WO2023109257A1 - Humanized bcma antibody and bcma-car-t/bcma-car-dnt cell - Google Patents

Abstract

The present application provides a humanized BCMA single-chain variable fragment (scFv), and also provides a chimeric antigen receptor constructed by using the humanized BCMA single-chain variable fragment. The chimeric antigen receptor comprises from the N-terminus to the C-terminus: (i) a single-chain variable fragment (scFv), (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain. The humanized BCMA-CAR-T/BCMA-CAR-DNT cell in the present application has specific killing activity, and can secrete IFN-γ when incubated together with a BCMA-positive target cell.

Description

Humanized BCMA antibody and BCMA-CAR-T/BCMA-CAR-DNT cells technical field

The present invention relates to a humanized BCMA antibody, humanized BCMA-CAR-T cells (PMC1497) and BCMA-CAR-DNT cells. The BCMA-CAR-T cells and BCMA-CAR-DNT cells specifically inhibit the growth of multiple myeloma, and can be used in the field of adoptive immune gene therapy for tumors.

Background technique

Immunotherapy is emerging as a very promising approach to cancer treatment. T cells, or T lymphocytes, are the armies of our immune system that constantly seek out foreign antigens and distinguish abnormalities (cancerous or infected cells) from normal cells. Genetic modification of T cells with CAR (chimeric antigen receptor) constructs is the most common approach to construct tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAAs) can be infused into patients (termed adoptive cell transfer or ACT), which is an effective immunotherapy approach [1,2]. Compared with chemotherapy or antibody drugs, the advantage of CAR-T technology is that the reprogrammed engineered T cells can proliferate and persist in the patient's body ("a living drug") [1,2].

CARs typically consist of a monoclonal antibody-derived single-chain variable fragment (scFv) located in the N-terminal portion, a hinge, a transmembrane domain, and a number of intracellular coactivation domains: (i) CD28, (ii) CD137 (4-1BB ), CD27 or other co-stimulatory domains, in tandem with the activating CD3-zeta domain. (Fig. 1) [2,3]. CARs have evolved from first-generation (without costimulatory domain) to second-generation (with one costimulatory domain) to third-generation CARs (with multiple costimulatory domains). Generation of CARs with two co-stimulatory domains (so-called third-generation CARs) resulted in increased activity of cytolytic CAR-T cells and improved persistence of CAR-T cells, thereby enhancing their antitumor activity. The fourth-generation CAR structure introduces a structure capable of inducing T cells to secrete cytokines on the basis of the third-generation CAR molecular components to enhance the biological activity and proliferation ability of CAR-T cells; the fifth-generation CAR follows the backbone of the second-generation CAR in CD3 and The IL-2 receptor domain is added between the CD28 signal region, and the YXXQ sequence is combined at the C-terminal CD3. The design of the fifth-generation CAR is based on simulating the activated state of T cells when they bind antigens, and at the same time activate TCR and costimulatory domains CD28, and cytokine triple signal, enhance the proliferation, survival and anti-tumor effect of CAR-T.

BCMA

B cell maturation antigen (BCMA) is a cell surface receptor, also known as CD269 and tumor necrosis factor receptor superfamily member 17 (TNFRSF17), encoded by the TNFRSF17 gene. This receptor is predominantly expressed on mature B lymphocytes and plasma cells and is in most cases overexpressed in multiple myeloma (MM) patients [4]. This makes it a new ideal target antigen for MM treatment, and it is also one of the most widely studied myeloma CAR targets. Current therapies targeting BCMA in MM include monoclonal antibodies, bispecific antibodies, T cell immunotherapy, and CAR-T therapy [4], [5].

Human BCMA protein consists of 184 amino acids: amino acids 1-54 are the extracellular domain; amino acids 55-77 are the transmembrane domain; amino acids 78-184 are the cytoplasmic domain. The amino acid sequence of BCMA is shown in FIG. 2 . BCMA lacks a signal peptide similar to the BAFF receptor and transmembrane activator and cyclophilin ligand interactor and calcium regulator (TACI) [5]. These receptors play a major role in the maturation and differentiation of B cells into plasma cells. Their ligands include BAFF and APRIL, which are highly expressed in MM patients [5]. Monoclonal antibodies interact through receptor-ligands, and CAR-T cell therapy kills MM cells by binding to BCMA protein. BCMA also interacts with TRAF1, 2, 3, 5 and 6.

As an ideal antigen target, BCMA is preferentially expressed on plasma cells rather than hematopoietic stem cells; binding of B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) to BCMA can promote the growth and proliferation of plasma cells in the bone marrow. Although the expression of BCMA is heterogeneous, it is ubiquitously present in all MM cells and its overexpression has important prognostic value. On March 26, 2021, the FDA approved Abecma (idecabtage nevicleucel; ide-cel) from BMS as the first directed chimeric antigen receptor (CAR) T cell immunotherapy targeting B cell maturation antigen (BCMA) for For the treatment of adult patients with relapsed or refractory multiple myeloma who have previously undergone four or more lines of therapy including immunomodulators, proteasome inhibitors and anti-CD38 monoclonal antibodies (this is the first FDA-approved marketed targeted CAR-T therapy for BCMA). On February 28, 2022, the CAR-T therapeutic product CARVYKTI (Cilta-cel, Cedargiolunsa) developed by Johnson & Johnson's Janssen and Legend Biotech targeting B-cell maturation antigen (BCMA) was also approved by the US FDA for marketing . Clinical data show that the effectiveness of CAR-T cell therapy for MM patients is as high as 90%. For MM patients, the deeper the degree of remission, the longer their survival time, so achieving a deep remission rate, including the strict complete remission rate (sCR) and minimal residual disease (MRD) negative conversion rate, is also an important treatment for patients Target. BCMA-CAR-T therapy can effectively improve the deep remission rate of patients and prolong the relapse time of patients, which has been strongly verified in patients with relapsed and refractory MM.

However, the currently marketed immune cell therapy products are all prepared from the patient's own cells. The preparation cost is expensive, and the preparation process is not easy to standardize. Patients have to wait for a long time for the preparation cycle, and are limited by the patient's physical condition and disease progression. In addition to the certain failure rate of CAR-T cells prepared from the patient's own cells, it has been reported that the virus with the CAR structure was transfected into the residual tumor cells in the patient's body, resulting in the death of the patient. In recent years, many companies at home and abroad have used cells from healthy donors to prepare general-purpose immune cell drugs for allogeneic infusion, such as Allogene (USA) and Crispr Therapeutics, which use gene editing technology to obtain general-purpose characteristics of cells. (Switzerland) and Caribou Biosciences (US), etc., as well as Fate Therapeutics (US, iPSC-CAR-NK cells), NKarta (US, CAR-NK) and Adicet Bio (US, CAR-γδT cells), etc. Such cell products can greatly reduce the production cost of CAR-T cells. Its standardized cell collection and reinfusion process is expected to fundamentally solve the problem of accessibility of CAR-T cell products. However, the technical development and clinical research of general-purpose CAR-T It is still in the stage of clinical trials and faces a series of challenges such as effective expansion in vivo, retention time in vivo, proportion of T cell subsets, and expansion of clinical indications. There is no approved marketed product globally.

Therefore, there is an urgent need in this area to develop a immune cell derived from a healthy donor, which has high safety (does not cause graft-versus-host disease (GvHD)), low immunogenicity (does not cause host-versus-graft immune response, and has a long retention time in vivo). Long), significant killing effect, simple and controllable production process, and low cost universal CAR-T cells in stock.

Contents of the invention

The present invention provides a humanized BCMA antibody and a humanized single-chain variable fragment (scFv) comprising a VH having the amino acid sequence of SEQ ID NO: 2 and a VH having the amino acid sequence of SEQ ID NO: 3 VL. The present invention also relates to a humanized BCMA chimeric antigen receptor fusion protein, which comprises from N-terminus to C-terminus: single-chain variable fragment (scFv) amino acid sequence SEQ ID NO: 1, transmembrane domain amino acid sequence SEQ ID NO:12, at least one co-stimulatory domain CD28 (amino acid sequence SEQ ID NO:19) or 41-BB (amino acid sequence SEQ ID NO:14) and activation domain CD3ζ (amino acid sequence SEQ ID NO:16). The present invention also provides a construction method of the humanized BCMA chimeric antigen receptor, and provides scFv gene and second-generation CAR structural gene.

The humanized BCMA-CAR chimeric antigen receptor in the present invention can construct an antigen receptor T cell on human immune cells through a carrier, including autologous CAR-T cells. The present invention also provides a construction method for preparing universal antigen chimeric receptor BCMA-CAR-DNT cells.

The first aspect of the present invention provides an anti-human BCMA antibody or an antigen-binding fragment thereof, comprising a VH having an amino acid of SEQ ID NO:2, and a VL having an amino acid of SEQ ID NO:3.

In another preferred embodiment, the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.

In another preferred example, the antibody is a diabody or a single-chain antibody.

In another preferred example, the antibody is a monoclonal antibody.

A second aspect of the present invention provides a single-chain variable fragment (scFv) of humanized BCMA, comprising a VH with an amino acid of SEQ ID NO: 2, and a VL with an amino acid of SEQ ID NO: 3.

In another preferred embodiment, the scFv further comprises a linker between VH and VL.

In another preferred embodiment, the scFv has the amino acid sequence of SEQ ID NO:1.

A third aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein, comprising from the N-terminus to the C-terminus:

(i) scFv according to the second aspect of the present invention,

(ii) transmembrane domain,

(iii) at least one co-stimulatory domain, and

(iv) Activation domain.

In another preferred example, the CAR has a structure as shown in formula I:

L-scFv-H-TM-C-CD3ζ (Formula I)

In the formula, L is nothing or a signal peptide sequence;

scFv is an antibody single-chain variable region sequence targeting humanized BCMA antigen;

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

In another preferred embodiment, said L is a signal peptide of a protein selected from the following group: CD8, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred example, said L is a signal peptide derived from CD8.

In another preferred embodiment, said H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

In another preferred example, said H is a hinge region derived from CD8.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM includes a transmembrane region derived from CD8, and/or a transmembrane region derived from CD28.

In another preferred example, said C is a co-stimulatory signal molecule selected from the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred example, the co-stimulatory domain is CD28 or 4-1BB.

In another preferred example, the activation domain is CD3ζ.

In another preferred embodiment, the CAR fusion protein has the amino acid sequence of SEQ ID NO:4.

The fourth aspect of the present invention provides a nucleic acid encoding the CAR according to the third aspect of the present invention or encoding the anti-human BCMA antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

In another preferred embodiment, the nucleic acid encodes the CAR described in the third aspect of the present invention, which has the nucleotide sequence shown in SEQ ID NO:17.

The fifth aspect of the present invention provides a vector, characterized in that the vector contains the nucleic acid molecule as described in the fourth aspect of the present invention.

In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

In another preferred example, the vector is a lentiviral vector.

The sixth aspect of the present invention provides an engineered immune cell modified to express the CAR as described in the third aspect of the present invention.

In another preferred example, the immune cells are DNT cells.

In another preferred example, the DNT cells are CD3+CD4-CD8- T cells.

In another preferred example, the immune cells are T cells.

In another preferred example, the T cells are CD3+CD4+ or CD3+CD8+ T cells.

In another preferred example, the immune cells are natural killer cells.

The seventh aspect of the present invention provides a method for preparing the engineered immune cell as described in the sixth aspect of the present invention, comprising the step of: using the nucleic acid as described in the fourth aspect of the present invention or the nucleic acid as described in the fifth aspect of the present invention The vector is transduced into immune cells.

In another preferred example, the method further includes the step of detecting the function and effectiveness of the CAR-expressing immune cells.

The eighth aspect of the present invention provides an antibody-drug conjugate, which contains:

(a) an antibody portion comprising the anti-human BCMA antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the scFv described in the second aspect of the present invention; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

In another preferred example, the antibody part is coupled to the coupling part through a chemical bond or a linker.

The ninth aspect of the present invention provides a preparation containing the anti-human BCMA antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the scFv described in the second aspect of the present invention, or the anti-human BCMA antibody described in the first aspect of the present invention. The chimeric antigen receptor (CAR) fusion protein of the third aspect, or the carrier of the fifth aspect of the present invention, or the engineered immune cell of the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

The tenth aspect of the present invention provides an anti-human BCMA antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, the scFv as described in the second aspect of the present invention, the scFv as described in the third aspect of the present invention The chimeric antigen receptor (CAR) fusion protein, the carrier as described in the fifth aspect of the present invention, or the engineered immune cell as described in the sixth aspect of the present invention, are prepared for the prevention and/or treatment of cancer or tumor Use in medicines and medicines for autoimmune diseases.

In another preferred example, the tumor is selected from the group consisting of blood tumors, solid tumors, or a combination thereof.

In another preferred example, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse massive B cell lymphoma (DLBCL), or a combination thereof.

In another preferred example, the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer , ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or its combination.

In another preferred example, the solid tumor is selected from the group consisting of ovarian cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, liver cancer, endometrial cancer, or a combination thereof. In the eleventh aspect of the present invention, there is provided a method for treating a disease, comprising administering a therapeutically effective amount of the anti-human BCMA antibody or antigen-binding fragment thereof as described in the first aspect of the present invention to a subject in need of treatment, The engineered immune cell according to the sixth aspect of the present invention, or the preparation according to the ninth aspect of the present invention.

In another preferred example, the disease is cancer or tumor and autoimmune disease.

Brief description of the drawings

Figure 1. The five-generation iterative structure of CAR. [3]

The amino acid sequence (SEQ ID NO:20) of Fig. 2.BCMA protein wherein the underline is extracellular domain.

Figure 3. Schematic diagram of the structure of humanized BCMA CAR: the second-generation BCMA-CAR uses 41BB as the co-stimulatory domain.

Figure 4. Differentiation flow diagram of non-transduced T cells and BCMA-CAR-T cell populations expanded in vitro.

Figure 5A. Proliferation curves of DNT cells and BCMA-CAR-DNT cells expanded at different times in vitro.

Figure 5B. Survival curves of DNT cells and BCMA-CAR-DNT cells expanded in vitro at different times.

Figure 6. The flow chart of the purity (CD3+CD4-CD8-) of DNT cells and BCMA-CAR-DNT cells on the 10th day of in vitro expansion.

Figure 7A. Flow diagram of cell population differentiation of BCMA-CAR-DNT and DNT cells on day 10 of in vitro expansion.

Figure 7B. Differentiation curves of BCMA-CAR-DNT cells expanded in vitro at different times.

Figure 8. Using anti-mouse F(ab)'2(mFAB), anti-human F(ab)'2(mFAB) and biotin-labeled BCMA protein to detect BCMA-CAR-T cells expanded on day 9 in vitro Flow chart of positive rate.

Figure 9. The flow chart of the positive rate of BCMA-CAR-DNT cells on the 10th day of in vitro expansion.

Figure 10A uses RTCA to detect the toxicity of humanized BCMA-CAR-T cells to CHO-BCMA cells.

Figure 10B. The toxicity of humanized BCMA-CAR-T cells to CHO cells was detected by RTCA.

Figure 11A. On the 10th day of in vitro expansion, the real-time tumor killing curve of DNT cells and BCMA-CAR-DNT cells against BCMA-CHO cell lines was detected by RTCA (effect-to-target ratio 2:1).

Figure 11B. On the 10th day of in vitro expansion, real-time tumor killing curves of DNT cells and BCMA-CAR-DNT cells against CHO cell lines were detected by RTCA (effect-to-target ratio 2:1).

Figure 12. Flow cytometry detection of the tumoricidal activity of DNT cells and BCMA-CAR-DNT cells on the 10th day of in vitro expansion against MM.IS/U266/ARP1 cell lines expressing BCMA antigens and K562 cell lines not expressing BCMA antigens (effect-to-target ratio 4:1).

Figure 13. Levels of secreted IFN-γ (pg/ml) after co-incubation of non-transduced BCMA-CAR T cells and humanized BCMA-CAR-T cells with CHO cells and CHO-BCMA cells, respectively.

Figure 14. ELISA method to detect the secretion of IFN-γ by DNT cells on the 10th day of in vitro expansion, BCMA-CAR-DNT cells and MM.IS/U266/ARP1/K562 cell lines (effect-to-target ratio 4:1) co-incubated for 3 hours level (pg/ml).

Figure 15A. The flow chart of the expression level of immunosuppressive receptors after co-incubation of humanized BCMA-CAR-T cells and Hela-BCMA cells.

Figure 15B. Statistical results of immunosuppressive receptor expression levels detected by flow cytometry after co-incubation of humanized BCMA-CAR-T cells and Hela-BCMA cells.

Figure 16A. Flow chromatogram of the expression level of immunosuppressive receptors after co-incubation of humanized BCMA-CAR-DNT cells and CHO-BCMA cells.

Fig. 16B. Statistical results of flow cytometric detection of expression levels of immunosuppressive receptors after co-incubation of humanized BCMA-CAR-DNT cells and CHO-BCMA cells.

Detailed description of the invention

the term

As used herein, “Chimeric Antigen Receptor (CAR), is a receptor protein that has been engineered to confer a novel ability on T cells to target specific proteins. Such receptors are chimeric in that they incorporate antigen The binding and T cell activation functions are combined in one receptor. CAR is a fusion protein that includes an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain.” Chimeric Antigen Receptor ( CAR) protein is sometimes referred to as "chimeric receptor", "T body" or "chimeric immune receptor (CIR) fusion. "Antigen-binding extracellular domain" refers to any protein that can bind to an antigen Oligopeptide or polypeptide. "Intracellular domain" refers to any known oligopeptide or polypeptide as a domain that transmits signals to cause cellular activation or inhibition of biological processes.

As used herein, "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

As used herein, "single-chain variable fragment (scFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind an antigen. One method of constructing scFv involves antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of the heavy (H chain) and light (L chain) chain fragments of an immunoglobulin are linked by a spacer sequence. Various methods for engineering scFv are known to those skilled in the art.

As used herein, "tumor antigen" refers to an antigenic biomolecule that is highly expressed in tumor cells.

The inventors humanized BCMA scFv, which was originally derived from the heavy and light chain variable regions of a mouse monoclonal antibody (clone 4C8A (WO2019/195017)). Mouse 4C8A antibody has strong selective binding ability to human BCMA antigen. The humanized BCMA antibody of the present invention also has strong selective binding ability to human BCMA antigen and has low immunogenicity.

The inventors prepared CAR-T cells/CAR-DNT cells based on the humanized BCMA ScFv sequence specifically targeting BCMA. The inventors prepared BCMA-CAR-T cells/BCMA-CAR-DNT cells for targeting tumor cells overexpressing BCMA tumor antigens. The humanized BCMA-CAR-T/BCMA-CAR-DNT cells of the present invention secrete high levels of cytokines against multiple myeloma cells, and kill CHO-BCMA positive target cells without killing control CHO cells.

The present invention relates to a humanized monoclonal anti-human BCMA antibody or antigen-binding fragment thereof (e.g., Fab, (Fab) ₂ , scFv), comprising a _VH having the amino acid of SEQ ID NO: 2 and having a VH having the amino acid of SEQ ID NO: 2, respectively. NO: _VL of 3 amino acids. In one embodiment, the humanized anti-human BCMA antibody is a single chain variable fragment (scFv). The ScFv can be _VH -linker- _VL or _VL -linker- _VH .

The present invention also relates to a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) single-chain variable fragment (scFv) against BCMA (invention), (ii) transmembrane structure domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain.

In some embodiments, the structure of the humanized BCMA CAR is shown in Figure 3.

In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP10 domains. Preferred co-stimulatory domains are CD28 or 4-1BB.

A preferred activation domain is CD3-zeta (CD3Z or CD3-).

Transmembrane domains can be derived from natural polypeptides or artificially designed. Transmembrane domains derived from natural polypeptides can be obtained from any membrane-bound or transmembrane protein. For example, T cell receptor alpha or beta chain, CD3 Zeta chain, CD28, CD38, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, Transmembrane domain of CD154 or GITR. Artificially designed transmembrane domains are polypeptides that primarily contain hydrophobic residues such as leucine and valine. A triplet of phenylalanine, tryptophan and valine is preferably designed at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, for example a linker with a length of 2-10 amino acids, can be placed between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence with a glycine-serine contiguous sequence can be used.

The invention provides a nucleic acid encoding BCMA-CAR. A nucleic acid encoding a CAR can be prepared from the amino acid sequence of a specific CAR by conventional methods. The base sequence encoding the amino acid sequence of each domain can be obtained from the NCBI RefSeq ID or GenBank accession number of the amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using standard molecular biology and/or chemical methods. For example, based on the base sequence, a nucleic acid can be synthesized, and by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR), the nucleic acid of the present invention can be prepared.

The nucleic acid encoding the CAR of the present invention can be inserted into a vector, and then the vector is introduced into cells. For example, retroviral vectors (including oncogenic retroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated viral (AAV) vectors, simian virus vectors, vaccinia virus vectors, or Sendai virus vectors, HIV Viral vectors such as Predstein-Barr virus (EBV) vectors and HSV vectors. It is preferred to use viral vectors that lack the ability to replicate and thus are unable to replicate themselves in infected cells.

For example, when a retroviral vector is used, appropriate packaging cells can be selected based on the LTR sequence and packaging signal sequence possessed by the vector, and retroviral particles can be prepared using the packaging cells. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. Retroviral particles can also be prepared using 293 cells or 293T cells, which have high transfection efficiency. Retroviral vectors and packaging cells useful for packaging retroviral vectors are commercially available from proprietary companies.

CAR-T cells bind to specific antigens through CAR and transmit signals to the cells, thereby activating the cells. Activation of CAR cells depends on the type of host cell and the intracellular domain of CAR, and can be confirmed based on, for example, release of cytokines, increase in cell proliferation speed, changes in cell surface molecules, etc. as indicators. For example, the release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells leads to the destruction of antigen-expressing target cells. In addition, released cytokines or changes in cell surface molecules induce other immune cells to participate in the immune response, such as B cells, dendritic cells, NK cells and macrophages.

Cells expressing CAR can be used as therapeutic agents for diseases. The therapeutic formulation comprises cells expressing the CAR as an active ingredient, and may also contain suitable excipients.

The inventors prepared humanized BCMA-ScFv-41-BB-CD3-CAR-T (BCMA-CAR-T) cells/BCMA-ScFv-41-BB targeting multiple myeloma cells (Multiple Myeloma, MM) - CD3-CAR-DNT (BCMA-CAR-DNT) cells. The BCMA-CAR-T cells/BCMA-CAR-DNT cells of the present invention secrete high levels of cytokines. Through cytotoxicity experiments, BCMA-CAR-T cells/BCMA-CAR-DNT cells specifically kill CHO-BCMA cells but not CHO cells, which indicates that CAR-T/CAR-DNT cells are specific to target cancer cells Sexual killing activity and its cytotoxic activity against tumor or viral antigens.

The construction method of the general-purpose antigen chimeric receptor BCMA-CAR-DNT cell in the present invention is to provide a general-purpose DNT cell derived from the peripheral blood of a healthy donor, without using gene editing methods to knock out the graft-versus-host disease TCR gene, high safety, low immunogenicity (does not cause host anti-graft immune response, long retention time in vivo), significant killing effect and low production cost of the off-the-shelf universal BCMA-CART cell preparation method.

DNT cells are derived from the peripheral blood of healthy people. DNT cells can kill tumor cells by releasing granzymes, perforin and various cytokines after binding to corresponding ligands on tumor cells through NKG2D and DNAM-1 receptors. DNT cells are part of the body's innate immune cells and a subtype of T cells. They can be expanded and prepared in vitro on a large scale without HLA matching, making them the preferred off-the-shelf universal cell drug and CAR-T/TCR- The carrier of T products. Chimeric B cell maturation antigen (BCMA) antibody to DNT cells can be prepared into a general-purpose BCMA-CAR-DNT cell therapy product, which can specifically kill multiple myeloma (MM) cells and achieve the treatment of relapsed and refractory multiple myeloma (MM). Purpose of myeloma.

The advantages of the humanized BCMA ScFv of the present invention include low immunogenicity and the humanized transformation of the ScFv sequence. Therefore, the BCMA antibody of the present invention is very effective and safe as a therapeutic drug in many clinical applications.

The humanized BCMAscFv antibody of the present invention can be used for immunotherapy: toxin/drug-binding antibody, monoclonal therapeutic antibody, humanized BCMA antibody, and CAR-T cell immunotherapy.

Humanized BCMA-CAR-T/BCMA-CAR-DNT cells using the humanized BCMA scFv of the present invention can be effectively used to target BCMA antigens in BCMA-positive cancer cell lines.

Humanized BCMA-CAR-T/BCMA-CAR-DNT cells can be used in combination with different therapies: immune checkpoint inhibitors, targeted therapy, small molecule inhibitors, antibody drugs.

Humanized BCMA-CAR-T/BCMA-CAR-DNT cells can be used clinically for BCMA-positive cancer cells and autoimmune diseases.

Modifications of coactivation domains (CD28, 41-BB, and others) can improve CAR effectiveness. The tag-coupled humanized BCMA scFv can be used in the preparation of CAR.

Humanized BCMA-CAR-T/BCMA-CAR-DNT cells can be fitted with safety switches (t-EGFR, RQR (rituximab-CD34-rituximab) and others).

Humanized BCMA scFv can be used in the third and newer generations of CAR-T or other co-activation signaling domains.

Humanized BCMA-CAR can be used in combination with CARs targeting other tumor antigens or tumor microenvironment (for example, VEGFR-1-3, PD-L1), and bispecific antibodies against BCMA and CD3 or other antigens can be prepared for Clinical treatment.

Humanized BCMA-CAR-T/BCMA-CAR-DNT cells can be used to kill chemotherapy-resistant and aggressive cancer stem cells.

Both humanized BCMA scFv or VH and VL of humanized BCMA can be combined with other antibodies (such as CD3 Scfv) as bispecific antibodies.

The following examples further illustrate the invention. These examples are only for illustrating the invention and should not be construed as limiting. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example

The present invention uses a lentiviral vector to express humanized BCMA-ScFv-CAR (CAR-PMC1497), the structure including human CD8 signal peptide, humanized BCMA scFv (VL-linker-VH), CD8 hinge, CD28 transmembrane, 41BB co-stimulatory domain and CD3ζ activation domain (Figure 3).

The lentivirus was produced in 293T cells, and the titer was determined by RT-PCR. Cells were then transduced with equal doses of lentivirus.

sequence

Example 1. Humanized BCMA VH, VL and scFv sequences

In the present invention, BCMA-positive hybridoma sequences are humanized to obtain BCMA scFv, and the structure of the humanized BCMA scFv is: VL-linker-VH.

The nucleotide sequence and amino acid sequence of the humanized BCMA scFv are as follows: VH is in bold, VL is underlined, and the nucleotide sequence of the linker is in italics in the middle.

Nucleotide sequence of humanized BCMA scFv (SEQ ID NO:5)

Humanized BCMA (PMC1497) scFv, amino acid sequence (SEQ ID NO: 1)

BCMA (PMC1497), _VH , amino acid sequence (SEQ ID NO: 2)

BCMA (PMC1497), _VL , amino acid sequence (SEQ ID NO: 3)

Amino acid sequence of linker (SEQ ID NO:6)

Example 2A.4-1BB as the humanized BCMA-CAR sequence of costimulatory domain (CAR-PMC1497)

The schematic diagram of the humanized BCMA-CAR structure is shown in Figure 3. The humanized scFv CAR sequence was cloned using a lentiviral vector with the EF1a promoter.

The CAR structure includes human CD8 signal peptide, humanized BCMA scFv (VL-linker-VH), CD8 hinge, CD28 transmembrane, 41BB co-stimulatory domain and CD3ζ activation domain, and its nucleic acid sequence is as follows: (Figure 3) .

<CD8 leader sequence with Nhe restriction site at the end>

Nucleotide sequence (SEQ ID NO:7)

Amino acid sequence (SEQ ID NO:8)

<Nhe I restriction enzyme site>

Nucleotide sequence

amino acid sequence

<Humanized BCMA (PMC1497) scFv> (see Example 1)

<Xho I restriction endonuclease site>

Nucleotide sequence

amino acid sequence

<CD8 hinge>

Nucleotide sequence (SEQ ID NO:9)

Amino acid sequence (SEQ ID NO: 10)

<space sequence>

Nucleotide sequence

amino acid sequence

<CD28 transmembrane region>

Nucleotide sequence (SEQ ID NO: 11)

Amino acid sequence (SEQ ID NO: 12)

<41BB co-stimulatory domain>

Nucleotide sequence (SEQ ID NO: 13)

Amino acid sequence (SEQ ID NO: 14)

<CD3ζ>

Nucleotide sequence (SEQ ID NO: 15)

Amino acid sequence (SEQ ID NO: 16)

Nucleotide sequence (SEQ ID NO: 17) of humanized BCMA-CAR protein (CAR-PMC1497)

Amino acid sequence of humanized BCMA-CAR protein (CAR-PMC1497), SEQ ID NO:4

Example 2B.CD28 as the humanized BCMA-CAR sequence of costimulatory domain

Only the 4-1BB co-stimulatory domain in the humanized Scfv-CAR structure was replaced by the CD28 co-stimulatory domain

<CD28/co-stimulatory domain>

Nucleotide sequence (SEQ ID NO: 18)

Amino acid sequence (SEQ ID NO: 19)

Materials and methods

Example 3. Lentiviral CAR constructs

The codon-optimized humanized BCMA ScFv sequence was used as a Gblock gene fragment, which was then subcloned into a second-generation CAR sequence with a 4-1BB co-stimulatory domain and a CD3 activation domain. CAR-T cells with 3TF tag without ScFv structure (Mock-CAR-T) were used as control.

Example 4. Lentivirus production

293T cells were cultured at 37°C in a 5% CO ₂ incubator, and the medium was DMEM+10% FBS. On the second day, when the cells reach 70-90% confluence, co-transfect with the target gene shuttle plasmid and packaging plasmid pMDL g/pRRE, pRSV-Rev, pMD2.0G, and add the appropriate molar ratio of mixed plasmids into the culture vessel , shake, mix well, and put into the incubator. The fresh medium was replaced the next day. After 48 hours, the medium containing lentiviral particles was harvested, and the floating and dead 293T cells and cell debris were removed by centrifugation at 2100g for 30 minutes. Then, the virus-containing medium was filtered, concentrated, purified, and separated. Store at -80°C and measure the titer.

Example 5. Preparation of CAR-T cells

PBMCs with a density of 1×10 ⁶ cells/ml were suspended in AIM V-Albummax medium (Thermo Fisher) containing 10% FBS and 10ng/mL IL-2 (Thermo Fisher), according to the cell: CD3/CD28 Dynabeads (Thermo Fisher) = 1:1 ratio was mixed to activate T cells. After 24-48 hours, add lentiviral particles with MOI=5-10 multiplicity of infection (MOI) into the medium for transduction, and then change the medium every 3 days to maintain the density of T cells and CAR-T cells at 1~ 2×10 ⁶ cells/ml, after 10-12 days of expansion, the cells were harvested and tested for transduction efficiency (CAR positive rate), cell differentiation, and cell tumoricidal activity.

Example 6. Preparation of BCMA-CAR-DNT cells

Collect 20-200ml of peripheral blood from healthy donors into sodium heparin tubes. Use Rossettsep kit (Stem Cell Technologies Inc) to deplete CD4 ⁺ , CD8 ⁺ T cells, or use CD4/CD8 magnetic beads to deplete CD4 ⁺ , CD8 ⁺ T cells, the cells thus obtained are CD4 ⁺ and CD8 ⁺ cells depleted DNT cell. The obtained purified DNT cells were divided into 2 parts, one part was transduced by adding lentiviral particles with MOI=1-10 after 3-5 days of in vitro expansion, and the other part was used as a control group without adding virus. Thereafter, the medium was supplemented every 2-3 days according to the cell density, and the BCMA-CAR-DNT cells were harvested 5-7 days after the virus transduction (for the preparation method, please refer to Patent No. 2021113062449, an in vitro efficient and stable transfer of amplified antigen-embedded cells. BCMA-CAR-DNT cell transduction efficiency (CAR positive rate), cell purity, cell differentiation, cell survival rate, cell proliferation multiple and cell tumoricidal activity were detected.

result

Example 7A. Characterization detection of BCMA-CAR-T cells of the present invention

Flow cytometric detection: The differentiation phenotype of BCMA-CAR-T cells was detected with fluorescein-labeled CD62L and CD45RA antibodies.

The results are shown in Figure 4, BCMA-CAR-T cells have a similar differentiation phenotype to untransduced T cells: the main cell population is central memory T cells and a lower proportion of Teff cells.

Example 7B. Characterization detection of BCMA-CAR-DNT cells of the present invention

Detection of viability: K2 dual fluorescent cell counter was used to detect the cell proliferation curve and cell viability of DNT cells and BCMA-CAR-DNT cells in different culture time in vitro by AO/PI staining.

Flow cytometry: On the 10th day of in vitro culture, use flow cytometry, use fluorescein-labeled CD3, CD4, CD8 antibodies to detect the purity of double-negative T cells in the culture system; use fluorescein-labeled CD62L, CD45RA antibodies to detect non-transduced DNT cells and the differentiation status of BCMA-CAR-DNT cells.

The results showed that the BCMA-CAR-DNT cells obtained after transduction of BCMA-CAR derived from the same donor had better performance in cell proliferation (Figure 5A), cell viability (Figure 5B), DNT cell purity (CD3 ⁺ CD4 ^- CD8 ^- cells, Figure 6) and cell differentiation (Figure 7A) are basically similar to those of untransduced DNT cells, indicating that the BCMA-CAR-DNT cells prepared after transduction of CAR molecules through lentivirus do not affect the above characteristics of DNT cells.

Figure 7B shows the curves of Tscm/Tcm/Tem/Teff cell ratio changes in BCMA-CAR-DNT cells on the 7th, 10th and 14th days of in vitro expansion using CD45RA/CD62L fluorescently labeled antibodies using flow cytometry. The results show that the BCMA-CAR-DNT cells obtained by using the expansion method of the invention patent No. 2021113062449 still have a high proportion of Tscm (>35%) and a low proportion of Teff cells even after being expanded to 14 days in vitro , so it has stronger self-renewal, differentiation and long-term survival ability, which can make BCMA-CAR-DNT cells exist for a long time in the body and play a long-term anti-tumor effect, and the CAR molecules are transduced into DNT cells by lentivirus to prepare BCMA-CAR-DNT cells did not affect the differentiation properties of DNT cells.

Example 8A. Humanized BCMA-CAR-T cells highly express BCMA-CAR

Flow cytometric detection: 2.5*10 ⁵ BCMA-CAR-T cells were added with 100 μL of buffer solution (PBS containing 2 mM EDTA and 0.5% BSA) and 1 μL of human serum and incubated on wet ice for 10 minutes. Incubate the diluted primary antibody biotin-anti-mouse F(ab)'2/biotin-anti-Human F(ab)'2/biotin-BCMA protein with cells at 4°C for 30 minutes, wash with buffer, add APC anti -Human CD3 antibody and PE-streptavidin diluted 1:100, incubated at 4°C for 30min. Cells were washed with 3 ml buffer, then stained with 7-AAD for 10 min, and suspended in FACS buffer. Analysis was performed on a BD flow cytometer. BCMA-CAR-T cell CAR expression detection was performed by analyzing CD3 ⁺ anti-(Fab)2 ⁺ or BCMA ⁺ in the 7-AAD negative cell population.

The results showed that lentivirus-transduced CAR-T cells had a high percentage of BCMA CAR-positive cells (Figure 8). Anti-mouse F(ab)'2 and biotin-labeled human BCMA protein were used to detect cells on the 9th day of in vitro amplification, and a relatively high positive rate was detected, among which anti-mouse F(ab)'2 detected up to 70 More than % of CAR positive cells (Figure 8).

Example 8B. Humanized BCMA-CAR-DNT cells highly express BCMA-CAR

Flow cytometry detection: the percentage of transduced CAR-DNT cells in BCMA-CAR-DNT cells was detected by flow cytometry and fluorescein-labeled human BCMA protein.

The results are shown in Figure 9, up to 27.8% of BCMA-CAR positive cells can be obtained on the 10th day of out-of-amplification.

Example 9A. Humanized BCMA-CAR-T cells kill CHO-BCMA cells but not CHO cells

Real-time cytotoxicity assay (RTCA) to detect the tumoricidal activity of effector cells: CHO-BCMA and CHO target cells (1× ¹⁰⁴ cells per well) were seeded into 96-well E-plates, and the impedance-based cell analysis instrument (RTCA ) xCELLigence system (Acea Biosciences) for real-time detection of killing activity. The next day, the medium was removed and replaced with effector cell medium (AIM V-AlbuMAX) containing 10% FBS, and CAR-T cells or non-transduced T cells were added according to the effect-to-target ratio of 10:1, with 3 replicate wells. After 24-48 hours, the tumor-killing activity of effector cells on target cells was calculated by the graph of the impedance of the RTCA device changing with time. The calculation formula is: target cell impedance - effector cell and target cell co-incubation impedance) × 100/target cell impedance.

The results are shown in Figure 10, humanized BCMA-CAR-T cells specifically kill CHO-BCMA cells (Figure 10A), but not CHO cells (Figure 10B). This indicates that humanized BCMA-CAR-T cells highly specifically target BCMA antigens and kill BCMA-positive target cells.

Example 9B. Humanized BCMA-CAR-DNT specifically targets BCMA-positive target cell lines

1. Tumoricidal activity against model cells expressing BCMA antigen

RTCA detection of tumoricidal activity of effector cells: CHO cells stably expressing human BCMA antigen (BCMA-CHO stable transfected cell line) were used as model target cells to evaluate the specific tumoricidal activity of BCMA-CAR-DNT cells, and CHO cells were used as negative control . The non-transduced DNT cells collected on the 10th day of in vitro culture and the BCMA-CAR-DNT cells transduced with humanized BCMA-CAR were used as effector cells. The RTCA method is used to monitor the killing situation in real time.

The results are shown in Figure 11A-B: Compared with non-transduced DNT cells, BCMA-CAR-DNT cells transduced with humanized BCMA-CAR showed specific killing of CHO-BCMA cells, and the maximum killing effect increased with The prolongation of the killing time can be sustained ( FIG. 11A ), and there is no significant difference in the killing of CHO cells between the above two types of cells ( FIG. 11B ). This shows that humanized BCMA-CAR-DNT cells can specifically target CHO-BCMA cells expressing BCMA antigen.

2. Tumoricidal activity against tumor cell lines naturally expressing BCMA molecules

Flow cytometric detection of tumoricidal activity of effector cells: the effective target ratio of BCMA-CAR-DNT cells on the 10th day of in vitro expansion to the target cell lines U266, ARP1 and MM.1S positively expressing PKH-26-labeled BCMA antigen was 4 Co-incubate for 3 hours under the condition of :1, use flow cytometry to detect its tumor killing activity respectively, and the K562 cell line that does not express BCMA antigen is used as a negative control;

The results are shown in Figure 12: after the MM.1S/U266/ARP1/K562 cell line was labeled with PKH-26, it was co-incubated with untransduced DNT cells and BCMA-CAR-DNT cells on the 10th day of expansion for 3 hours in vitro ( The effect-to-target ratio was 4:1), and the effect of BCMA-CAR-DNT cells on MM.1S/U266/ARP1 cell lines (BCMA-positive tumor cell lines) and K562 cells (BCMA-negative tumor cell lines) were detected by flow cytometry. Tumoricidal activity. The results showed that, compared with non-transduced DNT cells, BCMA-CAR-DNT cells transduced with humanized BCMA-CAR showed stronger tumoricidal activity on MM.1S/U266/ARP1 cells, but did not express There was no significant difference in the K562 cells of BCMA molecules (compared with the ARP1 cell line, since the myeloid leukemia cell line K562 is a relatively sensitive cell line of DNT cells, the tumor killing effect of BCMA-CAR-DNT cells on the two cell lines in this experiment The activities are basically the same, but the untransduced DNT cells are not sensitive to ARP1, and there is basically no killing under the conditions of this experiment). This shows that the humanized BCMA-CAR-DNT cells specifically target the target cells expressing BCMA antigen.

Example 10A. Co-incubation of humanized BCMA-CAR-T cells with CHO-BCMA cells positively expressing the target antigen to secrete high levels of IFN-γ

The secretion of IFN-γ was detected by ELISA method: CHO-BCMA cells, CHO target cells and effector cells (CAR-T cells or non-transduced T cells) were co-incubated in a V-bottom 96-well plate for 16 days according to the effector-target ratio of 1:1. After 2 hours, transfer the top 150 μl medium to a new V-bottom 96-well plate, centrifuge at 300 g for 5 min, and transfer the top 120 μl supernatant to a new 96-well plate to remove residual cells. IFN-A levels were analyzed by ELISA using a kit from R&D Systems.

The results showed that BCMA-CAR-T cells secreted high levels of IFN-γ after co-incubation with CHO-BCMA cells, and control CHO cells secreted low levels of IFN-γ (Figure 13), indicating that the humanized BCMA-CAR-T cells specificity of killing.

Example 10B. Co-incubation of humanized BCMA-CAR-DNT cells with cell lines positively expressing the target antigen to secrete high levels of IFN-γ

ELISA method to detect the secretion of IFN-γ: MM.1S/U266/ARP1/K562 cell line and DNT cells and BCMA-CAR-DNT cells on the 10th day of expansion have an effect-to-target ratio of 4:1 in a V-bottom 96-well plate After co-incubation for 3 hours, centrifuge at 300g for 5 minutes, transfer the top supernatant to a new 96-well plate, and analyze the IFN-γ level by ELISA.

The results showed that BCMA-CAR-DNT cells transduced with humanized BCMA-CAR secreted higher levels of IFN-γ after co-incubation with MM.1S/U266/ARP1 cells positively expressing the target antigen (Figure 14). Consistent with the in vitro tumoricidal activity results shown in Figure 12, it indicates the targeting specificity of humanized BCMA-CAR-DNT cells.

Example 11A. Co-incubation of humanized BCMA-CAR-T cells and Hela-BCMA cells leads to upregulation of expression of immunosuppressive receptors

Untransduced T cells or BCMA-CAR-T cells from two donors were co-incubated overnight in a 96-well plate alone or with Hela-BCMA target cells at a 2:1 effect-to-target ratio, and the cells were collected and labeled with fluorescein for anti-PD- 1. Lag-3 antibody, using flow cytometry to analyze the expression of immunosuppressive receptors on the surface of effector cells (CD3+7AAD- cells).

The results showed that after co-incubation with Hela-BCMA cells, the expressions of PD-1 and Lag-3 immunosuppressive receptors were significantly upregulated in BCMA-CAR-T cells from both donors compared with corresponding untransduced T cells (FIGS. 15A-15B).

Example 11B. Co-incubation of humanized BCMA-CAR-DNT cells and CHO-BCMA cells leads to upregulation of expression of immunosuppressive receptors

After co-incubating untransduced T cells and BCMA-CAR-DNT cells alone or with CHO-BCMA target cells in a 96-well plate at an effect-to-target ratio of 2:1 overnight, the cells were collected and labeled with anti-PD-1 and Lag with fluorescein -3 antibody, the expression of immunosuppressive receptors on the surface of effector cells (CD3+7AAD- cells) was analyzed by flow cytometry.

The results showed that after co-incubation with CHO-BCMA cells, the expression of PD-1 and Lag-3 immunosuppressive receptors was significantly upregulated in BCMA-CAR-DNT cells from both donors compared with corresponding untransduced DNT cells (FIGS. 16A-16B).

references

[1] M.V.Maus, A.R.Haas, G.L.Beatty, S.M.Albelda, B.L.Levine, X.Liu, Y.Zhao, M.Kalos, and C.H.June, T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 1 (2013) 26-31.

[2] M.V.Maus, S.A.Grupp, D.L.Porter, and C.H.June, Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 123(2014) 2625-35.

[3] V. Golubovskaya, and L. Wu, Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers (Basel) 8 (2016).

[4] S.A.Ali, V.Shi, I.Marie, M.Wang, D.F.Stroncek,].].Rose,].N.Brudno, M.Stetler-Stevenson, S.A.Feldman, B.G.Hansen, V.S.Fellowes, F.T.Hakim , R.E.Gress, and J.N.Kochenderfer, T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood 128(2016)1688-700.

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[6] R. Berahovich, H. Zhou, S. Xu, Y. Wei,]. Guan,]. Guan, H. Harto, S. Fu, K. Yang, S. Zhu, L. Li, L. Wu , and V. Golubovskaya, CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth. Cancers (Basel) 10 (2018).

Claims (16)

1. An anti-human BCMA antibody or an antigen-binding fragment thereof, comprising a VH with the amino acid of SEQ ID NO:2, and a VL with the amino acid of SEQ ID NO:3.
2. A single chain variable fragment (scFv) of humanized BCMA comprising a VH with the amino acid of SEQ ID NO:2, and a VL with the amino acid of SEQ ID NO:3.
3. The scFv of claim 2, further comprising a linker between VH and VL.
4. The scFv according to claim 3, which has the amino acid sequence of SEQ ID NO:1.
5. A chimeric antigen receptor (CAR) fusion protein comprising from N-terminus to C-terminus:

   (i) scFv according to claim 3,

   (ii) transmembrane domain,

   (iii) at least one co-stimulatory domain, and

   (iv) Activation domain.
6. The CAR fusion protein according to claim 5, said CAR has a structure as shown in formula I:

   L-scFv-H-TM-C-CD3ζ (Formula I)

   In the formula, L is nothing or a signal peptide sequence;

   scFv is an antibody single-chain variable region sequence targeting humanized BCMA antigen;

   H is none or hinge region;

   TM is the transmembrane domain;

   C is costimulatory domain;

   CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.
7. The CAR fusion protein according to claim 5, wherein the co-stimulatory domain is CD28 or 4-1BB.
8. The CAR fusion protein according to claim 5, wherein the activation domain is CD3ζ.
9. The CAR fusion protein according to claim 5, which has the amino acid sequence of SEQ ID NO:4.
10. A nucleic acid encoding the CAR according to any one of claims 5-8 or encoding the anti-human BCMA antibody or antigen-binding fragment thereof as claimed in claim 1.
11. A carrier, characterized in that the carrier contains the nucleic acid molecule according to claim 10.
12. An engineered immune cell modified to express the CAR according to any one of claims 5-9.
13. The engineered immune cell according to claim 12, wherein the immune cell is a DNT cell.
14. The engineered immune cell according to claim 12, wherein the immune cell is a T cell.
15. A method for preparing the engineered immune cell as claimed in claim 12, comprising the step of: transducing the nucleic acid as claimed in claim 10 or the vector as claimed in claim 11 into the immune cell.
16. An anti-human BCMA antibody or an antigen-binding fragment thereof according to claim 1, a scFv according to claim 2, a chimeric antigen receptor (CAR) fusion protein according to claim 5, a fusion protein according to claim 11 Use of the carrier or the engineered immune cell according to claim 12 in the preparation of drugs for preventing and/or treating cancer or tumors and drugs for autoimmune diseases.

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