimeric antigen receptor according to claim 33, wherein, The nucleotide sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

15.

35. A nucleic acid molecule, wherein, The nucleic acid molecule encodes the chimeric antigen receptor according to any one of claims 1-17 or the chimeric antigen receptor according to any one of claims 18-34.

36. The nucleic acid molecule according to claim 35, wherein, The nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1-17 is as shown in SEQ ID NO:

9.

37. The nucleic acid molecule according to claim 35, wherein, The nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 18-34 is as shown in SEQ ID NO:

15.

38. An expression vector, wherein, The expression vector contains the nucleic acid molecule according to any one of claims 35-37.

39. The expression vector according to claim 38, wherein, The vector is selected from a DNA vector, an RNA vector, a transposon vector or a CRISPR / Cas9 vector.

40. The expression vector according to claim 39, wherein, The DNA vector is a plasmid.

41. The expression vector according to claim 39, wherein, The RNA vector is a virus-derived vector.

42. The expression vector according to claim 41, wherein, The virus-derived vector is selected from a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector or a herpesviral vector.

43. An engineered host cell, wherein, The engineered host cell contains the nucleic acid molecule according to any one of claims 35-37 and / or the expression vector according to any one of claims 38-42.

44. The engineered host cell according to claim 43, wherein, The host cell is a mammalian cell.

45. The engineered host cell according to claim 44, wherein, The host cell is an immune cell.

46. The engineered host cell according to claim 45, wherein, The immune cell is selected from a T cell, a B cell, a NK cell, an iNKT cell, a γδT cell, a NK92 cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage, a neutrophil or any combination thereof.

47. The engineered host cell according to claim 46, wherein, The immune cell is a T cell.

48. The engineered host cell according to claim 47, wherein, The T cell is of human origin.

49. A derivative, wherein, The derivatives are selected from: (1) The chimeric antigen receptor according to any one of claims 1-17 and / or the chimeric antigen receptor according to any one of claims 18-34 and / or the nucleic acid molecule according to any one of claims 35-37 and / or the engineered host cell according to any one of claims 43-48, which contains a detectable label; (2) The chimeric antigen receptor according to any one of claims 1-17 and / or the chimeric antigen receptor according to any one of claims 18-34 and / or the nucleic acid molecule according to any one of claims 35-37 and / or the engineered host cell according to any one of claims 43-48, which confers antibiotic resistance; or (3) The chimeric antigen receptor according to any one of claims 1-17 and / or the chimeric antigen receptor according to any one of claims 18-34 and / or the nucleic acid molecule according to any one of claims 35-37 and / or the engineered host cell according to any one of claims 43-48, which is bound or conjugated to a therapeutic agent.

50. The derivative according to claim 49, wherein, The detectable label is selected from a fluorescent dye, colloidal gold, a chemiluminescent label, or a chemiluminescent catalyst.

51. The derivative according to claim 50, wherein, The chemiluminescent label is selected from luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, adamantane, rare earth elements, or ruthenium bipyridyl complexes.

52. The derivative according to claim 50, characterized in that, The chemiluminescent catalyst is selected from horseradish peroxidase or alkaline phosphatase.

53. The derivative according to claim 49, characterized in that, The gene for antibiotic resistance is selected from a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene.

54. The derivative according to claim 49, characterized in that, The therapeutic agent is selected from a radionuclide, a cytokine, gold nanoparticles, virus particles, liposomes, magnetic nanoparticles, a prodrug-activating enzyme, or a chemotherapeutic agent.

55. The derivative according to claim 54, characterized in that, The radionuclide is selected from 67 Ga, 68 Ga, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y, 90 Y, 89 Zr, 120 I, 123 I, 13 N, 15 O, 186 Re, 110 In or 111 In.

56. The derivative according to claim 54, characterized in that, The cytokine is selected from IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, or M-CSF.

57. The derivative according to claim 54, characterized in that, The magnetic nanoparticles are selected from chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), or vanadium(II).

58. The derivative according to claim 54, characterized in that, The chemotherapeutic agent is selected from cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, or leucovorin.

59. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor according to any one of claims 1-17, the chimeric antigen receptor according to any one of claims 18-34, the nucleic acid molecule according to any one of claims 35-37, the expression vector according to any one of claims 38-42, the engineered host cell according to any one of claims 43-48, and / or the derivative according to any one of claims 49-58.

60. A biological preparation, characterized in that, The biological agent comprises the chimeric antigen receptor according to any one of claims 1-17, the chimeric antigen receptor according to any one of claims 18-34, the nucleic acid molecule according to any one of claims 35-37, the expression vector according to any one of claims 38-42, the engineered host cell according to any one of claims 43-48, the derivative according to any one of claims 49-58, and / or the pharmaceutical composition according to claim 59.

61. A kit, characterized in that, The kit comprises the chimeric antigen receptor according to any one of claims 1-17, the chimeric antigen receptor according to any one of claims 18-34, the nucleic acid molecule according to any one of claims 35-37, the expression vector according to any one of claims 38-42, the engineered host cell according to any one of claims 43-48, and / or the derivative according to any one of claims 49-58.

62. A method for preparing the engineered host cell according to any one of claims 43-48, characterized in that, The method comprises the following steps: introducing the nucleic acid molecule according to any one of claims 35-37 or the expression vector according to any one of claims 38-42 into a host cell.

63. The method according to claim 62, characterized in that, The introduced method is selected from lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxviral vector, herpes simplex virus vector, adenoviral vector or adeno-associated viral vector.

64. An in vitro method for inhibiting the activity of BCMA and / or CD19 protein, characterized in that, The method includes the following steps: contacting the engineered host cell according to any one of claims 43-48, the derivative according to any one of claims 49-58, the pharmaceutical composition according to claim 59, and / or the biological agent according to claim 60 with somatic cells of an organism.

65. Any one of the following applications, the application comprising: (1) Use of the chimeric antigen receptor according to any one of claims 1-17, the chimeric antigen receptor according to any one of claims 18-34, the nucleic acid molecule according to any one of claims 35-37, the expression vector according to any one of claims 38-42, the engineered host cell according to any one of claims 43-48, the derivative according to any one of claims 49-58, the pharmaceutical composition according to claim 59, the biological agent according to claim 60, or the kit according to claim 61 in the preparation of a drug for treating BCMA- and / or CD19-related diseases; (2) Use of the chimeric antigen receptor according to any one of claims 1-17, the chimeric antigen receptor according to any one of claims 18-34, the nucleic acid molecule according to any one of claims 35-37, the expression vector according to any one of claims 38-42, the engineered host cell according to any one of claims 43-48, or the derivative according to any one of claims 49-58 in the preparation of a kit for preparing chimeric antigen receptor immune cells for treating BCMA- and / or CD19-related diseases; (3) Use of the chimeric antigen receptor according to any one of claims 1-17, the chimeric antigen receptor according to any one of claims 18-34, the nucleic acid molecule according to any one of claims 35-37, the expression vector according to any one of claims 38-42, the engineered host cell according to any one of claims 43-48, the derivative according to any one of claims 49-58, the pharmaceutical composition according to claim 59, or the kit according to claim 61 in the preparation of a biological agent for treating BCMA- and / or CD19-related diseases; (4) Use of the kit according to claim 61 in the preparation of chimeric antigen receptor immune cells for treating BCMA- and / or CD19-related diseases; (5) Use of the chimeric antigen receptor according to any one of claims 1-17 or the chimeric antigen receptor according to any one of claims 18-34 in the preparation of a nucleic acid molecule, an expression vector, an engineered host cell or a derivative; (6) Use of the nucleic acid molecule according to any one of claims 35-37 in the preparation of an expression vector, an engineered host cell or a derivative; (7) Use of the expression vector according to any one of claims 38-42 in the preparation of an engineered host cell or a derivative; Use of the engineered host cell according to any one of claims 43-48 in the preparation of a derivative; Use of the chimeric antigen receptor according to any one of claims 1-17 or the chimeric antigen receptor according to any one of claims 18-34 in the preparation of a chimeric antigen receptor immune cell for treating BCMA- and / or CD19-related diseases; The BCMA- and / or CD19-related diseases are selected from tumors or autoimmune diseases; The tumors are selected from multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, small lymphocyte lymphoma, follicular lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, megakaryocytic leukemia, Burkitt lymphoma, anaplastic large cell lymphoma or mucosa-associated lymphoid tissue lymphoma; The autoimmune diseases are selected from chronic Guillain-Barré syndrome, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, demyelinating lesions, autoimmune hemolysis, idiopathic thrombocytopenic purpura or idiopathic leukopenia.

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