Bispecific car-t cell targeting BCMA and GPRC5d

Abstract

Provided is a bispecific CAR-T cell targeting BCMA and GPRC5D. Specifically, provided is an autologous CAR-T cell targeting both BCMA and GPRC5D antigen molecules and expressing chimeric antigen receptors (CARs) in parallel. Also provided is a use of a CAR-T cell for adoptive T-cell therapy for diseases including multiple myeloma.

Description

Bispecific CAR-T cells targeting BCMA and GPRC5D Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more specifically, to CAR-T cells targeting BCMA and GPRC5D. Background Technology

[0002] Multiple myeloma is a plasma cell tumor that occurs in the bone marrow. This tumor can cause hypercalcemia, anemia, kidney dysfunction, osteonecrosis, and bone marrow failure. Currently, multiple myeloma is the second most common hematologic malignancy. Globally, the incidence-to-mortality ratio for multiple myeloma is 1.8:1.1, while in Asia it is 1.1:0.76, indicating a low survival rate among affected individuals. Multiple myeloma is almost incurable with commonly used chemotherapy, immunomodulatory agents, and monoclonal antibody therapies. Even with remission, there is a very high relapse rate, known as relapsed / refractory multiple myeloma (RRMM), with a five-year survival rate of only 51%.

[0003] In recent years, breakthroughs have been made in the treatment of multiple myeloma with bispecific T cell engagers (BiTEs) and adaptive T cell therapy (ACT). Targeted therapy products for multiple myeloma mainly target BCMA, CD38, CD138, and GPRC5D.

[0004] B cell maturation antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor family. Studies have shown that BCMA can bind to B cell activating factor receptor (BAFF) and B cell proliferation-inducing ligand (APRIL), promoting B cell survival at different developmental stages. Abnormal signaling can lead to abnormal B cell proliferation, resulting in autoimmune diseases and tumor formation (see Rickert et al., Immunological Reviews, 2011, Vol. 244: 115-133).

[0005] GPRC5D expression is highly specific, expressed only in plasma cells of myeloma patients, and almost not expressed in normal tissues. Further investigation revealed no correlation between BCMA expression and GPRC5D; although both are expressed in plasma cells, their expression profiles are relatively independent.

[0006] Taking T-cell bispecific antibody products as an example, products targeting BCMA mainly include REGN5458 developed by Regeneron Pharmaceuticals and Teclistamab developed by Johnson & Johnson. REGN5458 is currently in Phase I clinical trials. At the 2022 ASH conference, it was reported that in 54 patients receiving intravenous injection, the response rate was 71.4%, and the maximum response rate was 57.1%; in 51 patients receiving subcutaneous injection, the response rate was 60.4%, and the maximum response rate was 39.6%. Teclistamab was launched in October 2022, with an overall response rate of 61.8%, and 28.2% of patients achieving complete or strict remission. A representative product of GPRC5D-targeted bispecific antibodies is Talqulemab, which submitted a BLA for marketing authorization in December 2022. Completed Phase I / II clinical trials showed an overall response rate of 73% at a dose of 0.4 mg / kg, a maximum response rate of 58%, and a complete response rate greater than 29%. It has entered Phase III clinical trials.

[0007] For the treatment of multiple myeloma, simultaneous targeting of GPRC5D and BCMA may be a promising treatment option. Therefore, there is a need in the field to develop bispecific GPRC5D and BCMA-targeting drugs for the treatment of multiple myeloma. Summary of the Invention

[0008] The purpose of this invention is to provide a CAR-T cell that targets BCMA and GPRC5D.

[0009] In a first aspect of the invention, a bispecific engineered autologous chimeric antigen receptor (CAR)-T cell is provided, wherein the engineered autologous CAR-T cell expresses BCMA-CAR and GPRC5D-CAR.

[0010] The BCMA-CAR contains an antigen-binding domain that targets BCMA, and the antigen-binding domain that targets BCMA includes the following complementarity-determining region (CDR):

[0011] The amino acid sequence is shown in SEQ ID NO:1, which is the light chain CDR1 (LCDR1).

[0012] The amino acid sequence is shown in AAS for the light chain CDR2 (LCDR2).

[0013] The amino acid sequence is shown in SEQ ID NO:2 for the light chain CDR3 (LCDR3).

[0014] The amino acid sequence is shown in SEQ ID NO:3 for the heavy chain CDR1 (HCDR1).

[0015] The amino acid sequence is shown in SEQ ID NO:4 for the heavy chain CDR2 (HCDR2).

[0016] The amino acid sequence is shown in SEQ ID NO:5 for the heavy chain CDR3 (HCDR3);

[0017] The GPRC5D-CAR contains an antigen-binding domain targeting GPRC5D, and the antigen-binding domain targeting GPRC5D includes the following complementarity-determining region (CDR):

[0018] The amino acid sequence is shown in SEQ ID NO:8 for the heavy chain CDR1 (HCDR1).

[0019] The amino acid sequence is shown in SEQ ID NO:9 for the heavy chain CDR2 (HCDR2).

[0020] The amino acid sequence is shown in SEQ ID NO:10 for the heavy chain CDR3 (HCDR3).

[0021] In another preferred embodiment, the antigen-binding domain targeting BCMA is a single-chain antibody.

[0022] In another preferred embodiment, the amino acid sequence of the antigen-binding domain targeting BCMA is as shown in SEQ ID NO:6, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0023] In another preferred embodiment, the antigen-binding domain targeting GPRC5D is a single-domain antibody.

[0024] In another preferred embodiment, the amino acid sequence of the antigen-binding domain targeting GPRC5D is as shown in SEQ ID NO:11, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0025] Among them, any of the above amino acid sequences also includes a derivative sequence that has been optionally added, deleted, modified and / or substituted with at least one amino acid, and is capable of retaining the binding affinity of BCMA or GPRC5D.

[0026] In another preferred embodiment, the BCMA-CAR and GPRC5D-CAR are located on the cell membrane of the autologous CAR-T cells.

[0027] In another preferred embodiment, the structure of the BCMA-CAR is shown in Formula I:

[0028] L1-S1-H1-TM1-C1-CD3ζ (I)

[0029] In the formula, "-" represents a linking peptide or peptide bond;

[0030] L1 is either absent or represents the first signal peptide sequence;

[0031] S1 is the antigen-binding domain that targets BCMA;

[0032] H1 is either the absence of a hinge or the first hinge region;

[0033] TM1 is the first transmembrane domain;

[0034] C1 is either absent or the first co-stimulatory signal molecule;

[0035] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0036] In another preferred embodiment, the structure of the GPRC5D-CAR is shown in Formula II:

[0037] L2-S2-H2-TM2-C2-Z2 (II)

[0038] In the formula, "-" represents a linking peptide or peptide bond;

[0039] L2 is either absent or has a second signal peptide sequence;

[0040] S2 is the antigen-binding domain that targets GPRC5D;

[0041] H2 is the area without or with a second hinge;

[0042] TM2 is the second transmembrane domain;

[0043] C2 is the second co-stimulatory signaling molecule;

[0044] Z2 is a cytoplasmic signaling sequence that is absent or derived from CD3ζ.

[0045] In another preferred embodiment, L1 and L2 are each independently a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0046] In another preferred embodiment, L1 is a signal peptide derived from CD8α; preferably, its amino acid sequence is shown in SEQ ID NO:15.

[0047] In another preferred embodiment, L2 is a signal peptide derived from CD8α; preferably, its amino acid sequence is shown in SEQ ID NO:15.

[0048] In another preferred embodiment, H1 and H2 are each independently a hinge region of a protein selected from the group consisting of CD8, CD28, or a combination thereof.

[0049] In another preferred embodiment, H1 is a hinge region derived from CD8α; preferably, its amino acid sequence is shown in SEQ ID NO:17.

[0050] In another preferred embodiment, H1 is a hinge region derived from CD28; preferably, its amino acid sequence is shown in SEQ ID NO:31.

[0051] In another preferred embodiment, the H2 is a hinge region derived from CD8α; preferably, its amino acid sequence is shown in SEQ ID NO:17.

[0052] In another preferred embodiment, TM1 and TM2 are each independently a transmembrane region of a protein selected from the group consisting of: CD8, CD28, CD8a, CD33, CD37, CD8α, CD5, CD16, ICOS, CD9, CD22, CD134, CD137, CD154, CD19, CD45, CD4, CD3ε, or a combination thereof.

[0053] In another preferred embodiment, TM1 is a transmembrane region derived from CD8α; preferably, its amino acid sequence is shown in SEQ ID NO:19.

[0054] In another preferred embodiment, TM1 is a transmembrane region derived from CD28; preferably, its amino acid sequence is shown in SEQ ID NO:33.

[0055] In another preferred embodiment, TM2 is a transmembrane region derived from CD8α; preferably, its amino acid sequence is shown in SEQ ID NO:19.

[0056] In another preferred embodiment, C1 and C2 are each independently a co-stimulatory signaling molecule of a protein selected from the group consisting of: CD28, 4-1BB, CD30, CD40, CD70, CD134, LIGHT, DAP10, CDS, ICAM-1, OX40, or a combination thereof.

[0057] In another preferred embodiment, C1 is a co-stimulatory signaling molecule derived from 4-1BB; preferably, its amino acid sequence is shown in SEQ ID NO:22.

[0058] In another preferred embodiment, C2 is a co-stimulatory signaling molecule derived from 4-1BB; preferably, its amino acid sequence is shown in SEQ ID NO:22.

[0059] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:25.

[0060] In a second aspect of the present invention, a method for preparing engineered autologous CAR-T cells as described in the first aspect of the present invention is provided, comprising the following steps:

[0061] (A) Provide an autologous T cell to be modified; and

[0062] (B) The autologous T cells are modified to express the BCMA-CAR and GPRC5D-CAR, thereby obtaining engineered autologous CAR-T cells as described in the first aspect of the present invention.

[0063] In another preferred embodiment, step (A) further includes isolating and / or activating the autologous T cells to be modified.

[0064] In another preferred embodiment, step (B) includes the following steps:

[0065] (B1) Introducing the autologous T cells with a polynucleotide encoding BCMA-CAR, or a vector containing the polynucleotide; and

[0066] (B2) Introduce the autologous T cells with a polynucleotide encoding GPRC5D-CAR or a vector containing the polynucleotide.

[0067] The step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

[0068] In another preferred embodiment, in step (B), a polynucleotide encoding a protein as shown in formula (III), or a vector containing said polynucleotide, is introduced into the autologous T cells.

[0069] CAR1-2A-CAR2 (III)

[0070] In the formula, CAR1 is BCMA-CAR;

[0071] CAR2 is a GPRC5D-CAR;

[0072] 2A is a self-cutting sequence.

[0073] In another preferred embodiment, the self-cutting sequence is selected from P2A, T2A, or a combination thereof.

[0074] In another preferred embodiment, the self-cleaving sequence amino acid sequence is shown in SEQ ID NO:27.

[0075] In another preferred embodiment, the self-cleaving sequence nucleotide sequence is as shown in SEQ ID NO:26.

[0076] In another preferred embodiment, the amino acid sequence of the protein represented by formula (III) is shown in SEQ ID NO:29.

[0077] In another preferred embodiment, the nucleotide sequence encoding the protein shown in formula (III) is shown in SEQ ID NO:28.

[0078] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

[0079] In another preferred embodiment, the method further includes a step of testing the function and effectiveness of the obtained engineered autologous T cells.

[0080] In a third aspect of the invention, a pharmaceutical composition is provided comprising engineered autologous CAR-T cells as described in the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0081] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0082] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection.

[0083] In another preferred embodiment, the concentration of the engineered autologous CAR-T 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

[0084] In another preferred embodiment, the pharmaceutical composition also contains other drugs for treating cancer or tumors (such as emerging antibody drugs, other CAR-T drugs, or chemotherapy drugs).

[0085] In a fourth aspect of the invention, there is provided the use of engineered autologous CAR-T cells as described in the first aspect of the invention or pharmaceutical compositions as described in the third aspect of the invention for the preparation of medicaments or formulations for the prevention and / or treatment of diseases.

[0086] In another preferred embodiment, the disease is an autoimmune disease, a tumor, or cancer.

[0087] In another preferred embodiment, the tumor comprises a tumor that highly expresses GPRC5D and / or BCMA.

[0088] In another preferred embodiment, the tumor comprises a tumor that simultaneously expresses GPRC5D and BCMA.

[0089] In another preferred embodiment, the tumor is selected from the group consisting of hematologic malignancies, solid tumors, or combinations thereof.

[0090] In another preferred embodiment, the tumor is a plasma cell malignant tumor or a B-cell malignant tumor, preferably a myeloma.

[0091] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma (MM), myelodysplastic syndrome, or combinations thereof.

[0092] In another preferred embodiment, the solid tumor is selected from the group consisting of: prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin's lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, bile duct cancer, ovarian cancer, gastric cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell carcinoma, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma, breast cancer, nasopharyngeal carcinoma, thymic carcinoma, or combinations thereof.

[0093] In another preferred embodiment, the tumor that highly expresses GPRC5D and / or BCMA is a multiple myeloma.

[0094] In another preferred embodiment, the tumor that highly expresses GPRC5D and / or BCMA is a relapsed and / or refractory multiple myeloma.

[0095] In some embodiments, the autoimmune disease is an antibody-mediated autoimmune disease, preferably including systemic lupus erythematosus, myasthenia gravis, neuromyelitis optica, multiple sclerosis, and lupus nephritis.

[0096] In a fifth aspect of the invention, a method for inhibiting tumor cells in vitro is provided, comprising:

[0097] Tumor cells are contacted with engineered autologous T cells as described in the first aspect of the invention, or with a pharmaceutical composition as described in the third aspect of the invention, thereby inhibiting the tumor cells.

[0098] In another preferred embodiment, the tumor cells are myeloma cells.

[0099] In a sixth aspect of the invention, a method for preventing and / or treating a disease is provided, comprising:

[0100] Administer a safe and effective amount of engineered autologous T cells as described in the first aspect of the invention, or a pharmaceutical composition as described in the third aspect of the invention, to the subject requiring treatment.

[0101] In another preferred embodiment, the object includes a human or a non-human mammal.

[0102] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, and rabbits) and primates (such as monkeys).

[0103] In another preferred embodiment, the method further includes administering other drugs for treating autoimmune diseases, cancer, or tumors to the subject requiring treatment.

[0104] In another preferred embodiment, the other drugs include CAR-T drugs.

[0105] In another preferred embodiment, the disease is an autoimmune disease, cancer, or tumor.

[0106] In another preferred embodiment, the tumor comprises a tumor that highly expresses GPRC5D and / or BCMA.

[0107] In another preferred embodiment, the tumor comprises a tumor that simultaneously expresses GPRC5D and BCMA.

[0108] In another preferred embodiment, the tumor that highly expresses GPRC5D and / or BCMA is a multiple myeloma.

[0109] In another preferred embodiment, the tumor that highly expresses GPRC5D and / or BCMA is a relapsed and / or refractory multiple myeloma.

[0110] 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

[0111] Figure 1 shows a schematic diagram of the structure of each CAR-T cell in this application.

[0112] Figure 2 shows the transfection efficiencies of BCMA, GPRC5D single CAR-T, and dual CAR-T cells on BCMA and GPRC5D CAR, respectively.

[0113] Figure 3 shows the degranulation effects of BCMA, GPRC5D single CAR-T and dual CAR-T cells on various target cells.

[0114] Figure 4 shows the cytotoxic effects of BCMA, GPRC5D single CAR-T and dual CAR-T cells on various target cells.

[0115] Figures 5A-5C show the antitumor efficacy of BCMA and GPRC5D with different structures in NPG mice with single and double CAR-T cells in different tumor-bearing models. Figure A shows the H929-lucBCMAGPRC5D double-positive model, Figure B shows the H929-CMAKO-luc GPRC5D single-positive model, and Figure C shows the CCRFBCMA-luc BCMA single-positive model.

[0116] Figure 6 shows the survival rates of single and double CAR-T cells corresponding to BCMA and GPRC5D with different structures in NPG mice with different tumor-bearing models. Figure A is the H929-lucBCMAGPRC5D double-positive model, Figure B is the H929-CMAKO-luc GPRC5D single-positive model, and Figure C is the CCRFBCMA-luc BCMA single-positive model.

[0117] In each figure, RD118 is a GPRC5D CAR-T cell constructed using clone 39 GPRC5D (see patent application: PCT / CN2022 / 140769); B26 is a BCMA CAR-T cell constructed using clone B26 BCMA (see patent CN201980002363.X); and RD140B is a bispecific CAR-T cell constructed using clone 39 GPRC5D and clone B26 BCMA in parallel expression. Detailed Implementation

[0118] Through extensive and in-depth research and numerous screenings, the inventors obtained CAR-T cells targeting BCMA and GPRC5D. The bispecific CAR-T cells of this invention exhibited significant killing effects on tumor cells that were single-positive or double-positive for both BCMA and GPRC5D. In vivo experiments showed that they could effectively inhibit tumor growth and prolong the survival of tumor-bearing mice. Based on these findings, this invention was completed.

[0119] the term

[0120] 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. Other definitions are set forth throughout the application.

[0121] 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.

[0122] The term “giving” 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, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0123] 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.

[0124] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0125] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0126] Antibodies are immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). These foreign substances are correspondingly called antigens. The basic structure of an antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. Based on the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The interaction between the variable region of one heavy chain (HCVR, also known as VH) and the variable region of one light chain (LCVR, also known as VL) forms the antigen-binding site (Fv). Within the variable region, the composition and sequence of amino acid residues in certain areas are more variable than in other areas (backbone regions, FR), and these are called hypervariable regions (HVR). Hypervariable regions are actually the key sites for antibody-antigen binding. Because these hypervariable region sequences are complementary to the antigenic determinants, they are also called complementarity-determining regions (CDRs). Both the heavy and light chains have three complementarity-determining regions, designated HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen.

[0127] A single-chain antibody (scFv) is composed of the variable regions of the heavy and light chains of an antibody linked together by short peptides to form a single peptide chain. Through proper folding, the variable regions from the heavy and light chains interact non-covalently to form the Fv segment, thus allowing scFv to retain its affinity for the antigen relatively well.

[0128] "Single domain antibody (sdAb, or VHH)" and "nanobody" have the same meaning: they refer to the cloning of the variable region of the antibody heavy chain to construct a nanobody consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Usually, an antibody that is naturally missing from the light chain and the heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0129] As used herein, when referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence uniformity") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Generally, sequence alignment is performed and gaps (if any) are introduced before calculating the percentage of similarity between two amino acid or nucleotide sequences. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, sequence identity is obtained by dividing the number of matched positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and / or gap lengths are also taken into account. For the purposes of this invention, the publicly available alignment software BLAST (available at ncbi.nlm.nih.gov) can be used to obtain optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using default settings. In some embodiments, the “at least 90% sequence identity” of the present invention includes, but is not limited to, at least 95%, at least 98%, at least 99%, or even 100% sequence identity.

[0130] Chimeric antigen receptor (CAR)-T cells

[0131] As used herein, a "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide from the extracellular domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a particular antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0132] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0133] Connectors can be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR.

[0134] As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular or cytoplasmic domain of a polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2-100 amino acids, and most preferably 3-50 amino acids. More preferably, the linker is a flexible linker, for example, (G4S)n, where n is 1-4.

[0135] The CAR of this invention, when expressed in T cells, is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, resulting in a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with one or more intracellular domains derived from co-stimulatory molecules and the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a 4-1BB and / or CD28 signaling domain and a CD3ζ signaling domain.

[0136] As used in this article, "antigen-binding domain" refers to Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibody fragments, or single-domain antibody fragments that have antigen-binding activity.

[0137] The bispecific CAR-T cells provided by this invention are autologous CAR-T cells, that is, CAR-T cells extracted and isolated from the patient's own body and engineered to express parallel chimeric antigen receptors.

[0138] Method for preparing autologous CAR-T cells in this invention

[0139] The present invention also provides a method for preparing autologous CAR-T cells of the present invention, comprising the following steps:

[0140] (A) Provide an autologous T cell to be modified; and

[0141] (B) The autologous T cells are modified to express the BCMA-CAR and GPRC5D-CAR, thereby obtaining the engineered autologous CAR-T cells of the present invention.

[0142] The method of the present invention involves simultaneously or sequentially introducing polynucleotides encoding BCMA-CAR and GPRC5D-CAR into autologous T cells. The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a known vector containing the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0143] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0144] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0145] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0146] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0147] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vectors can be readily introduced into host cells by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0148] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Calcium phosphate transfection is a preferred method for introducing polynucleotides into host cells.

[0149] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0150] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0151] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0152] In the case of using a non-viral delivery system, genome editing technologies such as CRISPR-Cas9, ZFN, or TALEN are used exemplary to accomplish the present invention.

[0153] Therapeutic applications

[0154] The engineered autologous CAR-T cells of this invention can induce CAR-mediated T cell responses.

[0155] Therefore, the present invention also provides a method for stimulating a T-cell-mediated immune response to a target cell population or tissue in a mammal, comprising the step of administering the CAR-T cells of the present invention to a mammal.

[0156] In one embodiment, the present invention includes a class of cell therapies in which autologous T cells are genetically modified to express the CAR of the present invention, and the CAR-T cells are infused into desired recipients. The infused cells are able to kill tumor cells in the recipient's body. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting efficacy that can lead to sustained tumor control.

[0157] In one embodiment, the autologous CAR-T cells of the present invention can undergo robust in vivo T cell expansion and sustain for an extended period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR.

[0158] Therapeutic indications include cancers or tumors associated with BCMA and / or GPRC5D, such as BCMA and / or GPRC5D-positive tumors or cancers. These cancers or tumors can include solid tumors and hematologic malignancies, particularly multiple myeloma.

[0159] The CAR-modified autologous 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 target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers 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 compositions of the present invention are preferably formulated for intravenous administration.

[0160] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0161] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The 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 patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0162] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0163] 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 therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded autologous T cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0164] The main advantages of this invention include:

[0165] This invention's parallel-structured CAR-T cells target both BCMA and GPRC5D tumor markers, expanding the therapeutic scope of CAR-T cells and preventing off-target escape from single targets. Experiments show that the bispecific CAR-T cells of this invention exhibit significant killing effects on tumor cells that are single-positive or double-positive for BCMA and GPRC5D.

[0166] 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.

[0167] Example 1: Detection of CAR molecule expression on the surface of CAR-T cells

[0168] Based on previous research, the applicant constructed GPRC5D-CAR and BCMA-CAR single-expression and co-expression CAR-T cells. Among them, RD118 is a GPRC5D CAR-T cell constructed using the GPRC5D antibody clone No. 39 (see patent application: PCT / CN2022 / 140769); B26 is a BCMACAR-T cell constructed using the B26 BCMA antibody clone (see patent CN201980002363.X); RD140B is a bispecific CAR-T cell constructed using the GPRC5D antibody clone No. 39 and the B26 BCMA antibody clone in parallel expression.

[0169] After constructing each CAR structural vector, the CAR vectors were packaged into lentiviruses. Binding to BCMA antigen and specific anti-GPRC5D antibody was detected 5–7 days after T cell transfection with the CAR lentivirus. The structure of the CAR is shown in Figure 1.

[0170] The simplified experimental steps for detecting the binding of BCMA antigen and specific anti-GPRC5D antibody are as follows:

[0171] 1) Take 2×10 5 Take two wells of each type of CAR-T / T cell, wash once with PBS, centrifuge at 300g for 5 minutes, and discard the supernatant.

[0172] 2) Resuspend the cell pellet in 100 μL / well PBS, add FITC-BCMA antigen and PE-GPRC5D anti-antibody (developed by Nanjing Reindeer) to each CAR, and incubate at 4°C in the dark for 30 minutes.

[0173] 3) Wash twice with PBS and centrifuge at 300g for 5 minutes.

[0174] 4) Resuspend in 200 μL PBS and perform flow cytometry analysis.

[0175] The main materials and reagents are as follows:

[0176] FITC-BCMA,Acro,Cat.No.BCA-HF254

[0177] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0178] Experimental results:

[0179] The results are shown in Figure 2, which shows the binding of CAR-T cells corresponding to various CAR structures with BCMA antigen and specific anti-GPRC5D antibody. There was no significant difference between the BCMACAR expression of single-target CAR-T cell B26 and the GPRC5D CAR expression of single-target CAR-T cell RD118 and the BCMACAR and GPRC5D CAR expression of dual-target parallel CAR-T cell RD140B.

[0180] Example 2. CD107a threshing experiment

[0181] Experimental principle and purpose:

[0182] CD107a is a marker of intracellular microvesicles. When granzyme-loaded microvesicles fuse with the cell membrane, the amount of CD107a on the cell membrane increases. When monensin (purchased from BioLegend) is used to block its reabsorption, the intensity of microvesicle release can be quantitatively reflected. When CAR-T cells are stimulated by target antigens on target cells, granzyme release occurs, and the increase in CD107a can be detected by flow cytometry to determine the activation status of T cells.

[0183] Brief experimental steps for threshing CD107a:

[0184] 1) Centrifuge the CAR-T cells to be tested and the target cells separately at 300g for 5 min at room temperature, discard the supernatant, and resuspend them in 1640 medium + 10% FBS to a concentration of 2x10. 6 cells / mL;

[0185] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate, and mix well.

[0186] 3) Add 1.5 μL of PE / Cy7 anti-human CD107a antibody and 0.2 μL of monensin to each well of cells, and then incubate in a cell culture incubator at 37°C and 5% CO2 for 4 hours;

[0187] 4) After incubation, centrifuge at 300g for 5 minutes at 4℃, discard the supernatant, and wash the cells twice with 200ul PBS;

[0188] 5) Resuspend the cells in 100 μL PBS, and add 1.5 μL of BV421 anti-human CD8 and FITC-BCMA antigen or PE-GPRC5D anti-antibody respectively. Mix well and incubate on ice in the dark for 20 min.

[0189] 6) After incubation, wash the cells three times with 200 μL PBS; resuspend them in 100 μL PBS and then detect them by flow cytometry.

[0190] The main samples and reagents are as follows:

[0191] Target cells: 8226 (BCMA+GPRC5D+), U266 (BCMA+GPRC5D+); MM1.s (BCMA+GPRC5D+); H929 (BCMA+GPRC5D+); H929BCMAKO (BCMA-GPRC5D+); CCRFBCMA (BCMA+GPRC5D-); H929GPRC5DKO (BCMA+GPRC5D-);

[0192] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0193] Monensin,BioLegend,Cat.No.420701;

[0194] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0195] BV421 mouse anti-human CD8,BD,Cat.No.301036;

[0196] FITC-BCMA,Acro,Cat.No.BCA-HF254

[0197] Experimental results:

[0198] CAR-T cells were obtained via lentiviral transduction and cultured in vitro for 9-12 days before CD107a degranulation. The CAR-T cells to be tested, target cells, monensin, and CD107a antibody were co-incubated for 4 hours, with both CAR-T cell and target cell densities at 2 × 10⁶ cells / year. 5 Cells / mL. CAR+ samples were then labeled with CD8 antibody, FITC-BCMA antigen, or PE-GPRC5D anti-antibody before flow cytometry analysis. In Flowjo software, the viable cell gate (P1) was selected from the scatter plot, and cell debris was removed. Cells in the P1 gate were further analyzed to select a single dispersed cell gate (P2). Then, CD8-positive cells were further selected from the P2 gate (P3). Finally, in the P3 gate, the proportion of CD107a-positive cells among FITC-BCMA antigen or PE-GPRC5D anti-antibody-positive cells (i.e., CAR-positive cells) was analyzed to determine their degranulation level.

[0199] As shown in Figure 3, the degranulation level of single-target BCMACAR-T cells B26 was significantly increased only after co-incubation with BCMA-positive cells, while no significant increase was observed after co-incubation with BCMA-knockout H929BCMA KO cells. The degranulation level of single-target GPRC5D CAR-T cells RD118 was significantly increased only after co-incubation with GPRC5D-positive cells, while no significant increase was observed after co-incubation with GPRC5D-knockout H929GPRC5D KO cells or CCRF BCMA cells overexpressing BCMA on BCMA-negative cells. The degranulation level of dual-target CAR-T cells RD140B was significantly increased after incubation with both GPRC5D and BCMA-positive and double-positive target cells. Compared with the corresponding single-target CAR-T cells, the degranulation level of dual-target CAR-T cells was higher or comparable.

[0200] Example 3. In vitro cell killing experiment

[0201] Experimental objective and principle:

[0202] In vitro cell killing experiments used 8226, U266, MM1.s, and H929 cells as double-positive target cells for BCMA and GPRC5D, H929 GPRC5D KO cells with GPRC5D knockout as single-positive target cells for BCMA, and H929 BCMA KO cells with BCMA knockout as single-positive cells for GPRC5D to evaluate the antigen-specific killing ability of dual CAR cells with different structures.

[0203] In this study, target cells were obtained through lentiviral transduction to stably express firefly luciferase. Therefore, the luciferase activity in the sample can reflect the number of target cells. CAR-T cells and target cells were co-incubated. When target cells were killed by CAR-T cells, luciferase was released and quickly inactivated (firefly luciferase has a half-life of approximately 0.5 hours). If target cells were not killed or inhibited by CAR-T cells, more luciferase would be produced as target cells proliferated and luciferase expression continued. Therefore, luciferase activity can be used to detect the CAR-T cell-killing effect.

[0204] Brief experimental steps for in vitro cell killing:

[0205] 1) Centrifuge the target cells at 500g for 5 min at room temperature, discard the supernatant, and resuspend them in 1640+10% FBS medium to a concentration of 1x10⁻¹. 5 Cells / mL; Add 100 μL of target cells to each well of a 96-well plate;

[0206] 2) Based on the CAR positivity rate and effector-to-target ratio of the CAR-T samples to be tested, add 100 μL of CAR-T cells to each well of a 96-well plate and mix them with the target cells; then incubate in a carbon dioxide incubator for 24 h.

[0207] 3) Use a luciferase assay kit to detect the luciferase activity in each well sample.

[0208] The main samples and reagents are as follows:

[0209] Target cells: 8226 (BCMA+GPRC5D+), U266 (BCMA+GPRC5D+); MM1.s (BCMA+GPRC5D+); H929 (BCMA+GPRC5D+); H929BCMAKO (BCMA-GPRC5D+); CCRFBCMA (BCMA+GPRC5D-); H929GPRC5DKO (BCMA+GPRC5D-);

[0210] Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

[0211] Experimental results:

[0212] CAR-T cell samples and a fixed number of target cells (1×10⁶) were used. 4CAR-T cells were mixed at different effector-to-target ratios (E:T) and incubated for 24 hours. Then, the luciferase activity (RLU) in the samples was measured. Since luciferase activity reflects the number of target cells in the sample, changes in luciferase activity can indicate the CAR-T cells' ability to kill / inhibit target cells. A lower luciferase activity (RLU) reading indicates that more target cells have been killed.

[0213] As shown in Figure 4, the single-target BCMACAR-T B26 only significantly killed BCMA-positive cells, but did not significantly kill H929 BCMA KO cells with BCMA knockout. The single-target GPRC5DCAR-T RD118 only significantly killed GPRC5D-positive cells, but did not significantly kill H929GPRC5D KO cells with GPRC5D knockout. The dual-target CAR-T RD140B significantly killed both GPRC5D and BCMA-positive and double-positive target cells. Compared with the corresponding single-target CAR-T, the dual-target CAR-T has a higher or comparable killing ability.

[0214] Example 4. In vivo antitumor efficacy experiment in NPG tumor-bearing mice

[0215] To further verify the antitumor efficacy of dual CARRD140B in mice, the H929-Luc tumor cell line expressing firefly luciferase (ffLuc) was used. H929 is a naturally occurring cell line that is highly positive for both BCMA and GPRC5D. H929-Luc cells were injected via tail vein into immunodeficient NCG mice. After tumor formation, candidate clone CAR-T cells were injected to verify their antitumor effect (Figures 5A and 6A). H929 BCMA KO-Luc cells were injected via tail vein into immunodeficient NCG mice. After tumor formation, candidate clone CAR-T cells were injected to verify the antitumor effect of various CAR cells in a GPRC5D single-positive tumor model. CCRF BCMA-Luc cells were injected via tail vein into immunodeficient NPG mice. After tumor formation, candidate clone CAR-T cells were injected to verify the antitumor effect of various CAR cells in a BCMA single-positive tumor model.

[0216] Brief experimental procedure:

[0217] a) Establish the double-positive cell line H929 and the single-positive cell line H929BCMAKO and CCRF-BCMA, respectively, overexpressing firefly luciferase (ffLuc), and identify that they can express firefly luciferase normally.

[0218] b) After culturing the cells to the logarithmic growth phase, collect the cells;

[0219] c) Purchase NPG mice, feed them for 1 week, inject the corresponding target cells into the tail vein, and observe the general condition of the mice every day after injection.

[0220] d) When the tumors grew to a suitable size, mice were divided into groups and injected via the tail vein with CAR-T cells, control untransfected Mock-T cells, and cryopreservation solution, respectively.

[0221] e) Regularly perform imaging observations, weigh the mice, observe their general condition, and record the data.

[0222] Experimental results:

[0223] The in vivo efficacy of the drug in mice is shown in Figures 5A-5C. As shown in Figure 5A, the tumor burden continued to increase after PBS and mock T cell infusion, while the tumor burden of single CAR B26 was slightly lower than that of PBS and mock T. The tumor burden of single CAR RD118 and dual CAR RD140B was significantly inhibited, and the 28-day survival rate of mice was greater than 80% (Figures 5A and 6A).

[0224] The in vivo efficacy of GPRC5D monopositive tumor model is shown in Figures 5B and 6B. As shown in Figure 5B, tumor burden continued to increase after infusion of PBS, mock T, and B26 cells. Both dual CAR RD140B and single CAR 118 significantly inhibited tumor burden, and the 49-day survival rate of mice was 100%. The in vivo efficacy of BCMA monopositive tumor model is shown in Figures 5C and 6C. Tumor burden continued to increase after infusion of PBS, mock T, and RD118 cells. Single CAR B26 had a certain inhibitory effect on tumors, while dual CAR RD140B significantly inhibited tumor burden, and was stronger than BCMA single CAR B26. The 42-day survival rate of mice in this model was 100%.

[0225] sequence list

[0226] 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. A bispecific engineered autologous chimeric antigen receptor (CAR)-T cell, characterized in that, The engineered autologous CAR-T cells express BCMA-CAR and GPRC5D-CAR. The BCMA-CAR contains an antigen-binding domain that targets BCMA, and the antigen-binding domain that targets BCMA includes the following complementarity-determining region (CDR): The amino acid sequence is shown in SEQ ID NO:1, which is the light chain CDR1 (LCDR1). The amino acid sequence is shown in AAS for the light chain CDR2 (LCDR2). The amino acid sequence is shown in SEQ ID NO:2 for the light chain CDR3 (LCDR3). The amino acid sequence is shown in SEQ ID NO:3 for the heavy chain CDR1 (HCDR1). The amino acid sequence is shown in SEQ ID NO:4 for the heavy chain CDR2 (HCDR2). The amino acid sequence is shown in SEQ ID NO:5 for the heavy chain CDR3 (HCDR3); The GPRC5D-CAR contains an antigen-binding domain targeting GPRC5D, and the antigen-binding domain targeting GPRC5D includes the following complementarity-determining region (CDR): The amino acid sequence is shown in SEQ ID NO:8 for the heavy chain CDR1 (HCDR1). The amino acid sequence is shown in SEQ ID NO:9 for the heavy chain CDR2 (HCDR2). The amino acid sequence is shown in SEQ ID NO:10 for the heavy chain CDR3 (HCDR3).

2. The engineered autologous CAR-T cells as described in claim 1, characterized in that, The antigen-binding domain targeting BCMA is a single-chain antibody, and / or the antigen-binding domain targeting GPRC5D is a single-domain antibody.

3. The engineered autologous CAR-T cells as described in claim 1, characterized in that, The amino acid sequence of the antigen-binding domain targeting BCMA is as shown in SEQ ID NO:6, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

4. The engineered autologous CAR-T cells as described in claim 1, characterized in that, The amino acid sequence of the antigen-binding domain targeting GPRC5D is as shown in SEQ ID NO:11, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

5. The engineered autologous CAR-T cells as described in claim 1, characterized in that, The structure of the BCMA-CAR is shown in Formula I: L1-S1-H1-TM1-C1-CD3ζ (I) In the formula, "-" represents a linking peptide or peptide bond; L1 is either absent or represents the first signal peptide sequence; S1 is the antigen-binding domain that targets BCMA; H1 represents the area with no hinge or the first hinge region; TM1 is the first transmembrane domain; C1 is either absent or the first co-stimulatory signal molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

6. The engineered autologous CAR-T cells as described in claim 1, characterized in that, The structure of the GPRC5D-CAR is shown in Formula II: L2-S2-H2-TM2-C2-Z2 (II) In the formula, "-" represents a linking peptide or peptide bond; L2 is either absent or has a second signal peptide sequence; S2 is the antigen-binding domain that targets GPRC5D; H2 is the area without or with a second hinge; TM2 is the second transmembrane domain; C2 is the second co-stimulatory signaling molecule; Z2 is a cytoplasmic signaling sequence that is absent or derived from CD3ζ.

7. A method for preparing engineered autologous CAR-T cells as described in claim 1, characterized in that, Includes the following steps: (A) Provide an autologous T cell to be modified; and (B) The autologous T cells are modified to express the BCMA-CAR and GPRC5D-CAR, thereby obtaining the engineered autologous CAR-T cells as described in claim 1.

8. The method as described in claim 7, characterized in that, In step (B), a polynucleotide encoding the protein shown in formula (III), or a vector containing said polynucleotide, is introduced into the autologous T cells into CAR1-2A-CAR2 (III). In the formula, CAR1 is BCMA-CAR; CAR2 is a GPRC5D-CAR; 2A is a self-cutting sequence.

9. The method as described in claim 8, characterized in that, The amino acid sequence of the protein shown in formula (III) is shown in SEQ ID NO:

29.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains engineered autologous CAR-T cells as described in claim 1, and pharmaceutically acceptable carriers, diluents, or excipients.

11. Use of an engineered autologous CAR-T cell as described in claim 1 or a pharmaceutical composition as described in claim 10, characterized in that, Used to prepare medicines or preparations for the prevention and / or treatment of diseases.

12. The use as described in claim 11, characterized in that, The disease mentioned is an autoimmune disease, tumor, or cancer.

13. The use as described in claim 11, characterized in that, The tumor is a plasma cell malignant tumor or a B-cell malignant tumor, preferably a myeloma.

14. A method for inhibiting tumor cells in vitro, characterized in that, include: Tumor cells are contacted with engineered autologous T cells as described in claim 1, or with the pharmaceutical composition as described in claim 10, thereby inhibiting the tumor cells.

15. A method for preventing and / or treating a disease, characterized in that, include: Administer a safe and effective amount of the engineered autologous T cells as described in claim 1, or the pharmaceutical composition as described in claim 10, to the subject requiring treatment.

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