Application of BCMA-CD19 bispecific CAR-immune cell in treatment of membranous nephropathy

Abstract

The invention provides an application of a BCMA-CD19 bispecific CAR (Chimeric Antigen Receptor)-immune cell in the treatment of membranous nephropathy. Specifically, the invention provides a BCMA-CD19 bispecific CAR or a coding nucleic acid thereof, or a carrier thereof, or an application of a CAR-immune cell thereof, and the BCMA-CD19 bispecific CAR or the coding nucleic acid thereof, or the carrier thereof, or the CAR-immune cell thereof is used for (i) preparing a medicine for preventing and / or treating membranous nephropathy (MN); (ii) preparing a medicine for reducing the urine protein level; (iii) preparing a medicine for reducing the PLA2R antibody titer; (iv) preparing a medicine for improving the serum albumin level; and / or (v) preparing a medicine for improving the renal function eGFR level. The invention also provides a method for reducing urine protein level, reducing PLA2R antibody titer, increasing serum albumin level and / or increasing renal function eGFR level and a method for preventing and / or treating membranous nephropathy (MN) by using the BCMA-CD19 bispecific CAR or a coding nucleic acid thereof, or a carrier thereof, or a CAR-immune cell thereof, or a pharmaceutical composition. The BCMA-CD19 bispecific CAR-immune cell provided by the invention has very good safety and effectiveness, and can achieve the purpose that patients with high-risk, recurrent or refractory membranous nephropathy can continuously obtain clinical deep relief treatment.

Description

Technical Field

[0001] This invention relates to the field of cell therapy, and more specifically to the application of a BCMA-CD19 bispecific CAR-immune cell in the treatment of membranous nephropathy. Background Technology

[0002] Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults.

[0003] Phospholipase A2 receptor (PLA2R) antibodies are important biomarkers for the diagnosis, monitoring of disease activity, prognosis, and treatment decisions in proteinuria (PMN). Decreased phospholipase A2 receptor antibody levels are associated with remission of proteinuria, while high levels of anti-phospholipase A2 receptor antibodies are a risk factor for poor prognosis.

[0004] Immunosuppressive therapy remains the primary treatment for MN. Immunosuppressants such as cyclophosphamide (CTX) combined with glucocorticoids are the standard treatment for MN. Standard treatment is associated with an increased risk of severe infections, advanced malignancy, infertility, and other serious adverse events. Rituximab is an anti-CD20 chimeric monoclonal antibody that triggers B-cell death through apoptosis, complement-mediated cytotoxicity, and antibody-dependent cytotoxicity. Rituximab (used off-label) appears to be safer and allows for approximately 60% initial clinical remission, which is associated with long-term preservation of renal function. However, approximately 40% of MN patients remain rituximab-refractory, representing a significant unmet need.

[0005] Therefore, more effective treatment methods are still needed for the clinical treatment of membranous nephropathy, especially for patients with high-risk, refractory, or relapsed membranous nephropathy. Summary of the Invention

[0006] The purpose of this invention is to provide an application of BCMA-CD19 bispecific CAR or its encoded nucleic acid, or its vector, or its CAR-immune cells in the treatment of membranous nephropathy.

[0007] In a first aspect of the invention, there is provided a use of a BCMA-CD19 bispecific CAR or its encoded nucleic acid, or its vector, or its CAR-immune cells, for:

[0008] (i) To prepare drugs for the prevention and / or treatment of membranous nephropathy (MN);

[0009] (ii) Prepare drugs to reduce urinary protein levels;

[0010] (iii) Prepare drugs to reduce PLA2R antibody titers;

[0011] (iv) Preparation of drugs to increase serum albumin levels; and / or

[0012] (v) Prepare drugs that improve renal function eGFR levels.

[0013] In another preferred embodiment, the membranous nephropathy is idiopathic membranous nephropathy (IMN) or secondary membranous nephropathy.

[0014] In another preferred embodiment, the secondary membranous nephropathy is secondary to an autoimmune disease, infection, tumor, drug or toxin, or a combination thereof.

[0015] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjögren's syndrome, or a combination thereof.

[0016] In another preferred embodiment, the infection is selected from the group consisting of hepatitis B, hepatitis C, syphilis, HIV, or a combination thereof.

[0017] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, colon cancer, or a combination thereof.

[0018] In another preferred embodiment, the drug or poison is selected from the group consisting of: nonsteroidal anti-inflammatory drugs, probenecid, penicillamine, or combinations thereof.

[0019] In another preferred embodiment, the drug for reducing urinary protein levels reduces urinary protein levels to <3.5g / 24h, more preferably <2g / 24h, more preferably <1g / 24h, and most preferably <0.5g / 24h.

[0020] In another preferred embodiment, the drug for reducing PLA2R antibody titer reduces the PLA2R antibody titer to ≤20 U / ml, preferably ≤10 U / ml, more preferably ≤5 U / ml, most preferably ≤1 U / ml, such as ≤0.5 U / ml, ≤0.1 U / ml, ≤0.05 U / ml, ≤0.01 U / ml, or 0 U / ml.

[0021] In another preferred embodiment, the drug for increasing serum albumin levels increases serum albumin levels to ≥20 g / L, more preferably ≥25 g / L or ≥30 g / L, more preferably ≥35 g / L or ≥40 g / L or ≥45 g / L or ≥50 g / L or ≥55 g / L; preferably, the drug for increasing serum albumin levels increases serum albumin levels to the normal range of serum albumin: 35-55 g / L.

[0022] In another preferred embodiment, the drug that improves renal function eGFR levels increases eGFR levels to ≥30 ml / min, preferably ≥40 ml / min, ≥50 ml / min, or ≥60 ml / min, more preferably ≥65 ml / min, ≥70 ml / min, ≥75 ml / min, ≥80 ml / min, ≥85 ml / min, ≥90 ml / min, ≥95 ml / min, or ≥100 ml / min; preferably, the drug that improves renal function eGFR levels increases eGFR levels to the normal reference value for renal function eGFR: >90 ml / min.

[0023] In another preferred embodiment, the BCMA-CD19 bispecific CAR has the structure shown in Formula I:

[0024] SP1-B1-L-SP2-B2-H-TM-C-CD3ζ(I)

[0025] In the formula,

[0026] SP1 and SP2 are each independently either signal peptide sequences or have no signal peptide.

[0027] B1 and B2 are the first binding element for targeting the first target protein and the second binding element for targeting the second target protein, respectively.

[0028] L represents a flexible peptide;

[0029] H represents the area without hinges;

[0030] TM stands for transmembrane region;

[0031] C is a co-stimulatory signaling molecule;

[0032] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0033] "-" indicates a linking peptide or peptide bond.

[0034] In another preferred embodiment, SP1 and SP2 are signal peptide sequences of immune cell surface molecules commonly used in the art, preferably signal peptide sequences selected from the following histones: CD8, CD28, GM-CSF, CD4, CD137, NKG2D, or combinations thereof.

[0035] In another preferred embodiment, SP1 and SP2 are CD8 signal peptide sequences.

[0036] In another preferred embodiment, the amino acid sequence of the CD8 signal peptide sequence is shown in SEQ ID NO:1.

[0037] In another preferred embodiment, SP1 is a CD8 signal peptide sequence and SP2 is absent.

[0038] In another preferred embodiment, B1 and B2 are antigen-binding domains (scFv) of antibodies targeting the first or second target protein, and the structure of the scFv is shown in formula A or formula B below:

[0039] VH-VL(A)

[0040] VL-VH(B)

[0041] In the formula,

[0042] VH stands for the variable region of the antibody heavy chain;

[0043] VL is the variable region of the antibody light chain;

[0044] "-" indicates a linking peptide or peptide bond.

[0045] In another preferred embodiment, the first target protein is BCMA and the second target protein is CD19; or, the first target protein is CD19 and the second target protein is BCMA; preferably, the first target protein is BCMA and the second target protein is CD19.

[0046] In another preferred embodiment, the amino acid sequence of the linker peptide is shown in SEQ ID NO:3.

[0047] In another preferred embodiment, when the target protein is BCMA, the amino acid sequence of VH is as shown in SEQ ID NO:9, and the amino acid sequence of VL is as shown in SEQ ID NO:10.

[0048] In another preferred embodiment, when the target protein is CD19, the amino acid sequence of VH is as shown in SEQ ID NO:11, and the amino acid sequence of VL is as shown in SEQ ID NO:12.

[0049] In another preferred embodiment, B1 is BCMA scFv and B2 is CD19 scFv; or, B1 is CD19 scFv and B2 is BCMA scFv; preferably, B1 is BCMA scFv and B2 is CD19 scFv.

[0050] In another preferred embodiment, the amino acid sequence of the BCMA scFv is shown in SEQ ID NO:2.

[0051] In another preferred embodiment, the amino acid sequence of the CD19 scFv is shown in SEQ ID NO:4.

[0052] In another preferred embodiment, the L is a (GGGGS)4 sequence, the amino acid sequence of which is shown in SEQ ID NO:3.

[0053] In another preferred embodiment, H is a hinge region selected from the following histones: CD8, CD28, CD137, NKG2D, IgG, or a combination thereof.

[0054] In another preferred embodiment, H is the hinge region of the CD8 protein.

[0055] In another preferred embodiment, the TM is a transmembrane region of a commonly used immune cell surface molecule in the art, preferably a transmembrane region selected from the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, NKG2D, DAP10, DAP12, or a combination thereof.

[0056] In another preferred embodiment, the TM is the transmembrane region of the CD8 protein.

[0057] In another preferred embodiment, the sequence of “H-TM” in Formula I is a sequence composed of the hinge region and the transmembrane region of the CD8 protein, and its amino acid sequence is shown in SEQ ID NO:5.

[0058] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, CD137 (4-1BB), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, DAP10, DAP12, or a combination thereof.

[0059] In another preferred embodiment, C is a co-stimulatory signaling molecule of the CD137(4-1BB) protein.

[0060] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO:6.

[0061] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:7.

[0062] In another preferred embodiment, the amino acid sequence of the BCMA-CD19 bispecific CAR is selected from the group consisting of:

[0063] (i) The amino acid sequence as shown in SEQ ID NO:8;

[0064] (ii) A sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8;

[0065] (iii) A sequence obtained by optionally adding, deleting, modifying and / or substituting at least one (e.g., 1-3) amino acids based on the amino acid sequence shown in SEQ ID NO:8.

[0066] In another preferred embodiment, the amino acid sequence of the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:8.

[0067] In another preferred embodiment, the BCMA-CD19 bispecific CAR encodes RNA (including mRNA) or DNA (including cDNA).

[0068] In another preferred embodiment, the encoding nucleic acid of the BCMA-CD19 bispecific CAR further contains, flanking the ORF of the BCMA-CD19 bispecific CAR, an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0069] In another preferred embodiment, the encoding nucleic acid of the BCMA-CD19 bispecific CAR further includes a promoter operatively linked to the ORF sequence of the BCMA-CD19 bispecific CAR.

[0070] In another preferred embodiment, the promoter is a constitutive promoter or an inducible promoter.

[0071] In another preferred embodiment, the promoter is a strong promoter.

[0072] In another preferred embodiment, the promoter is a tissue-specific promoter.

[0073] In another preferred embodiment, the promoter is selected from the group consisting of: RSV promoter, EF-1α promoter, lac promoter, CMV promoter, U6 promoter, 35S promoter, T7 phage promoter, Ubiquitin promoter, Actin1 promoter, CsVMV promoter, or a combination thereof; preferably, the promoter is RSV promoter, EF-1α promoter, lac promoter, or a combination thereof.

[0074] In another preferred embodiment, the coding nucleic acid sequence of the BCMA-CD19 bispecific CAR is selected from the group consisting of:

[0075] (i) A nucleotide sequence as shown in SEQ ID NO:13;

[0076] (ii) A sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO:13;

[0077] (iii) A sequence obtained by optionally adding, deleting, modifying and / or substituting at least one (e.g., 1-3) nucleotides based on the nucleotide sequence shown in SEQ ID NO:13.

[0078] In another preferred embodiment, the encoding nucleic acid sequence of the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:13.

[0079] In another preferred embodiment, the vector for the BCMA-CD19 bispecific CAR is selected from the group consisting of viral vectors, plasmids, transposons, nanovectors, other gene transfer systems, or combinations thereof.

[0080] In another preferred embodiment, the viral vector is selected from the group consisting of lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0081] In another preferred embodiment, the vector further includes elements selected from the group consisting of: promoters, transcriptional enhancing elements (WPREs), long terminal repeat sequences (LTRs), selectable markers (such as reporter genes or resistance genes), polyA elements, etc., or combinations thereof.

[0082] In another preferred embodiment, the vector is selected from the group consisting of: pCDH vector, pTomo vector, plenti vector, pLVTH vector, pLJM1 vector, pHCMV vector, pLBS.CAG vector, pHR vector, pLV vector, etc.

[0083] In another preferred embodiment, the CAR-immune cells are selected from the group consisting of CAR-T cells, CAR-NK cells, CAR-NKT cells, or combinations thereof.

[0084] In another preferred embodiment, the CAR-immune cell is a CAR-T cell.

[0085] In another preferred embodiment, the CAR-T cells are selected from the group consisting of CAR-αβT cells, CAR-γδT cells, or combinations thereof.

[0086] In another preferred embodiment, the dosage form of the drug is selected from the group consisting of: injections and lyophilized preparations.

[0087] In another preferred embodiment, the drug is an injectable preparation with a strength of 1-100 mL, more preferably 10-50 mL, and even more preferably 20-30 mL.

[0088] In another preferred embodiment, the drug is an injectable preparation in a 20 mL volume.

[0089] In another preferred embodiment, the content of CAR-immune cells in the drug is 1×10⁻⁶. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 cells / mL, preferably 1×10⁻⁶. 5 -1×10 7 cells / mL, for example 2.5 × 10⁻⁶ cells / mL 5 cells / mL, 5×10 5 7.5 × 10⁻⁶ cells / mL 5 cells / mL, 2.5 × 10 6 cells / mL, 5×10 6 7.5 × 10⁻⁶ cells / mL 6 Cells / mL, etc.

[0090] In another preferred embodiment, the dosage of CAR-immune cells in the drug is 1 × 10⁻⁶. 3 -1×10 9 Cells / kg body weight, preferably 1×10⁻⁶ 4 -1×10 8 Cells / kg body weight, preferably 1×10 5 -1×10 7 Cells / kg body weight, e.g., 3×10 5 Cells / kg body weight, 6×10 5 Cells / kg body weight, 1×10 6 Cells / kg body weight, 3×10 6 Cells / kg body weight, 6×10 6 Cells per kg of body weight, etc.

[0091] In another preferred embodiment, the drug further includes other drugs for the prevention and / or treatment of membranous nephropathy.

[0092] In another preferred embodiment, the drug further includes: glucocorticoids, cyclophosphamide, tacrolimus, tripterygium wilfordii, cyclosporine, dapagliflozin, rituximab, methylprednisolone, prednisone, or combinations thereof.

[0093] In another preferred embodiment, the drug further includes components selected from the group consisting of immune checkpoint inhibitors, immunomodulators, chemotherapeutic drugs, lipid metabolism modulators, glucose metabolism modulators, adrenocortical hormones, proteasome inhibitors, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), or combinations thereof.

[0094] In another preferred embodiment, the immune checkpoint is selected from the group consisting of CD20, CD38, PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc., or combinations thereof.

[0095] In another preferred embodiment, the immune checkpoint inhibitor is an anti-CD20 antibody.

[0096] In another preferred embodiment, the anti-CD20 monoclonal antibody is rituximab.

[0097] In another preferred embodiment, the chemotherapeutic agent includes an alkylating agent.

[0098] In another preferred embodiment, the glucose metabolism regulating drug includes glucocorticoids.

[0099] In another preferred embodiment, the proteasome inhibitor comprises bortezomib.

[0100] In another preferred embodiment, the drug further includes a diagnostic reagent for diagnosing membranous nephropathy or determining urinary protein levels and / or PLA2R antibody titers.

[0101] In another preferred embodiment, the drug comprises:

[0102] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0103] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0104] In another preferred embodiment, the drug comprises 0.01 to 99.99% of BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentage is a percentage by mass of the drug.

[0105] In a second aspect of the invention, a method is provided for reducing urinary protein levels, reducing PLA2R antibody titers, increasing serum albumin levels, and / or increasing renal function eGFR levels, the method comprising the steps of: administering BCMA-CD19 bispecific CAR or its encoded nucleic acid, or its vector, or its CAR-immune cells to a desired subject;

[0106] Alternatively, the method includes the step of: administering a pharmaceutical composition to a desired object, the pharmaceutical composition comprising:

[0107] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0108] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0109] In another preferred embodiment, the method is an in vitro method.

[0110] In another preferred embodiment, the method is for non-disease treatment purposes or non-disease diagnosis purposes.

[0111] In another preferred embodiment, the BCMA-CD19 bispecific CAR has the structure shown in Formula I above.

[0112] In another preferred embodiment, the object is a cell.

[0113] In another preferred embodiment, the object is a human or a non-human mammal.

[0114] In another preferred embodiment, the object is untreated membranous nephropathy.

[0115] In another preferred embodiment, the subject is a patient with membranous nephropathy who has undergone treatment (conventional treatment) but has experienced ineffective or inefficient treatment or relapse.

[0116] In another preferred embodiment, the treatment includes, but is not limited to: autologous hematopoietic stem cell transplantation, administration of lipid metabolism regulating drugs, administration of glucose metabolism regulating drugs (such as glucocorticoids), administration of adrenocortical hormones, administration of chemotherapy drugs (such as alkylating agents), administration of immunomodulators (such as immunosuppressants), administration of immune checkpoint inhibitors (such as anti-CD20 monoclonal antibodies), administration of proteasome inhibitors, administration of angiotensin-converting enzyme inhibitors (ACEIs), administration of angiotensin receptor antagonists (ARBs), or combinations thereof.

[0117] In another preferred embodiment, the anti-CD20 monoclonal antibody is rituximab.

[0118] In another preferred embodiment, the treatment is selected from: glucocorticoids, cyclophosphamide, tacrolimus, tripterygium wilfordii, cyclosporine, dapagliflozin, rituximab, methylprednisolone, prednisone, or combinations thereof.

[0119] In another preferred embodiment, the subject is a patient with high-risk, refractory, or relapsed membranous nephropathy.

[0120] In another preferred embodiment, the method includes use in conjunction with other methods.

[0121] In another preferred embodiment, the other methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0122] In another preferred embodiment, the method further includes the steps of: determining the level of urinary protein and / or the PLA2R antibody titer.

[0123] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0124] In another preferred embodiment, the method further includes the step of administering other drugs for treating membranous nephropathy.

[0125] In another preferred embodiment, the method further includes the step of administering other drugs for reducing urinary protein levels, reducing PLA2R antibody titers, increasing serum albumin levels, and / or increasing renal function eGFR levels.

[0126] In a third aspect of the invention, a method for preventing and / or treating membranous nephropathy (MN) is provided, the method comprising the steps of: administering a BCMA-CD19 bispecific CAR or its encoded nucleic acid, or its vector, or its CAR-immune cells to a desired subject;

[0127] Alternatively, the method includes the step of: administering a pharmaceutical composition to a desired object, the pharmaceutical composition comprising:

[0128] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0129] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0130] In another preferred embodiment, the object is a cell.

[0131] In another preferred embodiment, the object is a human or a non-human mammal.

[0132] In another preferred embodiment, the subject is an untreated patient with membranous nephropathy.

[0133] In another preferred embodiment, the subject is a patient with membranous nephropathy who has undergone treatment (conventional treatment) but has experienced ineffective or inefficient treatment or relapse.

[0134] In another preferred embodiment, the treatment includes, but is not limited to: autologous hematopoietic stem cell transplantation, administration of lipid metabolism regulating drugs, administration of glucose metabolism regulating drugs (such as glucocorticoids), administration of adrenocortical hormones, administration of chemotherapy drugs (such as alkylating agents), administration of immunomodulators (such as immunosuppressants), administration of immune checkpoint inhibitors (such as anti-CD20 monoclonal antibodies), administration of proteasome inhibitors, administration of angiotensin-converting enzyme inhibitors (ACEIs), administration of angiotensin receptor antagonists (ARBs), or combinations thereof.

[0135] In another preferred embodiment, the anti-CD20 monoclonal antibody is rituximab.

[0136] In another preferred embodiment, the treatment is selected from: glucocorticoids, cyclophosphamide, tacrolimus, tripterygium wilfordii, cyclosporine, dapagliflozin, rituximab, methylprednisolone, prednisone, or combinations thereof.

[0137] In another preferred embodiment, the subject is a patient with high-risk, refractory, or relapsed membranous nephropathy.

[0138] In another preferred embodiment, the method includes use in conjunction with other methods.

[0139] In another preferred embodiment, the other methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0140] In another preferred embodiment, the method further includes the steps of: determining the level of urinary protein and / or the PLA2R antibody titer.

[0141] In another preferred embodiment, the method further includes the step of diagnosing membranous nephropathy.

[0142] In another preferred embodiment, membranous nephropathy is diagnosed using a method selected from the group consisting of: renal biopsy, PLA2R antibody titer detection, serum creatinine value, eGFR estimation, serum albumin, 24-hour urine protein / creatinine ratio, 24-hour urine protein quantification, tumor screening (excluding secondary factors), or a combination thereof.

[0143] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0144] In another preferred embodiment, the method further includes the step of administering other drugs for the prevention and / or treatment of membranous nephropathy.

[0145] In a fourth aspect of the invention, a kit is provided for (i) reducing urinary protein levels, reducing PLA2R antibody titers, increasing serum albumin levels, and / or increasing renal function eGFR levels; or (ii) preventing and / or treating membranous nephropathy (MN), said kit comprising:

[0146] (Z1) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; or

[0147] (Z2) A pharmaceutical composition comprising:

[0148] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0149] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0150] In another preferred embodiment, the kit further includes other medications for the prevention and / or treatment of membranous nephropathy (MN).

[0151] In another preferred embodiment, the kit further includes: other drugs for reducing urinary protein levels, reducing PLA2R antibody titers, increasing serum albumin levels, and / or increasing renal function eGFR levels.

[0152] In another preferred embodiment, the kit further includes components selected from the group consisting of immune checkpoint inhibitors, immunomodulators, chemotherapeutic drugs, lipid metabolism modulators, glucose metabolism modulators, adrenocortical hormones, proteasome inhibitors, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), or combinations thereof.

[0153] In another preferred embodiment, the immune checkpoint is selected from the group consisting of CD20, CD38, PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc., or combinations thereof.

[0154] In another preferred embodiment, the immune checkpoint inhibitor is an anti-CD20 antibody.

[0155] In another preferred embodiment, the anti-CD20 monoclonal antibody is rituximab.

[0156] In another preferred embodiment, the chemotherapeutic agent includes an alkylating agent.

[0157] In another preferred embodiment, the glucose metabolism regulating drug includes glucocorticoids.

[0158] In another preferred embodiment, the proteasome inhibitor comprises bortezomib.

[0159] In another preferred embodiment, the kit further includes detection reagents for diagnosing membranous nephropathy (MN) or determining urinary protein levels and / or PLA2R antibody titers.

[0160] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0161] Figure 1 The structure of BCMA-CD19 bispecific CAR-T cells was shown.

[0162] Figure 2 The pCDH-CAR plasmid map is displayed.

[0163] Figure 3 The baseline and concomitant medications of two patients (Subject 1 and Subject 2) with a follow-up period of more than 6 months are shown.

[0164] Figure 4 The study showed the efficacy of treatment based on changes in 24-hour urinary protein quantification in two patients (Subject 1 and Subject 2) with a follow-up period of more than 6 months.

[0165] Figure 5 The study showed the efficacy of changes in PLA2R antibody titers in two patients (Subject 1 and Subject 2) with a follow-up period of more than 6 months.

[0166] Figure 6 The study showed the efficacy of serum albumin level changes in two patients (Subject 1 and Subject 2) with a follow-up period of more than 6 months.

[0167] Figure 7 The study showed the efficacy of changes in renal function eGFR in two patients (Subject 1 and Subject 2) with a follow-up period of more than 6 months. Detailed Implementation

[0168] Through extensive and in-depth research and screening, the inventors have made a groundbreaking discovery for the first time: dual-target CAR-T cells simultaneously targeting CD19 and BCMA can effectively reduce 24-hour urinary protein levels, significantly decrease PLA2R antibody titers until PLA2R antibody becomes negative, and are accompanied by a sustained increase in serum albumin, complete improvement of hypoalbuminemia, and sustained improvement in renal function eGFR, thereby achieving clinical improvement and remission in patients, with excellent safety and efficacy. Furthermore, the BCMA-CD19 bispecific CAR-T cells of this invention can achieve very good clinical remission in subjects who have responded poorly, have low response, or have relapsed to conventional treatments (such as glucocorticoids, cyclophosphamide, tacrolimus, tripterygium wilfordii, cyclosporine, dapagliflozin, rituximab, etc., and combinations thereof). This invention was completed based on these findings.

[0169] the term

[0170] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0171] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0172] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0173] As used in this article, the term "24-hour urinary protein quantification" refers to the 24-hour urinary protein excretion rate. It involves collecting all urine over 24 hours to measure the protein content and calculate the total protein amount over 24 hours. Normal urine contains very little protein, with normal levels below 150 mg / 24h. In kidney disease and certain physiological conditions such as strenuous exercise, urinary protein levels can increase significantly. Dynamic assessment of 24-hour urinary protein quantification is an important indicator for evaluating the severity and remission of myocardial infarction (MN).

[0174] As used in this article, the term "PLA2R" refers to the M-type phospholipase A2 receptor (PLA2R), which is the pathogenic target antigen of membranous nephropathy (IMN). PLA2R antibodies can be detected in the peripheral blood of glomerular podocyte membranes in 70%–80% of IMN patients. Clinically, PLA2R antibody titers are widely used to monitor disease progression, treatment response, and renal prognosis in membranous nephropathy.

[0175] As used in this article, the term "eGFR" refers to estimated glomerular filtration rate. For patients diagnosed with MN, assessing their eGFR can determine the degree of renal impairment and the stage of chronic renal failure. eGFR level is also one of the indicators of MN disease remission.

[0176] As used in this article, the term "serum albumin" refers to the most common plasma protein produced by hepatocytes, which plays an important role in maintaining osmotic pressure in the human body. Patients with membranous nephropathy (MN) clinically present with nephrotic syndrome, characterized by massive proteinuria, hypoalbuminemia, and severe edema. Hypoalbuminemia is one of the important clinical manifestations, and similarly, in the assessment of clinical remission of MN, the reduction in serum hypoalbuminemia along with decreasing proteinuria is an important assessment indicator.

[0177] As used in this article, the term "ECOG" stands for Eastern Cooperative Oncology Group. The ECOG score, or ECOG scoring standard, is an indicator of a patient's general health status and tolerance to treatment based on their physical capabilities. It is divided into six levels, from 0 to 5. Level 0 indicates that the patient's activity level is completely normal, the same as before the onset of the disease, without any limitations; Level 1 means that the patient can move freely and can perform general light physical labor, but cannot perform heavy physical labor; Level 2 means that the patient can walk freely, but can no longer perform any labor activities; Level 3 means that the patient can partially take care of themselves, mainly using a wheelchair or being bedridden; Level 4 means that the patient is bedridden and unable to take care of themselves; Level 5 means that the patient has died.

[0178] As used in this article, the term "ALT" refers to Alanine Transaminase, which is mainly found in liver cells and is an important indicator of liver disease. When the liver is damaged, a large amount of ALT is released into the bloodstream, leading to elevated blood ALT levels. Therefore, detecting blood ALT levels can be used to diagnose liver diseases such as hepatitis and cirrhosis.

[0179] As used in this article, the term "AST" refers to Aspartate Transaminase, which is widely present in many tissues of the body, such as the liver, heart, and muscles. When these tissues are damaged, AST is released into the bloodstream. Therefore, AST testing can be used to diagnose liver diseases, as well as heart and muscle diseases.

[0180] As used in this article, the term "INR" refers to the International Normalized Ratio, which is a value derived from prothrombin time and the international sensitivity index of the assay reagent. Clinically, this indicator is often used to measure a patient's coagulation function.

[0181] As used in this article, the term "APTT" refers to activated partial thromboplastin time. The APTT test is the most commonly used sensitive screening test in clinical practice to reflect the coagulation activity of the intrinsic coagulation system. APTT is an indicator used in clinical practice to reflect coagulation function.

[0182] As used in this article, the term "CTCAE" refers to the Common Terminology Criteria for Adverse Events, which classifies each adverse event into severity levels from 1 to 5.

[0183] Membranous nephropathy (MN)

[0184] Membranous nephropathy (MN) is an antibody-mediated autoimmune disease. Autoantigens such as PLA2R located on podocytes are presented to T cells and B cells, activating an immune response that produces autoantibodies. These autoantibodies form immune complexes that deposit on glomerular epithelial cells, leading to the formation of membranous attack complexes, which increase basement membrane permeability and damage podocytes. Based on etiology, it can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy. Epidemiological surveys show that the former accounts for approximately 80% and the latter approximately 20%. Clinical manifestations often include massive proteinuria, hypoalbuminemia, severe edema, hyperlipidemia, or asymptomatic presentation. The pathological features of idiopathic membranous nephropathy include subepithelial immune complex deposition in the glomerular capillary loops and diffuse thickening and spike formation of the basement membrane. It is one of the most common primary glomerular diseases, and its incidence has been rising rapidly in the past decade, from 15% to 30%–40%. Approximately one-third of patients progress to end-stage renal disease.

[0185] Currently, the main medications for treating myasthenia gravis (MN) include glucocorticoids, immunosuppressants, biologics, and ACEIs / ARBs. Immunosuppression is a crucial method in MN treatment. Extensive evidence demonstrates that hormones alone are ineffective in treating MN patients; combination with immunosuppressants is necessary for a certain level of efficacy. The classic clinical treatment regimen is immunosuppressant combined with hormones, such as cyclophosphamide combined with methylprednisolone, or cyclosporine A combined with low-dose prednisone. However, long-term use of immunosuppressants can lead to adverse reactions such as infection, liver damage, bone marrow suppression, gastrointestinal reactions, and menstrual disorders. Furthermore, 20-50% of MN patients experience relapse within one year after discontinuing the medication. For many years, there has been a lack of approved innovative treatments for MN; currently, rituximab, a commonly used drug, is used off-label. Therefore, more effective treatment methods are still needed clinically for MN.

[0186] Inclusion criteria for subjects with membranous nephropathy

[0187] Subjects must meet all inclusion criteria and not meet any exclusion criteria to be enrolled:

[0188] (1) Subjects must personally sign an informed consent form approved by the ethics committee before the start of the study;

[0189] (2) The subjects were ≥18 years old and <70 years old;

[0190] (3) Tissue biopsy diagnosis of aPLA2R-related membranous nephropathy and clinical criteria for high-risk or relapsed / refractory membranous nephropathy:

[0191] 1) High-risk membranous nephropathy patients:

[0192] High-risk is defined as meeting any of the following criteria: ① normal eGFR, urine protein > 3.5 g / d, ACEI / ARB treatment for 6 months with a decrease in urine protein < 50%, combined with serum albumin < 25 g / L or aPLA2R.

[0193] >50RU / ml; ②eGFR<60ml / min / 1.73m 2 And / or urine protein > 8g / d for more than 6 months;

[0194] 2) Patients with refractory / relapsed membranous nephropathy:

[0195] Refractory is defined as: resistance to previous immunosuppressant therapy (persistent proteinuria ≥3.5g / d and a decrease of <50% compared to pre-treatment levels);

[0196] Relapse is defined as: a recurrence of proteinuria ≥3.5g / d after previous immunosuppressant therapy achieved complete or partial remission;

[0197] 3) eGFR ≥ 45 ml / min / 1.73 m 2 ;

[0198] (4) Expected survival ≥ 12 weeks;

[0199] (5) ECOG score ≤ 2 points;

[0200] (6) Female subjects of childbearing age should agree to use effective contraception from the date of signing the informed consent form until 365 days after reinfusion. Effective contraception is defined as: abstinence or using a contraceptive method with an annual failure rate of <1% as specified in the protocol;

[0201] (7) Subjects must have adequate organ function and meet all of the following examination results before enrollment:

[0202] a) Absolute neutrophil count ≥ 1.0 × 10⁻⁶ 9 / L[Allows use of granulocyte colony-stimulating factor (G-)]

[0203] CSF support is required, but the individual must not have received supportive treatment within 7 days prior to the examination.

[0204] b) Platelet count ≥50×10 9 / L [You must not have received any blood transfusions (including component transfusions) or treatments aimed at increasing platelet counts, such as thrombopoietin (TPO), within 7 days prior to the examination.]

[0205] [Treatment];

[0206] c) Hemoglobin ≥ 8.5 g / dl [must not have received blood transfusion support (including component transfusion) within 7 days prior to the test];

[0207] d) Bilirubin level ≤ 1.5 × upper limit of normal (ULN) (excluding bile duct obstruction caused by tumor compression)

[0208] outside);

[0209] e) ALT or AST ≤ 2.5 × upper limit of normal (ULN) (≤ 5 times the upper limit of normal for those with liver involvement);

[0210] f) Echocardiography results showed a cardiac ejection fraction ≥50% and no obvious pericardial effusion;

[0211] g) Stable coagulation function: INR≤1.5, APTT≤1.2×Upper limit of normal (ULN) (except for tumor-related anticoagulation therapy);

[0212] h) Baseline blood oxygen saturation >95% in indoor natural air environment.

[0213] Exclusion criteria for subjects with membranous nephropathy

[0214] Subjects meeting any of the following criteria will be excluded:

[0215] 1. Subjects who have received the following prior treatments:

[0216] 1.1 Individuals who had received gene therapy prior to enrollment;

[0217] 1.2 Subjects who received a live vaccine within 4 weeks prior to enrollment;

[0218] 1.3 Patients who received other interventional clinical trial drug treatments within 12 weeks prior to apheresis;

[0219] 2. Having an active malignant tumor within the past 5 years, unless it is a curable tumor that has been clearly cured, such as basal or squamous cell carcinoma, cervical or breast carcinoma in situ, etc.

[0220] 3. Subjects with positive hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (HBcAb) and abnormal peripheral blood HBV DNA test (abnormal HBV DNA test is defined as: quantitative HBV DNA test higher than the lower limit of detection of the testing center or higher than the normal reference range of the testing center or positive qualitative HBV DNA test); subjects with positive hepatitis C virus (HCV) antibody and positive peripheral blood hepatitis C virus (HCV) RNA; subjects with positive human immunodeficiency virus (HIV) antibody; subjects with positive cytomegalovirus (CMV) DNA test; subjects with positive RPR for syphilis detection;

[0221] 4. Presence of uncontrollable active infections (except <CTCAE> grade 2 genitourinary and upper respiratory tract infections);

[0222] 5. Severe heart diseases: including but not limited to unstable angina, myocardial infarction (within 6 months before screening), congestive heart failure (New York Heart Association [NYHA] classification ≥ III), severe arrhythmia;

[0223] 6. Subjects with hypertension or diabetes that cannot be controlled by drug treatment;

[0224] 7. Previous treatment toxic reactions not resolved to baseline or ≤ grade 1 (except for hair loss and laboratory test abnormalities without clinical significance, according to NCI-CTCAE v5.0);

[0225] 8. Major surgery within 2 weeks before enrollment, or planned surgery during the waiting period for reinfusion or within 12 weeks after receiving study treatment (except for planned local anesthesia surgery);

[0226] 9. Subjects with solid organ transplantation;

[0227] 10. Pregnant or lactating women;

[0228] 11. Subjects with previous central nervous system diseases (such as cerebral aneurysm, epilepsy, stroke, Alzheimer's disease, psychosis, etc.) or conscious disorders;

[0229] 12. Other systematically diseases judged by the investigator as unstable: including but not limited to severe liver, kidney, gastrointestinal or metabolic diseases that require drug treatment;

[0230] 13. Known life-threatening allergic reactions, hypersensitivity reactions or intolerance to BCMA-CD19 bispecific CAR-T cell preparation or its components;

[0231] 14. Patients judged by researchers to have bleeding, severe thrombosis, or have hereditary / acquired bleeding and severe thrombosis (including hemophilia, coagulation disorders, thrombocytopenia, hypersplenism, etc.), or who are currently receiving thrombolytic or anticoagulant therapy;

[0232] 15.6 months after receiving any B-cell depletion therapy or non-depletion B-cell targeted therapy;

[0233] 16. Has received methylprednisolone pulse therapy (cumulative dose >1.5g) or CTX pulse therapy within the past month;

[0234] 17. The investigator determined that the patient's condition did not allow for the discontinuation of other immunosuppressants one week prior to the apheresis, or that the patient had received treatment with more than 5 mg / day of prednisone (or an equivalent dose of other corticosteroids);

[0235] 18. Patients with membranous nephropathy who have used immunosuppressants (including CsA / CNI, cyclophosphamide) within 3 months prior to enrollment;

[0236] 19. Other situations deemed unsuitable for enrollment by the researchers.

[0237] Single-sample standard

[0238] (1) No infection requiring systemic anti-infective treatment prior to single-donor apheresis;

[0239] (2) Within one day prior to apheresis, a routine blood test showed a neutrophil count ≥1.0 × 10⁶. 9 / L [Granocyte colony-stimulating factor (G-CSF) support is permitted, but no supportive therapy must have been received within 7 days prior to the examination], lymphocyte count ≥0.3×10 9 / L, or as determined by the researcher, the number of mononuclear cells in the peripheral blood of the subject meets the requirements for CAR-T cell production;

[0240] (3) Platelet count ≥75×10⁶ within 1 day prior to apheresis. 9 / L [You must not have received blood transfusion support [including component blood transfusion] or treatment aimed at increasing platelets, such as thrombopoietin (TPO), within 7 days prior to the examination]; fibrinogen ≥1.0g / L; activated partial thromboplastin time ≤1.5×ULN; prothrombin time (PT) ≤1.5×ULN;

[0241] (4) Drug elution before single apheresis:

[0242] (4.1) No systemic use of therapeutic doses of hormones within one week prior to apheresis, but use of hormones within the physiological replacement therapy range (≤12 mg / m²) is permitted. 2 / day of hydrocortisone or other hormones within the same dose range after equivalent dose conversion) and topical and inhaled hormones;

[0243] (4.2) No immunosuppressive drugs (including monoclonal antibodies and small molecule drugs) were used within 2 weeks prior to apheresis, except for topical medications;

[0244] (4.3) No cytotoxic drugs, including low-dose maintenance chemotherapy or other drugs that may inhibit cell proliferation, were used 2 weeks prior to apheresis;

[0245] (4.4) No bevacizumab treatment was used within 20 days prior to apheresis;

[0246] (4.5) No other interventional clinical trial drug treatments were received within 12 weeks prior to apheresis;

[0247] (5) There are no other circumstances that researchers have determined are unsuitable for single-sample collection.

[0248] Shower pretreatment standards

[0249] (1) No infection requiring systemic anti-infective treatment prior to scrubbing;

[0250] (2) Within 7 days prior to administration of the medication to the subject, the subject's laboratory test results must meet the following conditions:

[0251] (2.1) Echocardiographic diagnosis of left ventricular ejection fraction (LVEF) ≥ 50%;

[0252] (2.2) Basal blood oxygen saturation >95% in indoor natural air environment;

[0253] (3) After the researchers assess blood routine, liver and kidney function, and coagulation function, the patient is able to undergo urinary tract cleansing.

[0254] (4) There are no other circumstances that researchers have determined are unsuitable for administering Qinglin medication.

[0255] Cell reinfusion standards

[0256] (1) No systemic anti-infective treatment is required before reinfusion;

[0257] (2) Blood oxygen saturation > 95%;

[0258] (3) >24 hours after rinsing pretreatment;

[0259] (4) Drug elution before reinfusion of BCMA-CD19 bispecific CAR-T cell injection:

[0260] (4.1) No therapeutic dose of hormones was used within 72 hours prior to infusion; use of hormones within the physiological replacement therapy dose range (≤12 mg / m²) is permitted. 2 / day of hydrocortisone or other hormones within the same dose range after equivalent dose conversion);

[0261] (4.2) No other immunosuppressive drugs (including monoclonal antibodies and small molecule drugs) were used in the two weeks prior to the infusion;

[0262] (4.3) Within 2 weeks prior to reinfusion or within 5 half-lives of the bridging therapy drug (whichever is shorter), the patient has not received chemotherapy (except for lymph node pretreatment) or radiotherapy;

[0263] (4.4) No PD-L1 / PD-1 or other immune checkpoint inhibitors were used within 8 weeks prior to reinfusion;

[0264] (5) There are no other circumstances that researchers have determined are unsuitable for cell reinfusion.

[0265] Relief Standard

[0266] MN treatment response assessment:

[0267] Complete remission (CR) is defined as: 24-hour proteinuria <0.5g / d; creatinine is normal or rises by no more than 10% from baseline;

[0268] Partial remission (PR) is defined as: a decrease in urinary protein of more than 50% and urinary protein quantification <3.5g / d; creatinine is normal or increases by no more than 10% from baseline;

[0269] Overall remission (OR) is defined as either complete remission or partial remission.

[0270] Relapse is defined as the recurrence of proteinuria ≥3.5g / d after achieving complete or partial remission.

[0271] In 2009, the M-type phospholipase A2 receptor (PLA2R) was first reported internationally as a pathogenic target antigen of membranous nephropathy (IMN), and PLA2R antibodies were detectable in the peripheral blood of glomerular podocyte membranes in 70%–80% of IMN patients. Studies have shown that PLA2R antibody titers are associated with disease progression, treatment response, and renal prognosis in membranous nephropathy. The 2021 Kidney Diseases: Improving Global Outcomes (KDIGO) guidelines recommend longitudinal monitoring of PLA2R antibody levels 6 months after the start of treatment to assess treatment response in IMN patients and to guide adjustments to treatment.

[0272] Drug / Drug Composition

[0273] As used herein, the present invention also provides the use of a medicament or pharmaceutical composition. The medicament or pharmaceutical composition typically contains a BCMA-CD19 bispecific CAR or its encoding nucleic acid, or a carrier thereof, or a CAR-immune cell thereof, and pharmaceutically acceptable carriers, diluents, or excipients. These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0274] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of a BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its carrier, or its CAR-immune cells (active ingredient), as well as pharmaceutically acceptable carriers, diluents, or excipients. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its carrier, or its CAR-immune cells, and the pharmaceutical compositions of the present invention can also be used with other therapeutic agents.

[0275] In a preferred embodiment, the drug / drug composition / drug formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The drug / drug composition / drug formulation is preferably formulated for intravenous administration.

[0276] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals, wherein this safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0277] The pharmaceutical composition may include 0.01 to 99.99% of BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable vector, wherein the percentages are percentages by mass of the pharmaceutical composition.

[0278] Preferably, the content of CAR-immune cells in the pharmaceutical composition is 1×10⁻⁶. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 cells / mL, preferably 1×10⁻⁶. 5 -1×10 7 cells / mL, for example 2.5 × 10⁻⁶ cells / mL 5 cells / mL, 5×10 5 7.5 × 10⁻⁶ cells / mL 5 cells / mL, 2.5 × 10 6 cells / mL, 5×10 6 7.5 × 10⁻⁶ cells / mL 6 Cells / mL, etc. Alternatively, the dosage of CAR-immune cells in the pharmaceutical composition is 1×10⁻⁶. 3 -1×10 9 Cells / kg body weight, preferably 1×10⁻⁶ 4 -1×10 8 Cells / kg body weight, preferably 1×10 5 -1×10 7 Cells / kg body weight, e.g., 3×10 5 Cells / kg body weight, 1×10 6 Cells / kg body weight, 3×10 6 Cells per kg of body weight, etc.

[0279] Therapeutic applications

[0280] In one embodiment, the present invention includes a type of cell therapy in which autologous T cells (or xenogeneic donor cells) of a subject or patient are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This approach results in an extremely low probability of graft-versus-host disease, and the antigens are recognized by the T cells in an MHC-free manner. In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and persist for months to years.

[0281] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may comprise CAR-T cells as described herein combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0282] The pharmaceutical compositions of the present invention can be administered in a manner suitable for treating (or preventing) the disease. The amount and frequency of administration will be determined by factors such as the condition of the subject / patient, the type and severity of the subject / patient's disease, or may be determined by clinical trials.

[0283] When dosages such as "pharmaceuticalally effective amount" or "therapeutic amount" are specified, the precise amount of the drug / pharmaceutical composition of the present invention to be administered can be determined by a physician, taking into account the subject's / patient's (object's) age, weight, individual differences in disease condition, PLA2R antibody level, etc. Pharmaceutical compositions including the CAR-immune cells (preferably CAR-T cells) described herein can be administered at a dose of 1 × 10⁻⁶. 3 -1×10 9 Cells / kg body weight, preferably 1×10⁻⁶ 4 -1×10 8 Cells / kg body weight, preferably 1×10 5 -1×10 7 Cells / kg body weight, e.g., 3×10 5 Cells / kg body weight, 1×10 6 Cells / kg body weight, 3×10 6 Cells / kg body weight, 6×10 6 Administered at a dose of cells / kg body weight. Alternatively, the CAR-immune cell content in the pharmaceutical composition of CAR-immune cells (preferably CAR-T cells) described herein may be 1×10⁻⁶ cells / kg body weight. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 cells / mL, preferably 1×10⁻⁶. 5 -1×10 7 cells / mL, for example 2.5 × 10⁻⁶ cells / mL 5 cells / mL, 5×10 5 7.5 × 10⁻⁶ cells / mL 5 cells / mL, 2.5 × 10 6 cells / mL, 5×10 6 7.5 × 10⁻⁶ cells / mL 6Cells / mL, etc. T-cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific subject / patient can be easily determined by medical professionals by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0284] Administration to subjects / patients / objects can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The drugs / drug compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the CAR-T cells of the present invention or the drug composition thereof are administered to a patient via intradermal or subcutaneous injection. In another embodiment, the CAR-T cells of the present invention or the drug composition thereof are preferably administered via intravenous injection.

[0285] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to a therapeutic level can be administered to a patient in conjunction with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as immunosuppressants like glucocorticoids, immunomodulators, cytotoxic drugs, and monoclonal antibodies including but not limited to CD20 monoclonal antibodies. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0286] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 3 -1×10 9 Cells / kg body weight, preferably 1×10⁻⁶ 4 -1×10 8 Cells / kg body weight, preferably 1×10 5 -1×10 7 Cells / kg body weight, e.g., 3×10 5 Cells / kg body weight, 1×10 6 Cells / kg body weight, 3×10 6 Cells / kg body weight, 6×10 6The CAR-T cells of the present invention, at a dose equal to 1 cell / kg body weight, are administered to the patient via, for example, intravenous infusion. Alternatively, the CAR-T cell content may be 1×10⁻⁶ cells / kg body weight. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 cells / mL, preferably 1×10⁻⁶. 5 -1×10 7 cells / mL, for example 2.5 × 10⁻⁶ cells / mL 5 cells / mL, 5×10 5 7.5 × 10⁻⁶ cells / mL 5 cells / mL, 2.5 × 10 6 cells / mL, 5×10 6 7.5 × 10⁻⁶ cells / mL 6 Cells / mL, etc.

[0287] Main advantages of the invention

[0288] This invention innovatively develops the preparation of BCMA-CD19 bispecific CAR-T cells and applies them to high-risk, relapsed, or refractory membranous nephropathy patients in high demand for improved treatment efficacy, achieving good results. After receiving the BCMA-CD19 bispecific CAR-T cell injection solution of this invention, patients experienced a sustained decrease in 24-hour urinary protein quantification, a significant reduction and seroconversion of PLA2 antibody titers, a sustained increase in serum albumin, complete improvement in hypoalbuminemia, and continuous improvement in renal function (eGFR), demonstrating excellent clinical improvement and remission in multiple aspects.

[0289] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0290] Sequence information

[0291] CD8 signal peptide sequence (SEQ ID NO:1):

[0292] MALPVTALLLPLALLLHAARP

[0293] BCMA scFv (SEQ ID NO:2):

[0294] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK

[0295] (GGGGS)4 sequence (SEQ ID NO:3):

[0296] GGGGSGGGGSGGGGSGGGGS

[0297] CD19 scFv (SEQ ID NO:4):

[0298] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0299] CD8 hinge region + CD8 transmembrane domain sequence (SEQ ID NO:5):

[0300] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0301] CD137 (4 - 1BB) co - stimulatory signaling molecule sequence (SEQ ID NO:6):

[0302] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0303] CD3ζ sequence (SEQ ID NO:7):

[0304] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0305] BCMA-CD19 bispecific CAR sequence (SEQ ID NO:8):

[0306] MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0307] Heavy chain variable region sequence of BCMA scFv (SEQ ID NO:9):

[0308] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS

[0309] The light chain variable region sequence of BCMA scFv (SEQ ID NO:10):

[0310] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK

[0311] The heavy chain variable region sequence of CD19 scFv (SEQ ID NO:11):

[0312] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0313] The light chain variable region sequence of CD19 scFv (SEQ ID NO:12):

[0314] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0315] The coding sequence of BCMA-CD19 bispecific CAR (SEQ ID NO:13):

[0316]

[0317] Example 1: Preparation of BCMA-CD19 bispecific CAR-T cells

[0318] BCMA-CD19 bispecific CAR-T cells were constructed and a BCMA-CD19 bispecific CAR-T cell injection solution was prepared using lentiviral infection of T cells. The BCMA-CD19 bispecific CAR-T cell injection solution is a gene-modified autologous T cell (CAR-T cell) immunotherapy targeting BCMA and CD19. This injection product strictly adheres to the "Technical Guidelines for Research and Evaluation of Cell Therapy Products (Trial)," "Self-Discipline Standards for Quality Management of Immunotherapy Cell Preparations," and "Quality Management Standards for CAR-T Cell Preparations (Draft for Comments)," among other standards and guidelines. Quality control and management are implemented for aspects such as cell number, cell viability, cell phenotype, functional molecule expression, cytokine secretion function, in vitro killing function, pH value, osmotic pressure, endotoxin, rapid aseptic testing, bacterial and fungal culture testing, mycoplasma culture testing, harmful residue testing, and cell preparation stability.

[0319] BCMA-CD19 bispecific CAR-T cells consist of an antigen-binding region, an extracellular region, a transmembrane region, and an intracellular signaling region that can activate T cells after binding to the antigen. BCMA-CD19 bispecific CAR-T cells are derived from the patient's autologous PBMCs and transduced using lentiviruses produced by a four-plasmid viral packaging system. The pCDH-CAR plasmid is used as the expression vector to encode a chimeric antigen receptor (CAR) targeting the B cell maturation antigen BCMA and the B lymphocyte surface marker CD19.

[0320] The pCDH-CAR vector is based on the HIV lentiviral vector backbone, including a 5' long terminal repeat (LTR), promoter, packaging signal; and (anti-BCMA and anti-CD19) CAR sequences, including a signal peptide, heavy chain variable region (VH), linker peptide, light chain variable region (VL), co-stimulatory domain, and CD3ζ cytoplasmic region, and a 3' LTR. The CAR-T cell structure is as follows: Figure 1 As shown, the pCDH-CAR plasmid map is as follows: Figure 2 As shown.

[0321] Example 2: Clinical trial in high-risk and refractory / relapsed membranous nephropathy subjects

[0322] In clinical trials, the inclusion criteria, exclusion criteria, apheresis criteria, lymphocyte pretreatment criteria, cell reinfusion criteria, and remission criteria for high-risk and refractory / relapsed membranous nephropathy subjects are as described above.

[0323] The clinical trial process includes: screening period, PBMC apheresis, pre-swallowing examination, pre-swallowing treatment, reinfusion of BCMA-CD19 bispecific CAR-T cell injection (with the day of reinfusion as D0), and primary follow-up period.

[0324] 1. Screening period (W-8~D-30):

[0325] Participants must personally sign an informed consent form in writing before commencing any study-related procedures and assessments. Participants who meet the inclusion criteria but not the exclusion criteria will be enrolled and assigned an enrollment number.

[0326] 2. PBMC single collection (D-30~D-18):

[0327] BCMA-CD19 bispecific CAR-T cells were prepared from mononuclear cells extracted from peripheral blood of the subjects.

[0328] 3. Pre-examination examination (7 days prior to exfoliation):

[0329] Subjects will be evaluated within 7 days prior to the pretreatment of the lymph node, including imaging examinations, disease marker examinations, and memory pain assessments. They will also undergo safety assessments, laboratory tests (complete blood count, blood biochemistry, coagulation function, urinalysis, etc.), and tests for inflammatory factors, ADA, and RCL.

[0330] 4. Shower pretreatment (D-7~D-2 for 3 consecutive days):

[0331] 7 to 2 days prior to BCMA-CD19 bispecific CAR-T cell infusion (day 0), subjects will receive lymph node pretreatment with the "fludarabine + cyclophosphamide" (FC) regimen, as follows:

[0332] Cyclophosphamide 300mg / m 2 / d, intravenous drip, once a day, for three consecutive days.

[0333] Fludarabine 30mg / m 2 / d, intravenous drip, once a day, for three consecutive days (infused on the same day as cyclophosphamide infusion).

[0334] 5. BCMA-CD19 bispecific CAR-T cell injection reinfusion (D0):

[0335] The infusion of BCMA-CD19 bispecific CAR-T cell injection must be performed at least 24 hours after the completion of chemotherapy pretreatment. According to the assigned dose group, the designated dose of BCMA-CD19 bispecific CAR-T cell injection is administered in a single infusion on day 0. Administration is by intravenous injection, completed within 30 minutes (single intravenous injection). If an adverse event occurs before reinfusion of BCMA-CD19 bispecific CAR-T cell injection after pretreatment, the investigator determines that the infusion should be paused, and the cell infusion time may be postponed until the AE has subsided to a state suitable for infusion. However, the investigator should reassess whether reinfusion is necessary after a second pretreatment with chemotherapy. The preferred infusion dose of BCMA-CD19 bispecific CAR-T cell injection is 1 × 10⁻⁶. 5 -1×10 7 Cells / kg body weight, e.g., 3×10 5 Cells / kg body weight, 6×10 5 Cells / kg body weight, 1×10 6 Cells / kg body weight, 3×10 6 Cells / kg body weight, 6×10 6 Cells per kg of body weight, etc.

[0336] 6. Master follow-up period (from cell reinfusion until meeting the exit criteria for master follow-up or completing the 2-year master follow-up visit [D720]):

[0337] The primary follow-up period is from the time the patient receives BCMA-CD19 bispecific CAR-T cell infusion until the criteria for exiting the primary follow-up are met. Efficacy assessments are conducted during the primary follow-up period at 28, 60, and 90 days, and then every 90 days until the end of the primary follow-up period.

[0338] The clinical trial results are summarized in the table below (the following are follow-up results up to January 2025; the complete clinical trial results are not shown due to space limitations):

[0339]

[0340] Clinical trial results show that BCMA-CD19 bispecific CAR-T cell injection has excellent clinical therapeutic effects on relapsed and refractory membranous nephropathy.

[0341] Of the 5 subjects, 2 patients who were followed up for more than 6 months achieved clinical remission. One of them (subject 2) achieved negative PLA2R antibody and clinical remission 1 month after infusion, and achieved complete remission (CR) after 6 months of follow-up.

[0342] Three patients (subjects 3, 4, and 5) are still under follow-up during the safe period. Two of them (subjects 3 and 4) have achieved PLA2R antibody negativity. Subject 5's PLA2R antibody titer decreased by more than 40% from baseline on day 14.

[0343] The baseline and concomitant medication information of the two patients (Subject 1 and Subject 2) with a follow-up period of more than 6 months are summarized as follows: Figure 3 It is evident that all patients were subjects with refractory membranous nephropathy who had not responded to multiple clinical treatments.

[0344] The efficacy outcomes of the two patients (i.e., Subject 1 and Subject 2) with a follow-up period of more than 6 months are summarized as follows: Figures 4-7 As shown:

[0345] Both patients showed a sustained decrease in 24-hour urinary protein levels and were in clinical remission. The second patient was assessed as having complete clinical remission at 6 months of follow-up (complete remission is defined as 24-hour urinary protein < 0.5 g / 24h). Figure 4 ).

[0346] Within 30 days after infusion, the PLA2R antibody titers in both patients became negative (PLA2R antibody titer reference range: 0-20 U / ml), indicating complete immunological remission. Figure 5 ).

[0347] In two patients, serum albumin levels remained elevated, gradually returning to the normal range of 35-55 g / L. Figure 6 ).

[0348] In both patients, the eGFR (elective Glycofiltration rate) of the kidneys continued to improve, gradually approaching the normal reference value for eGFR: >90 ml / min. Figure 7 ).

[0349] The above results show that with complete immunological remission, 24-hour urinary protein quantification continued to decrease, serum albumin continued to increase, hypoalbuminemia was completely improved, and renal function eGFR continued to improve.

[0350] In addition, the results of PLA2R antibody titer and 24-hour urinary protein quantification used as the primary diagnostic criteria during the clinical trials are summarized in the table below (as of December 2024):

[0351]

[0352] The above results indicate that BCMA-CD19 bispecific CAR-T cell injection can effectively reduce 24-hour urinary protein quantification and significantly reduce PLA2R antibody titer to seroconversion in patients with relapsed or refractory membranous nephropathy, thereby alleviating related clinical symptoms.

[0353] The results of Example 2 demonstrate that BCMA-CD19 bispecific CAR-T cell injection can achieve very good therapeutic effects in subjects who have failed conventional treatment, relapsed, or are refractory to treatment.

[0354] During the clinical trial, pharmacokinetic (PK), pharmacodynamic (PD), and immunogenicity studies were also conducted (the complete results of the clinical trial are not shown in full due to space limitations). The results all confirmed the safety and efficacy of BCMA-CD19 bispecific CAR-T cell injection.

[0355] discuss

[0356] For many years, there has been a lack of approved innovative treatments for membranous nephropathy (MN), especially for relapsed or refractory MN. Currently, rituximab, a commonly used drug, is used off-label. Therefore, more effective treatments are still needed in the clinical treatment of MN.

[0357] CAR-T products targeting CD19 and BCMA have already received FDA approval for the treatment of B-ALL, B-NHL, and multiple myeloma (MM). However, there are no reports of CAR-T products targeting CD19 and BCMA being effective in treating MN.

[0358] In this invention, the inventors have creatively discovered that the developed BCMA-CD19 bispecific CAR-T cell injection has excellent clinical efficacy in treating relapsed / refractory membranous nephropathy. Preliminary clinical results in 5 patients showed that the BCMA-CD19 bispecific CAR-T cell injection effectively reduced 24-hour urinary protein levels, significantly reduced and seroconverted PLA2 antibodies, and alleviated related clinical symptoms. Among the 5 subjects who had failed multiple treatments or relapsed, 2 patients with a follow-up period exceeding 6 months achieved clinical remission. One of these patients achieved PLA2R antibody seroconversion with clinical remission one month after infusion, and achieved complete remission (CR) after 6 months of follow-up. Three patients are still under follow-up during the safety period; 2 of these patients have achieved PLA2R antibody seroconversion, and one patient's PLA2R antibody titer decreased by more than 40% from baseline on day 14. Furthermore, after receiving the BCMA-CD19 bispecific CAR-T cell injection of the present invention, the patient's 24-hour urinary protein quantification continued to decrease, serum albumin continued to increase, hypoalbuminemia was completely improved, and renal function eGFR continued to improve, showing good clinical improvement and clinical remission effects in many aspects.

[0359] Therefore, the BCMA-CD19 bispecific CAR-T cells and their developed drugs of the present invention have broad application prospects in the treatment of relapsed and refractory membranous nephropathy (MN).

[0360] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a BCMA-CD19 bispecific CAR or its encoded nucleic acid, or its vector, or its CAR-immune cells, characterized in that, Used for: (i) To prepare drugs for the prevention and / or treatment of membranous nephropathy (MN); (ii) Prepare drugs to reduce urinary protein levels; (iii) Prepare drugs to reduce PLA2R antibody titers; (iv) Preparation of drugs to increase serum albumin levels; and / or (v) Prepare drugs that improve renal function eGFR levels.

2. The use as described in claim 1, characterized in that, The membranous nephropathy referred to is either idiopathic membranous nephropathy (IMN) or secondary membranous nephropathy.

3. The use as described in claim 2, characterized in that, The secondary membranous nephropathy is caused by autoimmune diseases, infections, tumors, drugs or toxins, or a combination thereof.

4. The use as described in claim 1, characterized in that, The BCMA-CD19 bispecific CAR has the structure shown in Formula I: SP1-B1-L-SP2-B2-H-TM-C-CD3ζ(I) In the formula, SP1 and SP2 are each independently either signal peptide sequences or have no signal peptide sequence. B1 and B2 are the first binding element for targeting the first target protein and the second binding element for targeting the second target protein, respectively. L represents a flexible peptide; H represents the area without hinges; TM stands for transmembrane region; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers); "-" indicates a linking peptide or peptide bond.

5. The use as described in claim 4, characterized in that, B1 is BCMA scFv and B2 is CD19 scFv; or, B1 is CD19 scFv and B2 is BCMA scFv; preferably, B1 is BCMA scFv and B2 is CD19 scFv.

6. The use as described in claim 5, characterized in that, The amino acid sequence of the BCMA scFv is shown in SEQ ID NO:2, and the amino acid sequence of the CD19 scFv is shown in SEQ ID NO:

4.

7. The use as described in claim 1, characterized in that, The amino acid sequence of the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:

8.

8. The use as described in claim 1, characterized in that, The encoding nucleic acid sequence of the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:

13.

9. The use as described in claim 1, characterized in that, The CAR-immune cells are selected from the following group: CAR-T cells, CAR-NK cells, CAR-NKT cells, or combinations thereof.

10. A method for reducing urinary protein levels, reducing PLA2R antibody titers, increasing serum albumin levels, and / or increasing renal function eGFR levels, characterized in that, The method includes the steps of: administering BCMA-CD19 bispecific CAR or its encoded nucleic acid, or its vector, or its CAR-immune cells to the desired subject; Alternatively, the method includes the step of: administering a pharmaceutical composition to a desired object, the pharmaceutical composition comprising: (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and (ii) Pharmaceutically acceptable carriers, diluents or excipients.

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