Use of BCMA-CD19 bispecific car-immune cells in treatment of amyloidosis

Abstract

Use of BCMA-CD19 bispecific CAR-immune cells in the treatment of amyloidosis. Specifically, provided is use of a BCMA-CD19 bispecific CAR or a nucleic acid encoding same, or a vector thereof, or CAR-immune cells thereof, for: (i) preparing a drug for preventing and / or treating amyloidosis, and / or (ii) preparing a drug for palliating or reversing amyloid deposition. Also provided are a method for palliating or reversing amyloid deposition by using a BCMA-CD19 bispecific CAR or a nucleic acid encoding same, or a vector thereof, or CAR-immune cells thereof, or a pharmaceutical composition, and a method for preventing and / or treating amyloidosis.

Description

Application of BCMA-CD19 bispecific CAR-immune cells in the treatment of amyloidosis 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 amyloidosis. Background Technology

[0002] Amyloidosis is a clinical syndrome caused by the deposition of amyloid material between cells in various organs, leading to the gradual decline of organ function. Systemic amyloidosis is a disease caused by the deposition of amyloid protein in the extracellular spaces throughout the body, thereby damaging cell and organ function. Systemic amyloidosis is caused by a variety of factors, including genetics, degeneration, and infection. These factors lead to abnormal protein folding, forming amyloid substances that deposit in the interstitial spaces of various tissues throughout the body.

[0003] Systemic light chain amyloidosis (AL amyloidosis) is the most common type of systemic amyloidosis, characterized by diverse clinical manifestations and challenging diagnosis and treatment. AL amyloidosis can clinically affect multiple organs. The kidneys and heart are the most commonly affected organs, while other affected organs include the liver, autonomic or peripheral nerves, digestive tract, and skin and soft tissues. Most clinical manifestations are nonspecific, but tongue hypertrophy and periorbital purpura are relatively specific clinical features of AL amyloidosis.

[0004] Alzheimer's disease (AL) is caused by the misfolding of monoclonal immunoglobulin light chains to form amyloid proteins, which deposit in tissues and organs, causing tissue damage, organ dysfunction, and progressive progression. It is primarily associated with the abnormal proliferation of clonal plasma cells. The treatment goal is to reduce the level of monoclonal immunoglobulin light chains in the body, prevent further deposition of amyloid proteins in vital organs, and alleviate or reverse organ dysfunction caused by amyloid protein deposition. The main method to achieve these treatment goals is to eliminate plasma cell or B cell clones that produce abnormal light chains. Currently used standard therapies show significant heterogeneity in patient efficacy and prognosis; some relapsed / refractory patients have a severe median survival of less than one year. Therefore, innovative treatment methods are urgently needed in clinical practice. Summary of the Invention

[0005] 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 amyloidosis.

[0006] 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:

[0007] (i) To prepare medicines for the prevention and / or treatment of amyloidosis; and / or

[0008] (ii) Prepare drugs to reduce or reverse amyloid deposition.

[0009] In another preferred embodiment, the amyloidosis is selected from the group consisting of: primary amyloidosis, secondary amyloidosis, dialysis-associated amyloidosis, hereditary amyloidosis, or a combination thereof.

[0010] In another preferred embodiment, the amyloidosis is light chain amyloidosis (AL Amyloidosis, ALA) or heavy chain amyloidosis (AH Amyloidosis).

[0011] In another preferred embodiment, the secondary amyloidosis is selected from the group consisting of: chronic inflammation-associated amyloidosis, chronic infection-associated amyloidosis, tumor-associated amyloidosis, or a combination thereof.

[0012] In another preferred embodiment, the chronic inflammation-associated amyloidosis is secondary to a chronic inflammatory disease selected from the group consisting of: rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis (vulgaris and arthropathy), Reiter syndrome, adult-onset Still's disease, Crohn's disease, or a combination thereof.

[0013] In another preferred embodiment, the chronic infection-associated amyloidosis is secondary to a chronic infection-associated disease selected from the group consisting of: leprosy, tuberculosis, bronchiectasis, pressure ulcers, osteomyelitis, Whipple's disease, hidradenitis suppurativa, chronic pyelonephritis, or a combination thereof.

[0014] In another preferred embodiment, the tumor-associated amyloidosis is secondary to a tumor selected from the group consisting of: Hodgkin's disease, hairy cell leukemia, renal cell carcinoma, intestinal cancer, lung cancer, genitourinary cancer, basal cell carcinoma, or a combination thereof.

[0015] In another preferred embodiment, the secondary amyloidosis is myeloma-associated amyloidosis.

[0016] In another preferred embodiment, the hereditary amyloidosis is familial Mediterranean fever-associated amyloidosis.

[0017] In another preferred embodiment, the amyloidosis is systemic amyloidosis or focal amyloidosis.

[0018] In another preferred embodiment, the systemic amyloidosis is systemic light chain amyloidosis.

[0019] In another preferred embodiment, the amyloidosis is a mixed type of amyloidosis.

[0020] In another preferred embodiment, the amyloidosis is light chain amyloidosis.

[0021] In another preferred embodiment, the light chain amyloidosis is λ light chain amyloidosis or κ light chain amyloidosis.

[0022] In another preferred embodiment, the light chain amyloidosis is recurrent and / or refractory light chain amyloidosis.

[0023] In another preferred embodiment, the light chain amyloidosis is either primary light chain amyloidosis or secondary light chain amyloidosis.

[0024] In another preferred embodiment, the secondary light chain amyloidosis is secondary to other plasma cell / B cell diseases selected from the group consisting of multiple myeloma, Waldenström macroglobulinemia, partially globulin-secreting mantle cell lymphoma, or combinations thereof.

[0025] In another preferred embodiment, the BCMA-CD19 bispecific CAR has the structure shown in Formula I:

[0026] SP1-B1-L-SP2-B2-H-TM-C-CD3ζ (I)

[0027] In the formula,

[0028] SP1 and SP2 are each independently either signal peptide sequences or have no signal peptide.

[0029] 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.

[0030] L represents a flexible peptide;

[0031] H represents the area with no hinge or no connection.

[0032] TM stands for transmembrane region;

[0033] C is a co-stimulatory signaling molecule;

[0034] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0035] "-" indicates a linking peptide or peptide bond.

[0036] 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.

[0037] In another preferred embodiment, SP1 and SP2 are CD8 signal peptide sequences.

[0038] In another preferred embodiment, the amino acid sequence of the CD8 signal peptide sequence is shown in SEQ ID NO:1.

[0039] In another preferred embodiment, SP1 is a CD8 signal peptide sequence and SP2 is absent.

[0040] 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:

[0041] VH-VL (A)

[0042] VL-VH (B)

[0043] In the formula,

[0044] VH stands for the variable region of the antibody heavy chain;

[0045] VL is the variable region of the antibody light chain;

[0046] "-" indicates a linking peptide or peptide bond.

[0047] 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.

[0048] In another preferred embodiment, the amino acid sequence of the linker peptide is shown in SEQ ID NO:3.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In another preferred embodiment, the amino acid sequence of the BCMA scFv is shown in SEQ ID NO:2.

[0053] In another preferred embodiment, the amino acid sequence of the CD19 scFv is shown in SEQ ID NO:4.

[0054] In another preferred embodiment, the L is a (GGGGS)4 sequence, the amino acid sequence of which is shown in SEQ ID NO:3.

[0055] In another preferred embodiment, H is a hinge region selected from the following histones: CD8, CD28, CD137, NKG2D, IgG, or a combination thereof.

[0056] In another preferred embodiment, H is the hinge region of the CD8 protein.

[0057] 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.

[0058] In another preferred embodiment, the TM is the transmembrane region of the CD8 protein.

[0059] 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.

[0060] 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.

[0061] In another preferred embodiment, C is a co-stimulatory signaling molecule of the CD137(4-1BB) protein.

[0062] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO:6.

[0063] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:7.

[0064] In another preferred embodiment, the amino acid sequence of the BCMA-CD19 bispecific CAR is selected from the group consisting of:

[0065] (i) The amino acid sequence as shown in SEQ ID NO:8;

[0066] (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;

[0067] (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.

[0068] In another preferred embodiment, the amino acid sequence of the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:8.

[0069] In another preferred embodiment, the BCMA-CD19 bispecific CAR encodes RNA (including mRNA) or DNA (including cDNA).

[0070] 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.

[0071] 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.

[0072] In another preferred embodiment, the promoter is a constitutive promoter or an inducible promoter.

[0073] In another preferred embodiment, the promoter is a strong promoter.

[0074] In another preferred embodiment, the promoter is a tissue-specific promoter.

[0075] 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.

[0076] In another preferred embodiment, the coding nucleic acid sequence of the BCMA-CD19 bispecific CAR is selected from the group consisting of:

[0077] (i) A nucleotide sequence as shown in SEQ ID NO:13;

[0078] (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;

[0079] (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.

[0080] In another preferred embodiment, the encoding nucleic acid sequence of the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:13.

[0081] 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.

[0082] In another preferred embodiment, the viral vector is selected from the group consisting of lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] In another preferred embodiment, the CAR-immune cell is a CAR-T cell.

[0087] In another preferred embodiment, the CAR-T cells are selected from the group consisting of CAR-αβT cells, CAR-γδT cells, or combinations thereof.

[0088] In another preferred embodiment, the dosage form of the drug is selected from the group consisting of: injections and lyophilized preparations.

[0089] 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.

[0090] In another preferred embodiment, the drug is an injectable preparation in a 20 mL volume.

[0091] 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.

[0092] 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 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.

[0093] In another preferred embodiment, the drug further includes other drugs for the prevention and / or treatment of amyloidosis.

[0094] In another preferred embodiment, the drug further includes other drugs for reducing or reversing amyloid deposition.

[0095] 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, or combinations thereof.

[0096] In another preferred embodiment, the immune checkpoint is selected from the group consisting of CD38, PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc., or combinations thereof.

[0097] In another preferred embodiment, the immune checkpoint inhibitor is an anti-CD38 antibody.

[0098] In another preferred embodiment, the anti-CD38 monoclonal antibody is daratumumab (DARA).

[0099] In another preferred embodiment, the chemotherapeutic agent includes an alkylating agent.

[0100] In another preferred embodiment, the glucose metabolism regulating drug includes glucocorticoids.

[0101] In another preferred embodiment, the proteasome inhibitor comprises bortezomib.

[0102] In another preferred embodiment, the drug further includes a diagnostic reagent for diagnosing amyloidosis or determining the level of amyloid protein deposition.

[0103] In another preferred embodiment, the drug comprises:

[0104] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0105] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0106] 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.

[0107] In a second aspect of the invention, a method for mitigating or reversing amyloid deposition is provided, the method comprising the steps of: administering a BCMA-CD19 bispecific CAR or its encoded nucleic acid, or a vector thereof, or a CAR-immune cell thereof to a subject in need;

[0108] Alternatively, the method includes the step of: administering a pharmaceutical composition to a desired object, the pharmaceutical composition comprising:

[0109] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0110] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0111] In another preferred embodiment, the method is an in vitro method.

[0112] In another preferred embodiment, the method is for non-disease treatment purposes or non-disease diagnosis purposes.

[0113] In another preferred embodiment, the BCMA-CD19 bispecific CAR has the structure shown in Formula I above.

[0114] In another preferred embodiment, the object is a cell.

[0115] In another preferred embodiment, the object is a human or a non-human mammal.

[0116] In another preferred embodiment, the subject is an untreated amyloidosis patient.

[0117] In another preferred embodiment, the subject is a patient with amyloidosis who has undergone treatment (conventional treatment) but has had no effect, low effect, or relapsed.

[0118] 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, administration of immune checkpoint inhibitors (such as anti-CD38 monoclonal antibodies), administration of proteasome inhibitors, or combinations thereof.

[0119] In another preferred embodiment, the anti-CD38 monoclonal antibody is daratumumab (DARA).

[0120] In another preferred embodiment, the treatment is selected from: administration of adrenocortical hormones (such as dexamethasone).

[0121] In another preferred embodiment, the treatment is selected from: a combination therapy regimen containing bortezomib or a combination therapy regimen containing anti-CD38 monoclonal antibody.

[0122] In another preferred embodiment, the treatment is selected from: bortezomib combined with dexamethasone (BD treatment), DARA combined with dexamethasone (DD treatment), or a combination thereof.

[0123] In another preferred embodiment, the subject is a patient with recurrent amyloidosis.

[0124] In another preferred embodiment, the method includes use in conjunction with other methods.

[0125] In another preferred embodiment, the other methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0126] In another preferred embodiment, the method further includes the step of determining the level of amyloid deposition.

[0127] 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.

[0128] In another preferred embodiment, the method further includes the step of administering other drugs for reducing or reversing amyloid deposition.

[0129] In a third aspect of the invention, a method for preventing and / or treating amyloidosis 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;

[0130] Alternatively, the method includes the step of: administering a pharmaceutical composition to a desired object, the pharmaceutical composition comprising:

[0131] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0132] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0133] In another preferred embodiment, the object is a cell.

[0134] In another preferred embodiment, the object is a human or a non-human mammal.

[0135] In another preferred embodiment, the subject is an untreated amyloidosis patient.

[0136] In another preferred embodiment, the subject is a patient with amyloidosis who has undergone treatment (conventional treatment) but has had no effect, low effect, or relapsed.

[0137] 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, administration of immune checkpoint inhibitors (such as anti-CD38 monoclonal antibodies), administration of proteasome inhibitors, or combinations thereof.

[0138] In another preferred embodiment, the anti-CD38 monoclonal antibody is daratumumab (DARA).

[0139] In another preferred embodiment, the treatment is selected from: administration of adrenocortical hormones (such as dexamethasone).

[0140] In another preferred embodiment, the treatment is selected from: a combination therapy regimen containing bortezomib or a combination therapy regimen containing anti-CD38 monoclonal antibody.

[0141] In another preferred embodiment, the treatment is selected from: bortezomib combined with dexamethasone (BD treatment), DARA combined with dexamethasone (DD treatment), or a combination thereof.

[0142] In another preferred embodiment, the subject is a patient with recurrent amyloidosis.

[0143] In another preferred embodiment, the method includes use in conjunction with other methods.

[0144] In another preferred embodiment, the other methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0145] In another preferred embodiment, the method further includes the step of determining the level of amyloid deposition.

[0146] In another preferred embodiment, the method further includes the step of diagnosing amyloidosis.

[0147] In another preferred embodiment, amyloidosis is diagnosed using a method selected from the group consisting of: tissue biopsy, amyloidosis typing, M protein detection, tumor screening, bone marrow fluorescence in situ hybridization (FISH) detection, or a combination thereof.

[0148] 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.

[0149] In another preferred embodiment, the method further includes the step of administering other drugs for the prevention and / or treatment of amyloidosis.

[0150] In a fourth aspect of the invention, a kit is provided for (i) mitigating or reversing amyloid deposition; or (ii) preventing and / or treating amyloidosis, the kit comprising:

[0151] (Z1) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; or

[0152] (Z2) A pharmaceutical composition comprising:

[0153] (i) BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector, or its CAR-immune cell, or a combination thereof; and

[0154] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0155] In another preferred embodiment, the kit further includes other medications for the prevention and / or treatment of amyloidosis.

[0156] In another preferred embodiment, the kit further includes other drugs for reducing or reversing amyloid deposition.

[0157] 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, or combinations thereof.

[0158] In another preferred embodiment, the immune checkpoint is selected from the group consisting of CD38, PD-1, PD-L1, CTLA-4, B7-H3, LAG-3, VISTA, CD47, TIM-3, TIGIT, BTLA, Siglec-15, etc., or combinations thereof.

[0159] In another preferred embodiment, the immune checkpoint inhibitor is an anti-CD38 antibody.

[0160] In another preferred embodiment, the anti-CD38 monoclonal antibody is daratumumab (DARA).

[0161] In another preferred embodiment, the chemotherapeutic agent includes an alkylating agent.

[0162] In another preferred embodiment, the glucose metabolism regulating drug includes glucocorticoids.

[0163] In another preferred embodiment, the proteasome inhibitor comprises bortezomib.

[0164] In another preferred embodiment, the drug further includes a diagnostic reagent for diagnosing amyloidosis or determining the level of amyloid protein deposition.

[0165] 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

[0166] Figure 1 shows the structure of BCMA-CD19 bispecific CAR-T cells.

[0167] Figure 2 shows the pCDH-CAR plasmid map.

[0168] Figure 3 shows the clinical trial process for subjects with relapsed / refractory light chain amyloidosis.

[0169] Figure 4 shows the hematological remission and progression criteria for systemic light chain amyloidosis, where iFLC represents serum affected free light chains; dFLC represents the difference in serum free light chains. Detailed Implementation

[0170] Through extensive and in-depth research and screening, the inventors have, for the first time, creatively discovered that dual-target CAR-T cells simultaneously targeting CD19 and BCMA can simultaneously eliminate plasma cells and their precursor cells that secrete misfolded immunoglobulins in patients with amyloidosis (such as those with systemic light chain amyloidosis), effectively reducing or reversing amyloid protein deposition, thereby achieving the goal of treating patients, and exhibiting very good safety and efficacy. Furthermore, the BCMA-CD19 bispecific CAR-T cells of this invention can maintain dFLC at a low level for a long period, achieving very good clinical remission in subjects who have not responded to, have had poor response to, or have relapsed after conventional treatments (such as bortezomib combined with dexamethasone (BD therapy) or DARA combined with dexamethasone (DD therapy)). This invention was completed based on these findings.

[0171] the term

[0172] 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.

[0173] 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.

[0174] 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.

[0175] As used in this article, the term "dFLC" refers to the difference of serum free light chain (dFLC), which is the difference between the FLC levels in affected serum and those in unaffected serum. When performing hematological assessments on patients with AL amyloidosis, appropriate criteria should be selected based on baseline dFLC levels.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] As used in this article, the term "NTproBNP" refers to N-terminal pro-B-type natriuretic peptide, also known as N-terminal pro-brain natriuretic peptide. It is a polypeptide composed of amino acid residues secreted by the heart, an important indicator used clinically to evaluate cardiac function, and a marker of heart failure.

[0180] As used in this article, the term "TNT" refers to troponin, and its value can reflect whether there are abnormalities in myocardial cells. It is an important indicator in cardiovascular diseases.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] As used in this article, the term "M protein" refers to Monoclonal Protein (MP), an abbreviation for monoclonal immunoglobulin. It is an abnormal monoclonal immunoglobulin produced by the abnormal monoclonal proliferation of B lymphocytes or plasma cells. It has a highly homogeneous molecular structure and lacks normal physiological function. Its characteristics include: identical amino acid sequences, spatial conformations, and electrophoretic properties. M protein screening includes serum protein electrophoresis (SPE) and serum immunofixation electrophoresis (IFE). In addition, it may include urine immunofixation electrophoresis, urine protein electrophoresis, quantitative detection of immunoglobulins, quantitative determination of free light chains and their ratio (FK / FL), and quantitative determination of light chains (K, L).

[0185] Systemic light chain amyloidosis (AL amyloidosis)

[0186] AL amyloidosis is classified based on the type of monoclonal light chains and the presence or absence of hematologic malignancies. According to the type of monoclonal light chain deposition in histopathological findings, AL amyloidosis can be divided into λ light chain type and κ light chain type. Clinically, the λ light chain type is predominant, accounting for approximately 85%. Based on the presence or absence of hematologic malignancies, AL amyloidosis can be classified as primary or secondary. Secondary AL amyloidosis is secondary to other plasma cell / B cell diseases such as multiple myeloma, Waldenström macroglobulinemia, and some globulin-secreting mantle cell lymphomas.

[0187] The Mayo Clinic 2012 staging system is routinely used for the clinical staging of AL amyloidosis. The Mayo Clinic 2004 staging system is recommended for patients with predominantly cardiac involvement. The renal prognostic staging system is recommended for assessing the progression of renal amyloidosis. The Nanjing prognostic staging system is recommended for patients with both renal and cardiac involvement.

[0188] The diagnosis of AL amyloidosis requires meeting the following criteria: (1) clinical manifestations, physical examination, laboratory or imaging examinations confirming involvement of tissues and organs; (2) pathological examination of tissue biopsy confirming amyloid protein deposition, and the precursor protein of amyloid protein is immunoglobulin light chain or heavy light chain. After AL amyloidosis is diagnosed, the involvement of various organs throughout the body can be judged by combining clinical, histological and imaging characteristics.

[0189] The tests for diagnosing amyloidosis generally include: 1. tissue biopsy site, 2. amyloidosis typing, 3. M protein detection and tumor screening, and 4. bone marrow fluorescence in situ hybridization (FISH) detection.

[0190] Once diagnosed, patients with AL amyloidosis should begin treatment as early as possible, based on prognostic staging, affected organ function, physical condition, and available medications. The treatment goal is to reduce the level of monoclonal immunoglobulin light chains in the body, prevent further deposition of amyloid protein in vital organs, and alleviate or reverse organ dysfunction caused by amyloid protein deposition. The main method to achieve these treatment goals is to eliminate plasma cell or B cell clones that produce abnormal light chains.

[0191] The main principles for treating AL amyloidosis are: (1) Patients who meet the criteria for autologous hematopoietic stem cell transplantation should be given the first choice for transplantation. Patients who refuse transplantation can also choose a combination of drugs such as glucocorticoids, alkylating agents, immunomodulators, proteasome inhibitors and anti-CD38 monoclonal antibodies; (2) For patients who do not meet the criteria for transplantation, a combination therapy regimen containing bortezomib is recommended. After every two cycles of treatment, patients should be reassessed to see if they meet the criteria for transplantation; (3) Patients who cannot achieve very good partial remission (VGPR) or above in hematology should be considered for consolidation therapy; patients who achieve VGPR or above can be considered for discontinuation of medication and observation; (4) For patients with relapsed or refractory AL amyloidosis, there is currently no suitable treatment method. Patients are recommended to participate in clinical trials.

[0192] Inclusion criteria for subjects with relapsed / refractory light chain amyloidosis

[0193] Subjects must meet all inclusion criteria and not meet any exclusion criteria to be enrolled:

[0194] (1) Subjects must personally sign a written informed consent form approved by the ethics committee before the start of the study;

[0195] (2) The subjects were ≥18 years old and <70 years old;

[0196] (3) Pathological examination confirms light chain amyloidosis, with at least one major organ involved (heart, kidney or liver);

[0197] (4) Clinical patients with relapsed / refractory light chain amyloidosis who do not respond to conventional treatment;

[0198] (5) dFLC > 50 mg / L;

[0199] (6) Expected survival ≥ 12 weeks;

[0200] (7) ECOG score ≤ 2 points;

[0201] (8) The following conditions must be met to diagnose AL amyloidosis: 1) Clinical manifestations, physical examination, laboratory or imaging examinations confirm involvement of tissues and organs. 2) Histopathological examination of tissue biopsy confirms the deposition of amyloid protein, and the precursor protein of amyloid protein is immunoglobulin light chain or heavy light chain; and the patient belongs to the relapsed and refractory type.

[0202] (9) 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;

[0203] (10) Subjects must have adequate organ function and meet all of the following examination results before enrollment:

[0204] (10.1) Absolute neutrophil count ≥ 1.0 × 10⁻⁶ 9 / L (Granocyte colony-stimulating factor (G-CSF) support is permitted, but the patient must not have received supportive treatment within 7 days prior to the examination);

[0205] (10.2) Platelet count ≥75×10 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);

[0206] (10.3) Hemoglobin ≥ 9 g / dl (must not have received blood transfusion support [including component blood transfusion] within 7 days prior to the test);

[0207] (10.4) Bilirubin level ≤ 1.5 × upper limit of normal (ULN) (except for bile duct obstruction caused by tumor compression);

[0208] (10.5) Creatinine clearance rate ≥40 ml / min;

[0209] (10.6) ALT or AST ≤ 2.5 × upper limit of normal (ULN) (≤ 5 times the upper limit of normal for those with liver involvement);

[0210] (10.7) Echocardiography results showed cardiac ejection fraction ≥50% and no obvious pericardial effusion;

[0211] (10.8)NTproBNP<1800pg / ml, TNT<0.06ng / ml;

[0212] (10.9) Stable coagulation function: INR≤1.5, APTT≤1.2×Upper limit of normal (ULN) (except for tumor-related anticoagulation therapy);

[0213] (10.10) Basal blood oxygen saturation >95% in indoor natural air environment.

[0214] Exclusion criteria for subjects with relapsed / refractory light chain amyloidosis

[0215] Subjects meeting any of the following criteria will be excluded:

[0216] (1) Subjects who have received the following prior treatments:

[0217] (1.1) Individuals who had received gene therapy prior to enrollment;

[0218] (1.2) Subjects who received a live vaccine within 4 weeks prior to enrollment;

[0219] (1.3) Received other interventional clinical trial drug treatment within 12 weeks prior to apheresis.

[0220] (2) Patients with central nervous system metastasis or complete intestinal obstruction.

[0221] (3) Patients with moderate or severe pleural and peritoneal effusion that are difficult to control with conventional treatment and require continuous catheter drainage.

[0222] (4) Having an active malignant tumor within the past 5 years, unless it is a curable tumor and has been clearly cured.

[0223] 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 value range of the testing center or positive qualitative HBV DNA test); subjects with positive hepatitis C virus (HCV) antibody and positive HCV RNA in peripheral blood; subjects with positive human immunodeficiency virus (HIV) antibody; subjects with positive cytomegalovirus (CMV) DNA test; subjects with positive RPR in syphilis test.

[0224] (6) Presence of uncontrolled active infection (except for <CTCAE> grade 2 genitourinary and upper respiratory tract infections).

[0225] (7) 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), and severe arrhythmia.

[0226] (8) Subjects with hypertension that cannot be controlled with medications.

[0227] (9) Previous treatment toxicities that have not been alleviated to baseline or ≤ grade 1 (except for hair loss and laboratory test abnormalities without clinical significance according to the NCI-CTCAE v5.0 version).

[0228] (10) 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).

[0229] (11) Subjects with solid organ transplantation.

[0230] (12) Pregnant or lactating women.

[0231] (13) Subjects with previous central nervous system diseases (such as cerebral aneurysm, epilepsy, stroke, Alzheimer's disease, psychosis, etc.) or consciousness disorders.

[0232] (14) Other systematically unstable diseases judged by the investigator: including but not limited to severe liver, kidney or metabolic diseases that require drug treatment.

[0233] (15) Known life-threatening allergic reactions, hypersensitivity reactions or intolerance to the BCMA-CD19 bispecific CAR-T cell preparation or its components.

[0234] (16) Subjects judged by the investigator to have bleeding, severe thrombosis or presence of hereditary / acquired bleeding and severe thrombosis (including hemophilia, coagulation disorders, thrombocytopenia, hypersplenism, etc.), or patients receiving thrombolytic or anticoagulant therapy.

[0235] (17) Other situations in which the researchers deem it unsuitable for enrollment.

[0236] Single-sample standard

[0237] (1) No infection requiring systemic anti-infective treatment prior to single-donor apheresis;

[0238] (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;

[0239] (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;

[0240] (4) Drug elution before single apheresis:

[0241] (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;

[0242] (4.2) No immunosuppressive drugs (including monoclonal antibodies and small molecule drugs) were used within 2 weeks prior to apheresis, except for topical medications;

[0243] (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;

[0244] (4.4) No bevacizumab treatment was used within 20 days prior to apheresis;

[0245] (4.5) No other interventional clinical trial drug treatments were received within 12 weeks prior to apheresis;

[0246] (5) There are no other circumstances that researchers have determined are unsuitable for single-sample collection.

[0247] Shower pretreatment standards

[0248] (1) No infection requiring systemic anti-infective treatment prior to scrubbing;

[0249] (2) Within 7 days prior to administration of the medication to the subject, the subject's laboratory test results must meet the following conditions:

[0250] (2.1) Echocardiographic diagnosis of left ventricular ejection fraction (LVEF) ≥ 50%;

[0251] (2.2) Basal blood oxygen saturation >95% in indoor natural air environment;

[0252] (3) After the researchers assess blood routine, liver and kidney function, and coagulation function, the patient is able to undergo urinary tract cleansing.

[0253] (4) There are no other circumstances that researchers have determined are unsuitable for administering Qinglin medication.

[0254] Cell reinfusion standards

[0255] (1) No systemic anti-infective treatment is required before reinfusion;

[0256] (2) Blood oxygen saturation > 95%;

[0257] (3) >24 hours after rinsing pretreatment;

[0258] (4) Drug elution before reinfusion of BCMA-CD19 bispecific CAR-T cell injection:

[0259] (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);

[0260] (4.2) No other immunosuppressive drugs (including monoclonal antibodies and small molecule drugs) were used in the two weeks prior to the infusion;

[0261] (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;

[0262] (4.4) No PD-L1 / PD-1 or other immune checkpoint inhibitors were used within 8 weeks prior to reinfusion;

[0263] (5) There are no other circumstances that researchers have determined are unsuitable for cell reinfusion.

[0264] Relief Standard

[0265] 1. Hematological assessment:

[0266] Appropriate criteria should be selected based on the patient's baseline serum free light chain difference (dFLC) level. All hematological responses can be evaluated in patients with dFLC > 50 mg / L. Complete remission (CR) and partial remission (PR) can be evaluated in patients with dFLC levels between 20 and 50 mg / L. Hematological efficacy cannot currently be evaluated in patients with dFLC < 20 mg / L.

[0267] Complete remission (CR): negative blood / urine immunofixation electrophoresis, and normal serum free light chain levels and ratios.

[0268] Very good partial remission (VGPR): dFLC decreased to <40 mg / L.

[0269] Partial remission (PR):

[0270] (1) Patients with dFLC > 50 mg / L: dFLC decrease > 50%;

[0271] (2) Patients with dFLC between 20 and 50 mg / L: dFLC < 10 mg / L.

[0272] Disease progression (PD):

[0273] (1) If CR is achieved, abnormal M protein or light chain ratio can be detected (the serum level of affected free light chain (iFLC) must be doubled);

[0274] (2) If PR is achieved, serum M protein increases by ≥50% and >5g / L; or urine M protein increases by ≥50% and >200mg / d;

[0275] (3) iFLC level increased by ≥50% and >100mg / L.

[0276] Stable disease (SD): did not achieve PR, nor does it meet the PD criteria.

[0277] 2. Organ remission assessment:

[0278] The disease criteria that can be assessed in organ response are: heart: NT-proBNP > 650 ng / L or brain natriuretic peptide (BNP) > 150 ng / L; kidney: urinary protein quantification > 0.5 g / 24 h; liver: alkaline phosphatase > 1.5 times the upper limit of normal.

[0279] (1) Heart:

[0280] CR: NT-proBNP ≤ 350 ng / L and BNP < 80 ng / L

[0281] VGPR: NT-ProBNP decreased by >60%

[0282] PR: NT-ProBNP decreased by 31% to 60%.

[0283] PD: NT-proBNP elevation >30% and >300 ng / L, or troponin elevation ≥33%, or ejection fraction decrease ≥10%.

[0284] (2) Kidneys:

[0285] CR: Urinary protein quantification ≤200mg / 24h; and eGFR decrease ≤25%.

[0286] VGPR: Proteinuria reduced by >60%

[0287] PR: Proteinuria reduced by 31%–60%

[0288] PD: Increase in urinary protein quantification by 50% (at least an increase of 1 g / day); or decrease in eGFR by >25% from baseline.

[0289] (3) Liver:

[0290] Relief: A decrease in alkaline phosphatase of more than 50%, and / or a reduction in liver volume of ≥2 cm

[0291] Progress: Alkaline phosphatase increased by more than 50%.

[0292] (4) Peripheral nerves:

[0293] Relief: Electromyography showed improved nerve conduction velocity.

[0294] Progression: Electromyography or nerve conduction velocity indicates lesion progression.

[0295] Drug / Drug Composition

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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 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.

[0302] Therapeutic applications

[0303] This invention includes therapeutic applications using cells (e.g., T cells) transduced with BCMA-CD19 bispecific CAR or its encoding nucleic acid, or its vector (such as a lentiviral vector). The transduced T cells can simultaneously clear plasma cells and their precursor cells that secrete misfolded immunoglobulins in patients with systemic light chain amyloidosis, effectively reducing or reversing light chain amyloid deposition, thereby achieving the goal of treating the patient.

[0304] 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 a non-MHC-restricted manner. Furthermore, the CAR-T cells can replicate in vivo, producing long-term persistence that leads to sustained control of light chain amyloid deposition. In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and persist for months to years.

[0305] 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.

[0306] The pharmaceutical compositions of the present invention can be administered in a manner suitable for treating (or preventing) a 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.

[0307] 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, dFLC 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 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 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 6 Cells / 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.

[0308] 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.

[0309] 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 combination with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with: glucocorticoids, alkylating agents, immunomodulators, proteasome inhibitors, anti-CD38 monoclonal antibodies, bortezomib-containing combination therapies (e.g., BD: bortezomib combined with dexamethasone), DARA combined with dexamethasone (dexamethasone is an adrenocortical hormone), etc. 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.

[0310] 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 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 The 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 5cells / 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.

[0311] Main advantages of the invention

[0312] 1. Based on the inventors' in-depth research into the pathogenesis of systemic light chain amyloidosis and the mechanism of action of CAR-T cells, this invention utilizes dual-target CAR-T cells that simultaneously target CD19 and BCMA to simultaneously eliminate plasma cells and their precursor cells that secrete misfolded immunoglobulins in amyloidosis patients (such as those with systemic light chain amyloidosis), thereby achieving the goal of treating patients. Compared with the therapeutic effects of existing technologies for patients with systemic light chain amyloidosis, the BCMA-CD19 bispecific CAR-T cell injection solution of this invention has very good safety and efficacy.

[0313] 2. For subjects who have not responded to, have had poor response to or have relapsed after conventional treatment (such as bortezomib combined with dexamethasone (BD therapy) or DARA combined with dexamethasone (DD therapy)), the BCMA-CD19 bispecific CAR-T cells of the present invention can achieve very good therapeutic effects.

[0314] 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.

[0315] Sequence information

[0316] CD8 signal peptide sequence (SEQ ID NO:1):

[0317] BCMA scFv (SEQ ID NO:2):

[0318] (GGGGS)4 sequence (SEQ ID NO:3):

[0319] CD19 scFv (SEQ ID NO:4):

[0320] CD8 hinge region + CD8 transmembrane domain sequence (SEQ ID NO:5):

[0321] CD137(4-1BB) co-stimulatory signaling molecule sequence (SEQ ID NO:6):

[0322] CD3ζ sequence (SEQ ID NO:7):

[0323] BCMA-CD19 bispecific CAR sequence (SEQ ID NO:8):

[0324] The heavy chain variable region sequence of BCMA scFv (SEQ ID NO:9):

[0325] The light chain variable region sequence of BCMA scFv (SEQ ID NO:10):

[0326] The heavy chain variable region sequence of CD19 scFv (SEQ ID NO:11):

[0327] The light chain variable region sequence of CD19 scFv (SEQ ID NO:12):

[0328] The coding sequence of BCMA-CD19 bispecific CAR (SEQ ID NO:13):

[0329] Example 1: Preparation of BCMA-CD19 bispecific CAR-T cells

[0330] 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.

[0331] 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.

[0332] The pCDH-CAR vector is based on the HIV lentiviral vector backbone, including a 5' long terminal repeat (LTR), a promoter, and a packaging signal; the (anti-BCMA and anti-CD19) CAR sequence includes a signal peptide, a heavy chain variable region (VH), a linker peptide, a light chain variable region (VL), a co-stimulatory domain, and a CD3ζ cytoplasmic region, and a 3' LTR. The CAR-T cell structure is shown in Figure 1, and the pCDH-CAR plasmid map is shown in Figure 2.

[0333] Preclinical studies have shown that the drug can effectively clear B cells and plasma cells in vitro and in animal models. It also shows that clearing plasma cells can effectively clear light chain amyloid deposits in patients, so it is expected to benefit subjects participating in subsequent clinical trials.

[0334] Example 2: Clinical trial in subjects with relapsed / refractory light chain amyloidosis

[0335] In clinical trials, the inclusion criteria, exclusion criteria, single apheresis criteria, lymph node pretreatment criteria, cell reinfusion criteria, and remission criteria for subjects with relapsed / refractory light chain amyloidosis are as described above.

[0336] 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.

[0337] 1. Screening period (W-8~D-30):

[0338] 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.

[0339] 2. PBMC single collection (D-30~D-18):

[0340] BCMA-CD19 bispecific CAR-T cells were prepared from mononuclear cells extracted from peripheral blood of the subjects.

[0341] 3. Pre-examination examination (7 days prior to exfoliation):

[0342] 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.

[0343] 4. Shower pretreatment (D-7~D-2 for 3 consecutive days):

[0344] 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:

[0345] Cyclophosphamide 300mg / m 2 / d, intravenous drip, once a day, for three consecutive days.

[0346] Fludarabine 30mg / m 2 / d, intravenous drip, once a day, for three consecutive days (infused on the same day as cyclophosphamide infusion).

[0347] 5. BCMA-CD19 bispecific CAR-T cell injection reinfusion (D0):

[0348] 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, 1×10 6 Cells / kg body weight, 3×10 6 Cells per kg of body weight, etc.

[0349] 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]):

[0350] 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.

[0351] The clinical trial results are summarized in the table below (the complete clinical trial results are not shown due to space limitations):

[0352] Clinical trial results show that BCMA-CD19 bispecific CAR-T cell injection has excellent clinical efficacy in treating AL amyloidosis. Of the six subjects, five achieved complete remission (CR), and one achieved very good partial remission (VGPR). These results are superior to existing technologies for treating AL amyloidosis.

[0353] In addition, the results of dFLC measurement during the clinical trial are summarized in the table below (the complete clinical trial results are not shown due to space limitations):

[0354] (Note: In the three tables above, the same serial number indicates the same subject. BD represents bortezomib combined with dexamethasone, and DD represents DARA combined with dexamethasone.)

[0355] The above results indicate that BCMA-CD19 bispecific CAR-T cell injection can effectively reduce or reverse light chain amyloid protein deposition in AL amyloidosis, and dFLC characterization also shows hematologic remission of AL amyloidosis.

[0356] Furthermore, according to the dFLC measurement results of Subject 1, after conventional BD and DD treatment, Subject 1's dFLC level remained high, and its light chain amyloid deposition was not effectively reduced. However, after administration of BCMA-CD19 bispecific CAR-T cell injection, its dFLC level significantly decreased and remained at a low level for a long period. Similarly, for Subjects 2 and 3, who relapsed before treatment with the BCMA-CD19 bispecific CAR-T cell injection of this invention, their dFLC levels significantly increased to high levels at the time of relapse. After administration of BCMA-CD19 bispecific CAR-T cell injection, their dFLC levels significantly decreased and remained at a low level for a long period.

[0357] The above results demonstrate that BCMA-CD19 bispecific CAR-T cell injection can achieve very good therapeutic effects for subjects who have not responded to, have had low efficacy or have relapsed after conventional treatment.

[0358] 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.

[0359] discuss

[0360] Treatment of amyloidosis, especially systemic light chain amyloidosis (AL amyloidosis), has always been a challenge in this field, particularly for patients who do not respond to conventional treatments, have low efficacy, or experience relapse and refractory disease. Currently, there has been some exploration in the field of cell therapy; for example, foreign researchers treated eight AL patients with CAR-T therapy (CAR-T cell infusion doses of 1.5 × 10⁻⁶ cells each). 8 4.5×10 8 8×10 8 CAR+cell therapy showed that approximately half of the patients did not achieve a remission period exceeding 6 months during follow-up. Therefore, further development of more effective and sustained remission strategies for Alzheimer's disease is needed.

[0361] CAR-T products targeting CD19 and BCMA have been approved by the FDA 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 the treatment of AL.

[0362] In this invention, the inventors creatively discovered that the developed BCMA-CD19 bispecific CAR-T cell injection can effectively reduce or reverse light chain amyloid deposition, demonstrating excellent efficacy in 6 AL patients. Five achieved complete remission (CR), and one achieved very good partial remission (VGPR), with no deaths reported. The dFLC characteristics of all 6 patients also showed hematological remission of AL-type amyloidosis, remaining at low levels long-term. Furthermore, among the 6 subjects were refractory AL patients whose dFLC levels remained high after conventional BD and DD treatments, as well as relapsed AL patients. After treatment with the BCMA-CD19 bispecific CAR-T cell injection of this invention, their dFLC levels significantly decreased and remained at low levels long-term, demonstrating excellent therapeutic effects. Compared to the aforementioned studies by foreign scholars, the BCMA-CD19 bispecific CAR-T cell injection of this invention showed significant advantages in the depth and durability of remission in the 6 patients treated.

[0363] Therefore, the BCMA-CD19 bispecific CAR-T cells and their developed drugs of the present invention have broad application prospects in the treatment of amyloidosis, especially systemic light chain amyloidosis (AL Amyloidosis).

[0364] 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. Use of a BCMA-CD19 bispecific CAR or a nucleic acid encoding the same, or a vector thereof, or a CAR-immune cell thereof, characterized in that, Use of: (i) a medicament for preventing and / or treating amyloidosis; and / or (ii) a medicament for reducing or reversing amyloid deposition.

2. Use according to claim 1, characterized in that, The amyloidosis is selected from the group consisting of primary amyloidosis, secondary amyloidosis, dialysis-related amyloidosis, hereditary amyloidosis, or a combination thereof.

3. Use according to claim 1, characterized in that, The amyloidosis is light chain type amyloidosis (AL Amyloidosis, ALA) or heavy chain type amyloidosis (AH Amyloidosis).

4. Use according to claim 3, characterized in that, The light chain type amyloidosis is relapsed and / or refractory light chain type amyloidosis.

5. The use according to claim 1, characterized in that, The amyloidosis is systemic light chain type amyloidosis.

6. The use according to claim 1, characterized in that, The BCMA-CD19 bispecific CAR has a structure shown in Formula I: SP1-B1-L-SP2-B2-H-TM-C-CD3ζ (I) In the formula, SP1 and SP2 are each independently null or a signal peptide sequence; B1 and B2 are respectively a first binding element targeting a first target protein, a second binding element targeting a second target protein; L is a flexible peptide; H is null or a hinge region; TM is a transmembrane region; C is a costimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or a mutant / modified version thereof); "-" is a connecting peptide or a peptide bond.

7. Use according to claim 6, characterized in that, The B1 is a BCMA scFv, and the B2 is a CD19 scFv; or, the B1 is a CD19 scFv, and the B2 is a BCMA scFv; preferably, the B1 is a BCMA scFv, and the B2 is a CD19 scFv.

8. Use according to claim 7, 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.

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

8.

10. The use according to claim 1, characterized in that, The nucleic acid sequence encoding the BCMA-CD19 bispecific CAR is shown in SEQ ID NO:

13.

11. Use according to claim 1, characterized in that, The CAR-immune cell is selected from the group consisting of a CAR-T cell, a CAR-NK cell, a CAR-NKT cell, or a combination thereof.

12. The use according to claim 1, characterized in that, The dosage form of the medicament is selected from the group consisting of an injection, a lyophilized agent.

13. The use according to claim 1, characterized in that, The content of CAR-immune cells in the drug is 1 x 10 3 -1 x 10 8 cells / mL.

14. The use according to claim 1, characterized in that, The medicament further comprises: another medicament for preventing and / or treating amyloidosis, or another medicament for reducing or reversing amyloid deposition.

15. A method of reducing or reversing amyloid deposition, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-14. The method comprises the step of: administering the BCMA-CD19 bispecific CAR or a nucleic acid encoding the same, or a vector thereof, or a CAR-immune cell thereof, to a subject in need thereof; Alternatively, the method comprises the step of: administering a pharmaceutical composition comprising: (i) a BCMA-CD19 bispecific CAR or a nucleic acid encoding the same, or a vector thereof, or a CAR-immune cell thereof, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

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