Anti-bcma antibodies and preparation and use thereof

Abstract

The application discloses an anti-BCMA antibody and preparation and use thereof. A heavy chain variable region of the anti-BCMA antibody comprises three complementarity determining regions (CDRs), wherein the CDRs comprise CDR1, CDR2 and / or CDR3; and amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The anti-BCMA antibody of the application exhibits affinity to human and monkey BCMA recombinant proteins, and CAR-M cells and TCE constructed by the anti-BCMA antibody exhibit target-dependent killing activity.

Description

[0001] This application claims priority to Chinese patent application 202511323177X, filed on 2025 / 09 / 16. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biomedical technology, specifically to an anti-BCMA antibody and its preparation and use. Background Technology

[0003] B cell maturation antigen (BCMA) has become a novel target for the treatment of multiple myeloma (MM) due to its highly selective expression in malignant plasma cells (PCs). Various BCMA-targeted therapies, including bispecific T-cell conjugates (BiTE), chimeric antigen receptor (CAR)-T cells, and antibody-drug conjugates (ADCs), have achieved significant clinical efficacy in relapsed and refractory MM patients. However, the large molecular weight (approximately 150 kDa) or weak affinity of traditional antibodies limits their penetration efficiency into solid tumors. Therefore, it is necessary to develop short fragments (Fab, scFv, VHH, or other nanobodies) or high-affinity antibodies with antibody function to overcome the limitations of traditional antibody therapies. Summary of the Invention

[0004] In order to provide an effective targeting antibody for the treatment of cancers expressing BCMA or autoimmune diseases containing BCMA-positive autoreactive B cells or plasma cells, the present invention provides an anti-BCMA antibody and its preparation and use.

[0005] In view of this, this invention uses recombinant BCMA antigen to immunize camels, constructs a VHH phage library, and obtains three anti-human and anti-monkey BCMA nanobodies after bio-panning. More specifically, this study uses recombinant BCMA antigen to immunize camels, constructs a VHH phage library, and obtains three BCMA-binding nanobodies after bio-panning. These nanobodies have an affinity (KD) for recombinant human and anti-monkey BCMA proteins between 0.1 and 10 nM, and a FACS affinity >90% for BCMA-positive cells NCI-H929 and RPMI8226.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] The first aspect of the present invention provides an anti-BCMA antibody or an active variant thereof or an antigen-binding fragment thereof, wherein the heavy chain variable region of the anti-BCMA antibody comprises three complementarity-determining regions (CDRs), wherein the CDRs comprise CDR1, CDR2 and / or CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0008] In some embodiments of the present invention, the framework region FR of the anti-BCMA antibody comprises FR1, FR2, FR3 and / or FR4; the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; or, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 29, SEQ ID NO: 20, SEQ ID NO: 30 and SEQ ID NO: 31, respectively.

[0009] In some preferred embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-BCMA antibody is shown in SEQ ID NO: 1 or SEQ ID NO: 28.

[0010] In some preferred embodiments of the present invention, the anti-BCMA antibody is a VHH or a nanobody.

[0011] In some embodiments of the present invention, the anti-BCMA antibody is a nanobody and also contains Fc.

[0012] In some preferred embodiments of the present invention, the Fc is the Fc of human or mouse IgG1, IgG2, IgG3 or IgG4 or a variant thereof; the variant has 80%-99.9% sequence identity with the sequence from which it is derived.

[0013] In some more preferred embodiments of the present invention, the active variant has 80%-99.9% identity with the amino acid sequence of the anti-BCMA antibody; for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity.

[0014] The antigen-binding fragment comprises the heavy chain variable region, light chain, and CH1 of the anti-BCMA antibody; CH1 is the CH1 of human or mouse IgG1, IgG2, IgG3, or IgG4, or a variant thereof; the variant has 80%-99.9% sequence identity with the sequence from which it is derived.

[0015] A second aspect of the present invention provides a chimeric antigen receptor, wherein the extracellular recognition domain of the chimeric antigen receptor comprises an anti-BCMA antibody as described in the first aspect of the present invention, or an active variant thereof, or an antigen-binding fragment thereof.

[0016] In some preferred embodiments of the present invention, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the anti-BCMA antibody, a hinge region and a transmembrane sequence, and an intracellular signal sequence; optionally, it also includes a signal peptide.

[0017] In some embodiments of the present invention, the chimeric antigen receptor is selected from one or more of the following:

[0018] The hinge region and transmembrane sequence are the hinge region and transmembrane sequence of CD8 or CD28; their amino acid sequences are, for example, as shown in SEQ ID NO: 8 or SEQ ID NO: 9;

[0019] The intracellular signal sequence is a CD3ζ or FcεR1γ intracellular signal sequence; its amino acid sequence is, for example, shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0020] The signal peptide may be optionally the CSF2Rα-SP signal peptide or the IL-10 signal peptide; its amino acid sequence is shown, for example, as SEQ ID NO: 7 or SEQ ID NO: 14.

[0021] In some preferred embodiments of the present invention, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:32 or SEQ ID NO:33.

[0022] A third aspect of the present invention provides a bispecific or multispecific antibody comprising an anti-BCMA antibody as described in the first aspect of the present invention, an active variant thereof, or an antigen-binding fragment thereof;

[0023] In some preferred embodiments of the present invention, the bispecific or multispecific antibody is a bispecific T cell conjugate.

[0024] In some preferred embodiments of the invention, the bispecific T-cell connector further comprises an antibody targeting a T-cell marker, such as CD3, CD2, CD4, or CD8.

[0025] In some embodiments of the present invention, the bispecific T cell connector comprises, from the N-terminus to the C-terminus, the anti-BCMA antibody or its dual tandem antibody, a G4S-linker sequence, and an antibody targeting CD3ε.

[0026] In some preferred embodiments of the present invention, the amino acid sequence of the G4S-linker sequence is shown in SEQ ID NO:16;

[0027] The antibody targeting CD3ε is the scFv sequence of the monoclonal antibody clone SP34 that targets the CD3ε chain; its amino acid sequence is shown, for example, as SEQ ID NO: 17.

[0028] In some preferred embodiments of the present invention, the amino acid sequence of the bispecific T cell conjugate is shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:25 or SEQ ID NO:26.

[0029] A fourth aspect of the present invention provides a polynucleotide selected from any one of the following groups:

[0030] (1) Encoding an anti-BCMA antibody as described in the first aspect of the present invention or an active variant thereof or an antigen-binding fragment thereof, or a bispecific or multispecific antibody as described in the third aspect of the present invention;

[0031] (2) Encoding the chimeric antigen receptor as described in the second aspect of the present invention.

[0032] A fifth aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector is selected from any one of the following groups:

[0033] (i) comprising (1) of the polynucleotide as described in the fourth aspect of the present invention, or expressing the anti-BCMA antibody as described in the first aspect of the present invention or its active variant or antigen-binding fragment or the bispecific or multispecific antibody as described in the third aspect of the present invention;

[0034] (ii) Contains the polynucleotide described in (2) as described in the fourth aspect of the invention, or expresses the chimeric antigen receptor described in the second aspect of the invention.

[0035] In some preferred embodiments of the present invention, the backbone plasmid of the recombinant expression vector is pFcIG or GS-CMV-100A-BspQI.

[0036] The sixth aspect of the present invention provides a recombinant host cell expressing the anti-BCMA antibody or its active variant or its antigen-binding fragment as described in the first aspect of the present invention, or a bispecific or multispecific antibody as described in the third aspect of the present invention; for example, the recombinant host cell contains (1) of the polynucleotide as described in the fourth aspect of the present invention, or contains (i) of the recombinant expression vector as described in the fifth aspect of the present invention.

[0037] In some preferred embodiments of the present invention, the original cell of the recombinant host cell is a prokaryotic cell or a eukaryotic cell.

[0038] In some more preferred embodiments of the present invention, the original cells are mammalian cells, Escherichia coli or yeast cells, such as Escherichia coli trans5α, Expi293F cells, CHO cells, HEK293F cells, and HEK293T cells.

[0039] The seventh aspect of the present invention provides a recombinant immune cell comprising or expressing a chimeric antigen receptor as described in the second aspect of the present invention; for example, the recombinant immune cell comprising (2) of the polynucleotide as described in the fourth aspect of the present invention, or comprising (ii) of the recombinant expression vector as described in the fifth aspect of the present invention.

[0040] In some preferred embodiments of the present invention, the recombinant immune cells originate from any one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, NKT cells, and iPSCs.

[0041] In some preferred embodiments of the present invention, the macrophages are human primary macrophages; the T cells are regulatory T cells, helper T cells, cytotoxic T cells or γδ T cells; and the monocytes are human monocytes, such as THP-1 cells.

[0042] The eighth aspect of the present invention provides a method for preparing an anti-BCMA antibody or its active variant or its antigen-binding fragment as described in the first aspect of the present invention, or a bispecific or multispecific antibody as described in the third aspect of the present invention, the method comprising: culturing recombinant host cells as described in the sixth aspect of the present invention to obtain the BCMA antibody or its active variant or its antigen-binding fragment or bispecific or multispecific antibody.

[0043] In some preferred embodiments of the present invention, the method includes the following steps: inserting a polynucleotide encoding such as the anti-BCMA antibody or its active variant or its antigen-binding fragment or a bispecific or multispecific antibody into an expression vector to obtain the recombinant expression vector;

[0044] The recombinant expression vector was transferred into host cells to obtain the recombinant host cells;

[0045] The recombinant host cells were cultured to obtain a culture.

[0046] Optionally, the process also includes purifying the culture to obtain the anti-BCMA antibody or its active variant or its antigen-binding fragment or bi- or multi-specific antibody.

[0047] The ninth aspect of the present invention provides an antibody-drug conjugate or a pharmaceutically usable salt thereof, comprising an anti-BCMA antibody as described in the first aspect of the present invention or an active variant thereof or an antigen-binding fragment thereof, or a bispecific or multispecific antibody as described in the third aspect of the present invention.

[0048] The tenth aspect of the present invention provides a pharmaceutical composition comprising: an anti-BCMA antibody as described in the first aspect of the present invention or an active variant thereof or an antigen-binding fragment thereof; a bispecific or multispecific antibody as described in the third aspect of the present invention; a recombinant immune cell as described in the seventh aspect of the present invention; or an antibody-drug conjugate as described in the ninth aspect of the present invention or a pharmaceutically acceptable salt thereof; and pharmaceutically acceptable excipients.

[0049] The eleventh aspect of the present invention provides a kit comprising an anti-BCMA antibody or its active variant or antigen-binding fragment as described in the first aspect of the present invention, a bispecific or multispecific antibody as described in the third aspect of the present invention, a polynucleotide as described in the fourth aspect of the present invention, a recombinant expression vector as described in the fifth aspect of the present invention, a recombinant host cell as described in the sixth aspect of the present invention, a recombinant immune cell as described in the seventh aspect of the present invention, an antibody-drug conjugate or its pharmaceutically usable salt as described in the ninth aspect of the present invention, and / or a pharmaceutical composition as described in the tenth aspect of the present invention; and a detection-acceptable reagent.

[0050] The twelfth aspect of the present invention provides a method for detecting BCMA, the method comprising contacting a sample to be tested with an anti-BCMA antibody as described in the first aspect of the present invention or an active variant thereof or an antigen-binding fragment thereof, or a bispecific or multispecific antibody as described in the third aspect of the present invention.

[0051] In some preferred embodiments of the present invention, the method is for non-diagnostic and / or therapeutic purposes.

[0052] The thirteenth aspect of this invention provides the use of anti-BCMA antibodies or active variants thereof or antigen-binding fragments thereof as described in the first aspect of this invention, chimeric antigen receptors as described in the second aspect of this invention, bispecific or multispecific antibodies as described in the third aspect of this invention, polynucleotides as described in the fourth aspect of this invention, recombinant expression vectors as described in the fifth aspect of this invention, recombinant host cells as described in the sixth aspect of this invention, recombinant immune cells as described in the seventh aspect of this invention, antibody-drug conjugates or pharmaceutically usable salts thereof as described in the ninth aspect of this invention, or pharmaceutical compositions as described in the tenth aspect of this invention in the preparation of medicaments for the prevention and / or treatment of cancer or kits for the detection of BCMA protein.

[0053] In some preferred embodiments of the present invention, the cancer is a BCMA-positive cancer or an autoimmune disease containing BCMA-positive autoreactive B cells or plasma cells.

[0054] In some more preferred embodiments of the present invention, the cancer is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, plasmacytoma, and melanoma; the autoimmune disease is selected from one or more of the following: systemic lupus erythematosus, multiple sclerosis, inflammatory myositis, antineutrophil cytoplasmic antibody vasculitis, rheumatoid arthritis, Sjögren's syndrome, antiphospholipid syndrome, myasthenia gravis, autoimmune thyroid disease, pemphigus / pemphigoid, and type 1 diabetes.

[0055] In some further preferred embodiments of the present invention, the myeloma is multiple myeloma or plasma cell myeloma; the leukemia is chronic lymphocytic leukemia or acute B-lymphocytic leukemia; the malignant lymphoma is non-Hodgkin lymphoma or Hodgkin lymphoma; the plasma cell tumor is solitary plasma cell tumor; and the autoimmune thyroid disease is Graves' disease or Hashimoto's thyroiditis.

[0056] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0057] The present invention provides an anti-BCMA antibody and its variants, or antigen-binding fragments thereof. The amino acid sequence of the anti-BCMA antibody is shown in Table 1, and it includes three complementarity-determining regions CDR1, CDR2 and CDR3 of VHH named CBCHC-25, as well as a frame region. The amino acid sequence is shown in Table 2.

[0058] Table 1: Amino acid sequence of CBCHC-25 nano-alpaca VHH

[0059]

[0060] For example, the antibodies and their variants, or antigen-binding fragments thereof, described herein are humanized variants or identical variants (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%).

[0061] Furthermore, the antibodies and their variants, or their antigen-binding fragments, described herein are selected from camel Ig, IgNAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bis-scFv, (scFv)2, microantibodies, double-chain antibodies, triple-chain antibodies, tetra-chain antibodies, disulfide-stabilized Fv proteins, and single-domain antibodies (sdAb, nanobodies), bispecific antibodies, or trispecific antibodies.

[0062] The present invention also provides a fusion protein comprising the antibody described herein and its variants, or an antigen-binding fragment thereof.

[0063] For example, the fusion protein described herein may further include a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (e.g., an epitope targeting or different from BCMA) or an additional antibody active fragment (e.g., an antibody or antibody active fragment targeting or the same epitope targeting BCMA, or a ligand capable of binding to BCMA).

[0064] The present invention also provides an antibody-drug conjugate comprising the antibody described herein and its variants, or an antigen-binding fragment thereof.

[0065] For the antibody-drug conjugates described herein, the drug is selected from the following: radiolabeled substances, 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, digitalisin, haptens, immunogenic proteins, nucleic acid molecules having sequences complementary to the target, or any combination thereof; or immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or any combination thereof.

[0066] The present invention also provides an isolated polynucleotide expressing an antibody or an active variant thereof or an antigen-binding fragment thereof, the polynucleotide being capable of expressing the antibodies and variants thereof described herein, or the antigen-binding fragment thereof; the polynucleotide being capable of expressing the fusion proteins described herein; the polynucleotide being capable of expressing the antibody-drug conjugates described herein.

[0067] The present invention also provides a vector comprising the polynucleotides described herein, preferably a plasmid vector.

[0068] The present invention also provides a host cell comprising the polynucleotide or the vector described in this disclosure, preferably a eukaryotic cell.

[0069] The present invention also provides a pharmaceutical composition comprising an antibody described herein and an active variant thereof, or an antigen-binding fragment thereof, comprising a fusion protein described herein, comprising an antibody-drug conjugate described herein, and optionally, comprising a pharmaceutically acceptable carrier.

[0070] The present invention also provides the use of the antibody described herein and its active variants, or antigen-binding fragments thereof, the fusion protein described herein, and the antibody-drug conjugate described herein in the preparation of medicaments for the treatment and / or prevention of BCMA-related diseases.

[0071] The present invention also provides a method for treating and / or preventing BCMA-related diseases and related symptoms, comprising administering an effective amount of the antibody described herein and its active variants, or antigen-binding fragments thereof, the fusion protein described herein, the antibody-drug conjugate described herein, or the pharmaceutical composition described herein to a subject.

[0072] The present invention also provides a method for detecting whether a sample contains T cells that highly express BCMA, comprising the steps of contacting the sample with the antibody described herein and its active variants, or antigen-binding fragments thereof, or the fusion protein described herein, optionally for diagnostic or non-diagnostic purposes.

[0073] The present invention also provides a detection product comprising the antibodies described herein and their active variants, or antigen-binding fragments thereof.

[0074] For example, the detection products described herein are selected from one or more of detection reagents, kits, chips, or test strips.

[0075] To achieve the above-mentioned objectives, the present invention further provides the following technical solutions:

[0076] A first aspect of the present invention provides an anti-BCMA antibody (e.g., a nanobody), wherein the heavy chain variable region of the anti-BCMA antibody (e.g., a nanobody) is composed of a framework region (FR) and a complementarity-determining region (CDR), the CDR comprising at least one of the following groups:

[0077] CDR1, CDR2, and CDR3 are shown in VHH with the name CBCHC-25.

[0078] For example, see Table 2 below.

[0079] Table 2: CDR and FR of CBCHC-25 nano alpaca VHH in the Kabat coding system

[0080]

[0081] Nanobodies (Nb) are variable regions of heavy chain antibodies (IgG2 and IgG3) found in camel-dwelling animals, and are considered the smallest antigen-binding fragments found in nature. Compared to traditional full-length monoclonal antibodies (mAb, approximately 150 KD), Nb offers advantages such as smaller molecular weight (12-15 KD), simpler structure, lower immunogenicity, higher tissue permeability, higher stability, higher solubility, lower aggregation, and ease of cloning. Furthermore, compared to similar mAb products, Nb has significantly lower production costs, making it accessible to most cancer patients. In September 2018, the European Medicines Agency approved the first nanobody drug, caplacizumab (trade name Cablivi), primarily for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults. Therefore, Nb holds promise for a wide range of applications in cancer treatment and diagnosis.

[0082] In this study, camels were immunized with recombinant BCMA antigen to construct a VHH phage library, and a functional anti-BCMA antibody was obtained after biopanning.

[0083] In the embodiments provided by the present invention, the frame region FR includes at least one of the following groups:

[0084] The VHH with the name CBCHC-25 indicates FR1, FR2, FR3, or FR4.

[0085] However, the frame region FR of this invention is not limited to the above sequence. Any sequence that can achieve its function is within the protection scope of this invention.

[0086] A second aspect of the present invention provides a polynucleotide that encodes the aforementioned anti-BCMA antibody (e.g., nanobody).

[0087] A third aspect of the present invention provides a recombinant expression vector comprising the aforementioned polynucleotides.

[0088] In the embodiments provided by the present invention, the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector.

[0089] A fourth aspect of the present invention provides a recombinant host cell comprising the above-mentioned polynucleotides or comprising the above-mentioned recombinant expression vector.

[0090] In the embodiments provided by the present invention, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0091] In the specific embodiments provided by the present invention, the host cell is selected from Escherichia coli or yeast cells.

[0092] In the specific embodiments provided by the present invention, the host cells are selected from HEK293T cells, HEK293F cells, Expi293F cells or CHO cells.

[0093] The fifth aspect of this invention provides a method for preparing the above-mentioned anti-BCMA antibody (e.g., nanobody), comprising the following steps:

[0094] The polynucleotide encoding the above-mentioned anti-BCMA antibody (e.g., nanobody) is inserted into the expression vector to obtain the recombinant expression vector;

[0095] The recombinant expression vector was transferred into host cells to obtain recombinant host cells;

[0096] Recombinant host cells are cultured to obtain a culture.

[0097] The culture is purified to obtain anti-BCMA antibodies (e.g., nanobodies).

[0098] In the embodiments provided by the present invention, purification is performed using Protein A agarose purification resin.

[0099] The sixth aspect of the present invention provides a bispecific antibody comprising the above-mentioned anti-BCMA antibody (e.g., nanobody) and a second antibody.

[0100] In the embodiments provided by the present invention, the second antibody includes, but is not limited to, 4-1BB nanobody, CD47 nanobody, VEGF nanobody, HER2 nanobody, EGFR nanobody, HER3 nanobody, B7H3 nanobody, TIGIT nanobody, OX-40 nanobody, CD40 nanobody or PD-L1 nanobody.

[0101] The seventh aspect of the present invention provides the use of the above-mentioned anti-BCMA antibody (e.g., nanobody) or bispecific or multispecific antibody in the preparation of drugs for the prevention and / or treatment of cancer and in the detection of BCMA protein.

[0102] In the embodiments provided by the present invention, cancers include, but are not limited to, lung cancer, stomach cancer, liver cancer, leukemia, kidney tumors, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumors, or bladder tumors.

[0103] The eighth aspect of the present invention provides a pharmaceutical composition comprising: the above-described anti-BCMA antibody (e.g., nanobody) or bispecific or multispecific antibody; and pharmaceutically acceptable excipients.

[0104] In the embodiments provided by the present invention, the dosage form of the pharmaceutical composition includes, but is not limited to, injection, powder for injection, tablet or capsule.

[0105] The ninth aspect of the present invention provides a kit for detecting BCMA protein, the kit comprising: the above-mentioned anti-BCMA antibody (e.g., nanobody) or bispecific or multispecific antibody; and a detection-acceptable reagent.

[0106] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0107] The reagents and raw materials used in this invention are all commercially available.

[0108] The positive and progressive effects of this invention are as follows: the anti-BCMA antibody of this invention has good affinity with human and monkey BCMA recombinant proteins, and CAR-M cells and TCE constructed from the anti-BCMA antibody exhibit target-dependent killing activity. Attached Figure Description

[0109] Figure 1 It is a BCMA CAR structure.

[0110] Figure 2 Capillary electrophoresis results for 8Z CAR-encoded mRNA.

[0111] Figure 3 Capillary electrophoresis results for 28F CAR-encoding mRNA.

[0112] Figure 4 For BCMA CAR expression.

[0113] Figure 5 The in vitro killing activity of BCMA CAR-M was demonstrated.

[0114] Figure 6 It is a BCMA / CD3 TCE structure.

[0115] Figure 7 The results are from BCMA / CD3 TCE SDS-PAGE gel electrophoresis.

[0116] Figure 8 This demonstrates the in vitro T cell-dependent cytotoxicity induced by BCMA / CD3 TCE. Detailed Implementation

[0117] This invention discloses an anti-BCMA antibody (e.g., a nanobody) and its biomaterials and products. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0118] Nanobodies (Nb) are naturally occurring antibodies lacking the light chain found in alpaca peripheral blood. These antibodies contain only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Unlike artificially engineered single-chain antibody fragments (scFv), they do not readily adhere to each other or aggregate. More importantly, the individually cloned and expressed VHH structure exhibits structural stability and antigen-binding activity comparable to the original heavy chain antibody, making it the smallest known unit capable of binding target antigens.

[0119] The framework region (FR) is the backbone region within the variable region of an antibody. Approximately 110 amino acid sequences near the N-terminus of the H and L chains of immunoglobulins exhibit significant variation, while the amino acid sequences of other parts remain relatively constant. Based on this, the light and heavy chains can be divided into variable (V) and constant (C) regions. The variable region contains the hypervariable region (HVR), also known as the complementarity-determining region (CDR), and the FR backbone region. The variability of the FR is less than that of the CDR. There are four FR molecules: FR1, FR2, FR3, and FR4. During antibody recognition, the four FR molecules coil, causing the CDR molecules to approach each other.

[0120] Complementarity determining region (CDR): The entire antibody molecule can be divided into two parts: constant region and variable region. Within the variable region, a small subset of amino acid residues exhibits particularly strong variations; these regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions. There are three hypervariable regions (HVRs) in the V region of the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions.

[0121] Bispecific antibodies are artificial antibodies containing two specific antigen-binding sites. They can bridge the gap between target cells and functional molecules (cells), stimulating a targeted immune response. They are a type of genetically engineered antibody and have become a hot topic in the field of antibody engineering, with broad application prospects in the immunotherapy of tumors.

[0122] The reagents, instruments, strains or biological materials used in this invention can all be obtained commercially.

[0123] The present invention will be further illustrated below with reference to the embodiments.

[0124] Example 1: Antigen preparation and animal immunization

[0125] Animal immunization: Multiple subcutaneous injections of recombinant human BCMA extracellular domain protein (hFc tag) were administered to the neck and back of one Bactrian camel. The absorption of the injected masses was monitored to confirm correct immunization. For the first immunization, 0.5 mg of antigen was mixed with Freund's complete adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at a volume of 1 mL per camel. For the second immunization, two weeks after the first immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at a volume of 1 mL per camel. For the third immunization, two weeks after the second immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at a volume of 1 mL per camel. For the fourth immunization, two weeks after the third immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at a volume of 1 mL per camel. Fifth immunization: Two weeks after the fourth immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously, with an injection volume of 1 mL per camel.

[0126] Serum processing and titer assay: One week after the fifth immunization, 2 mL of peripheral blood was collected, and serum was separated. The recombinant human BCMA extracellular protein (his tag) was coated onto a 96-well ELISA plate, and the antibody titer in the serum was determined using ELISA. ELISA results showed that the serum titer of the fifth immunization in camels was >1:32000, meeting the library construction criteria.

[0127] Example 2: Construction of a phage display immune antibody library

[0128] Since the serum titer of camels after the fifth immunization was >1:32000, it indicates the presence of high-affinity antibodies against human BCMA in the serum. Therefore, a phage display antibody library was constructed according to the following steps: ① Collect 50 mL of peripheral blood from camels after the fifth immunization and isolate PBMCs; take 2×10 7 Total RNA was extracted from PBMCs using an RNA extraction kit; an appropriate amount of RNA (e.g., 3-5 mg) was used to obtain cDNA using an RT-PCR reverse transcription kit. ② The variable region sequences of the IgG2 and IgG3 heavy chains (the VHH region of the heavy chain of the nanobody) were obtained stepwise by nested PCR. The experimental steps are as follows: 1) Design a pair of specific nested outer primers and perform the first round of PCR amplification using cDNA as a template. The amplified region is the Leader-CH2 region of the camel heavy chain antibody gene, and the product sizes are 700 bp and 900 bp. The 700 bp PCR product was recovered by gel electrophoresis. 2) Design nested inner primers (5 pairs of camel primers) and perform the second round of PCR amplification using the 700 bp first-round PCR product as a template. The amplified region is the VHH fragment of the camel heavy chain antibody variable region, and the product size is 400 bp. The second-round PCR product was purified and recovered using a PCR product purification kit. ③ The heavy chain variable region sequence was inserted into the enzyme-digested linearized phage vector VHH-LibTemplate via homologous recombination or enzyme digestion ligation to obtain the recombinant vector. After purification and recovery, it was transformed into supercompetent SS320 cells (containing helper phage M13K07). The transformed bacterial solution was resuspended in SOC medium and activated for 1 hour. A small amount of bacterial solution was serially diluted 10-fold to select an appropriate dilution titer and plated on LB / tet10 and LB / Carb50 culture plates. The plates were incubated overnight at 37°C and used for library volume calculation the next day. The remaining bacterial solution was transferred to a large volume of 2YT / Carb50 / Kan25 liquid medium and cultured overnight on a shaker at 37°C. The supernatant was harvested the next day, and 1 / 4 volume of PEG / NaCl solution was added to precipitate the phages. After precipitation, an appropriate amount of PBT solution was taken to resuspend the phages and diluted to the required concentration to obtain the phage display immune antibody library (stored at -80°C for later use). ④ Count the number of clones on LB / Carb50 plates and calculate the library size: The library size of the camel antibody library Lib BCMACamel is 3.69 × 10⁻⁶. 10 Twenty single clones were randomly selected from each plate for sequencing, and the results showed that the VHH insertion efficiency of the camel antibody library Lib BCMA Camel was 95%.

[0129] Example 3: Screening of antibody libraries

[0130] First, protein screening of Lib BCMA Camel was performed using recombinant BCMA extracellular region protein: 5 mg / mL of recombinant human BCMA extracellular region protein (his tag) was added to a 96-well plate (100 mL / well) and incubated overnight at 4°C; NEB5αF' E. coli was streaked onto 2YT / tet10 plates and incubated overnight at 37°C; the next day, NEB5αF' single clones were picked from the overnight 2YT / tet10 plates and added to 3 mL of 2YT / tet10 liquid medium, and cultured at 37°C until OD600 = 0.8; simultaneously, the antigen supernatant of the 96-well plate was removed, and 200 mL of 1% BSA was added to each well for blocking. 200 mL of 1% BSA was added to the blank wells as negative controls, and the plates were placed in a 3D rotary shaker at room temperature for 2 hours; afterwards, the supernatant of the protein wells and control wells was removed, and the plates were washed with 200 mL of PT, and 100 mL of PT was added to each well. A phage antibody library of mL was placed in a 3D rotary shaker at room temperature for 2 hours. The supernatant from the protein and control wells was removed, and the cells were washed with 200 mL of PT. 100 mL of 100 mM HCl was added to the wells, and the cells were incubated at room temperature for 5 minutes. The supernatant was aspirated and added to a 1.5 mL centrifuge tube, which was then neutralized with 1 M Tris-HCl. This mixture was added to a centrifuge tube containing 1 mL of NEB5αF' bacteria and incubated at 37°C for 1 hour. 20 mL of the culture was diluted appropriately and plated onto LB / Carb50 plates. The plates were incubated overnight at 37°C for titer and enrichment calculations the following day. 1 mL of helper phage M13K07 (final concentration 10) was added to the remaining culture medium. 10 Incubate the phages in a shaker at 37°C for 1 hour (number of phages per mL). Transfer the culture medium to 35 mL of 2YT / Carb50 / Kan25 medium and incubate overnight at 37°C. Collect the phages to form the antibody library for each round. Repeat the above steps 2-3 times until phage enrichment occurs. Enrichment is considered successful if the number of colonies in the antigen-binding wells on the LB / Carb50 plate is more than 10 times that of the negative control wells. In this experiment, after the second round of protein panning, the number of colonies in the antigen-binding wells of Lib BCMA Camel was 10 times that of the negative control wells, indicating successful enrichment.

[0131] Subsequently, BCMA-overexpressing 293F cells were used to pan-pick the enriched libraries obtained from protein screening: first, 2 × 10⁻⁶ cells were used. 7Mix the blank 293F cells with the phage library collected in the previous round (R1 round in this project) and place them in a 3D rotary shaker at 4°C for 2 hours. After the negative screening is completed, centrifuge at 300 g for 10 minutes to remove the cells. Take the phage from the supernatant and divide it into two portions: one portion with 1×10 g of phage. 7 Mixed with 293F cells overexpressing BCMA, incubated at 4°C in a 3D rotary shaker for 2 hours for positive screening; one copy was mixed with 1×10 7 Mix 293F blank cells and incubate at 4°C in a 3D rotary shaker for 2 hours as a negative control; then centrifuge at 300 g for 10 minutes to collect both cell types. After washing the cells 6 times with pre-chilled PBS, add the cells to a centrifuge tube containing 1 mL of NEB5αF' bacteria with OD600=0.8 and incubate at 37°C for 1 hour. Take 20 mL of the culture medium from the centrifuge tube, dilute it appropriately, plate it on an LB / c plate, and incubate overnight at 37°C. The next day, the titer and enrichment level will be calculated. Add 1 mL of helper phage M13K07 (final concentration 10) to the remaining culture medium. 10 Incubate the phages in a shaker at 37°C for 1 hour (phages / mL). Transfer the culture medium to 35 mL of 2YT / Carb50 / Kan25 medium and incubate overnight at 37°C. Collect the phages to form the antibody library for each round. Repeat the above steps 2-3 times until phage enrichment occurs. Enrichment is considered successful if the number of colonies in the 293F cell positive binding wells overexpressing BCMA on the LB / Carb50 plate is more than three times that in the blank 293F cell negative control wells. In this experiment, after the second round of cell screening, the number of colonies in the 293F cell positive binding wells overexpressing BCMA was six times that in the blank 293F cell negative control wells, indicating successful enrichment.

[0132] Clones from the cell enrichment rounds were randomly selected and cultured in 96-well plates. After centrifugation, the supernatant was used for Phage ELISA screening. Clones with OD values ​​binding to the recombinant human BCMA extracellular region protein (his tag) were selected: clones with OD values ​​>2 binding to the blocking solution were defined as positive clones, and sequenced and aligned to obtain unique sequences.

[0133] Example 4: Statistical Analysis of Positive Sequences

[0134] The results of statistical analysis of phage ELISA positive sequences are shown in Table 3.

[0135] Table 3: Amino acid sequence of VHH in CBCHC-25 nano-alpaca and CDR and FR in the Kabat coding system

[0136]

[0137] Example 5: Eukaryotic expression of nanobodies

[0138] The sequences in Table 1 of Example 4 were expressed in eukaryotic cells. The experimental steps were as follows: 1) The VHH fragments of these sequences were amplified by PCR, and the fragments were inserted into the eukaryotic expression vector pFcIG containing the hFc tag using homologous recombination or enzyme digestion ligation. The fragments were electroporated into E. coli trans5α host bacteria, and after bleomycin screening, the correct recombinant plasmids were obtained by sequencing of single clones. Then, the host bacteria containing the recombinant plasmids were expanded and cultured, and sterile endotoxin-free plasmids were obtained using an endotoxin-free kit. 2) HEK293F cells were cultured in serum-free medium. The recombinant expression plasmids were transfected into HEK293F cells for expression using Polyplus suspension cell transfection reagent. Feed was added 24 and 72 hours after transfection, and the supernatant was collected on day 5. The antibodies were purified using Protein A agarose resin and stored in PBS solution. The experimental results (Table 4) showed that the VHH-hFc recombinant antibody was transiently transfected and expressed in 293F cells. SDS-PAGE electrophoresis confirmed that the anti-human BCMA nanobody (structure: VHH-hFc) had a normal band size and a purity >95%. The melting temperature Tm of the antibody was detected by fluorescence spectroscopy (IF), and the aggregation temperature Tagg was detected by static light scattering (SLS). The results (Table 5) showed that the antibody sequence had good stability. Non-specific binding of the antibody was detected by surface plasmon resonance (SPR). The results (Table 6) showed that the antibody sequence did not bind non-specifically to Lysozyme, Trypsin inhibitor, Deactivated Carboxymethyl Dextran, or Carboxymethyl Dextran.

[0139] Table 4. Eukaryotic transient expression results of VHH-hFc recombinant antibody

[0140]

[0141] Table 5. Tm & Tagg assays for VHH-hFc recombinant antibodies.

[0142]

[0143] Table 6 Nonspecific binding detection of VHH-hFc recombinant antibodies

[0144]

[0145] Example 6: Affinity EC50 between nanobody and BCMA recombinant protein

[0146] The affinity of the VHH-hFc recombinant antibody for recombinant extracellular proteins of BCMA from different species was determined using ELISA: BCMA extracellular recombinant protein (his tag) was added to 96-well ELISA plates at 200 ng / well and incubated overnight at 4°C. The VHH-hFc recombinant antibody was diluted to different concentrations (0.014-10 mg / mL) and reacted with the antigen using ELISA. Color development was performed using HRP-labeled anti-hIgG1Fc secondary antibody, and the absorbance at 450 nm was measured using a microplate reader. The results (Tables 7 and 8) showed that the affinity (EC50) of the anti-BCMA nanobody (structure: VHH-hFc) for human BCMA extracellular recombinant protein was 0.240 mg / mL; and the affinity (EC50) for monkey BCMA extracellular recombinant protein was 0.102 mg / mL. Simultaneously, the antibody did not bind to BSA (Table 9).

[0147] Table 7. ELISA binding EC50 of VHH-hFc recombinant antibody with human BCMA (his tag)

[0148]

[0149] Table 8. ELISA binding EC50 of VHH-hFc recombinant antibody with monkey BCMA (his tag)

[0150]

[0151] Table 9. VHH-hFc recombinant antibody does not bind to BSA.

[0152]

[0153] Example 7: Affinity KD between nanobodies and BCMA recombinant protein

[0154] Antibodies were captured using Biacore's Protein A chip. Human / monkey / mouse BCMA solutions at concentrations of 50 nM, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 were used as analytes. Multi-cycle kinetic screening was performed: Association: 120 s, Disassociation: 300 s, Flow rate: 30 μL / min; regeneration was performed with 10 mM Glycine at pH 1.5 at a flow rate of 50 μL / min for 30 s. A 1:1 binding model and Fit local analysis were used to determine the fitting constants. The results are shown in Tables 10-12.

[0155] Table 10 Affinity KD values ​​of VHH-hFc recombinant antibody with human BCMA (his tag)

[0156]

[0157] Table 11 Affinity KD values ​​of VHH-hFc recombinant antibody with monkey BCMA (his tag)

[0158]

[0159] Table 12 Affinity KD values ​​of VHH-hFc recombinant antibody with mouse BCMA (his tag)

[0160]

[0161] Example 8: Binding assay with BCMA-positive cells

[0162] The affinity of the nanobody for BCMA-positive cells NCI-H929 and RPMI8226, and for BCMA-negative cells K562, was detected using flow cytometry: 1) Take 0.3 × 10⁻⁶ cells... 6 Cells (NCI-H929, RPMI8226, and K562, respectively) were washed twice with PBS and then resuspended in 100 mL PBS; 2) they were incubated with 10 mg / mL VHH-hFc recombinant antibody for 1 hour; 3) after washing the cells three times with PBS, the cells were resuspended in 100 mL PBS, and 0.2 mg / mL FITC-labeled Anti-hFc antibody was added, followed by incubation for 1 hour; 4) after washing the cells three times with PBS, the cells were resuspended in 300 mL PBS, and fluorescence was detected by flow cytometry. The results are shown in Table 13. The experimental results show that the nanobodies obtained by screening can bind to NCI-H929 and RPMI8226 cells, but not to K562 cells.

[0163] Table 13 Binding affinity of VHH-hFc recombinant antibody to different cells (positive rate %)

[0164]

[0165] Example 9: Construction of BCMA-targeted CAR molecules and preparation of mRNA

[0166] Design as Figure 1The 8Z CAR (SEQ ID NO: 5) and 28F CAR (SEQ ID NO: 6) molecules targeting the BCMA antigen shown are composed, from the N-terminus to the C-terminus, of the CSF2Rα-SP signal peptide (SEQ ID NO: 7), the VHH sequence of the BCMA single-domain antibody CBCHC-25 (SEQ ID NO: 1) or its humanized sequence (SEQ ID NO: 28), the hinge region and transmembrane sequence of CD8H+TM (SEQ ID NO: 8) or CD28H+TM (SEQ ID NO: 9), and the intracellular signal sequence of CD3ζ-ICD (SEQ ID NO: 10) or FcεR1γ-ICD (SEQ ID NO: 11). The specific sequences are shown in Table 14.

[0167] To test CAR molecule expression and function, a nucleic acid fragment containing a T7 promoter, 5'-UTR, the aforementioned CAR coding sequence (CDS), 3'-UTR, a poly(A) sequence containing 100 A nucleotide residues, and an IIS-type restriction endonuclease cleavage site was synthesized in vitro and cloned into an in vitro transcription vector (GS-CMV-100A-BspQI, Nanjing Genscript Biotech Co., Ltd.). The obtained vector was linearized by BspQI digestion and then transcribed in vitro using T7-RNA polymerase to produce mRNA molecules, with a 5' cap structure added. The 5' cap structure, through co-transcriptional capping, incorporates a cap analog as the first nucleotide into the transcript during in vitro transcription, directly producing mRNA molecules with a Cap1 structure. The resulting mRNA molecules were purified and resuspended in 1 mM sodium citrate. Quality control of the mRNA molecules was performed using capillary electrophoresis; the mRNA molecule length and integrity met the requirements (see...). Figure 2 and 3 ).

[0168] Table 14 BCMA CAR and its component sequence

[0169]

[0170]

[0171] Example 10: Preparation of BCMA-targeted CAR-M cells

[0172] This embodiment uses electroporation to transfect mRNA encoding CAR molecules with different structures into 293T or THP-1 cells. The translation efficiency of different BCMA CAR structures is identified by detecting the expression level of CAR molecules on the cell surface. The specific procedures are as follows: Lonza nuclear transfer working solutions were prepared: Supplement 1 was added to Cell Line Solution SF (Lonza, V4XC-2032) to prepare the nuclear transfer solution for 293T cells; Supplement 1 was added to Cell Line Solution SG (Lonza, V4XC-3032) to prepare the nuclear transfer solution for THP-1 cells, and kept on ice. Single-cell suspensions of 293T and THP-1 cells were collected, centrifuged at 300g for 5 min, and then diluted to a concentration of 1×10⁻⁶ cells using the corresponding nuclear transfer working solutions. 7 Cells / mL, keep on ice. Take a 1.5 mL low-adsorption EP tube from the ice bath and add 20 μL of cell suspension resuspended in nuclear transfer working solution (i.e., 2 × 10⁻⁶ cells / mL). 5 Add 2 μg mRNA to each cell and gently pipette 2-3 times to mix. Take a 20 μL Lonza 4D nuclear transfer cuvette and slowly transfer the entire nuclear transfer complex (approximately 22 μL) along the cuvette wall. Gently tap the cuvette wall several times to allow the liquid to settle to the bottom. Select the appropriate electroporation program and start electroporation immediately. After electroporation, transfer all cell suspension from the nuclear transfer cuvette to a 24-well plate pre-coated with complete culture medium (400 μL of preheated 37°C complete culture medium is added to each well). Incubate at 37°C, 5% CO2, and saturated humidity for 24 hours without moving the cells. After incubation, collect the cell suspension and perform flow cytometry staining to detect CAR expression levels.

[0173] Experimental results showed that in 293T cells, the positive expression rates of CAR in the 8Z-CAR and 28F-CAR groups reached 99.6% and 99.3%, respectively, both significantly higher than the negative control MOCK group. Furthermore, there was no significant difference in expression efficiency between the two groups, indicating that both BCMA CAR structures can achieve efficient translation and membrane surface expression in 293T cells (see...). Figure 4 A).

[0174] In THP-1 cells, the positive expression rate of 8Z-CAR was 51.1% in the 8Z-CAR group and 43.1% in the 28F-CAR group, both significantly higher than that of the MOCK control group. The expression efficiency of 8Z-CAR was slightly higher than that of 28F-CAR, suggesting that differences in CAR molecular structure affect its expression level in myeloid THP-1 cells (see...). Figure 4 B).

[0175] Example 11: In vitro functional study of BCMA-targeted CAR-M cells

[0176] This example aims to evaluate the biological activity of the gene expression of the CAR molecule designed in this invention. Using human primary macrophages (hMDM) as a cell model, CAR mRNA transfection was mediated by electroporation, and combined with cell killing experiments to evaluate its targeted killing ability against BCMA-positive target cells. The specific method is as follows: Frozen PBMCs were resuscitated, resuspended in PBS, passed through a 40 μm cell sieve, and centrifuged at 300 g for 5 min. Following the instructions of Miltenyi CD14 MicroBeads-human Kit (Micro,human 130-050-201), high-purity CD14⁺ monocytes were separated using magnetic bead sorting. The CD14⁺ monocyte density was adjusted to 1 × 10⁶ cells / cells. 6 Cells were seeded at a density of 10 cells / mL into 6-well plates. Macrophage colony-stimulating factor M-CSF (Sinobiological, 11792-HNAH) at a concentration of 50 ng / mL was added to induce differentiation into mature macrophages. The medium was changed halfway every three days, and mature macrophages were harvested after 6-7 days of culture. Mature macrophages were digested, prepared into single-cell suspensions, and centrifuged at 300g for 5 min. Electroporation was performed using the Primary Cell Nucleofection Solution Kit (Lonza, V4XP-3032). After electroporation, all cell suspensions were transferred to 6-well plates pre-coated with complete culture medium (900 μL of pre-warmed 37℃ complete culture medium was added to each well). The plates were then incubated at 37℃, 5% CO2, and saturated humidity for 24 hours without movement. After culture, CAR-M cells were obtained. The cell suspension was collected, and 2 × 10⁶ cells were taken from each group. 5 Flow cytometry staining was performed on the cells, and the expression level of CAR was detected by flow cytometry. A suspension of target cells RPMI8226 was prepared and the density was adjusted to 1×10⁻⁶. 5 Cells were seeded at 100 μl / well in 96-well plates (BIOLAND, CCP06-096W) at a density of 1 × 10⁶ cells / mL. 4For each well, additional CAR-M cells and target cells RPMI8226 were co-incubated at an E:T ratio of 10 / 3 / 1. After 24 hours of culture, the white 96-well plate was removed from the incubator and allowed to equilibrate to room temperature for 10 minutes. After centrifugation at 350g for 5 minutes, ONE-Glo™ Luciferase Assay System (Promega, E6110) was added, and the plate was incubated at room temperature in the dark for 3 minutes. The cytotoxic function of CAR-M cells was then assessed by detecting the luciferase (luc) activity value.

[0177] Experimental results showed that in all detection gradients with an E:T ratio of 10 / 3 / 1, the target cell killing rate of the 8Z-CAR-M group was significantly higher than that of the MOCK control group; the MOCK group showed only a very low level of non-specific killing, confirming that 8Z-CAR-M cells have high targeting specificity in killing BCMA-positive target cells (see...). Figure 5 ).

[0178] Example 12: Design of BCMA / CD3 Bispecific T Cell Connector (TCE)

[0179] Design as Figure 6 The BCMA / CD3 TCE molecules DD003-3 (SEQ ID NO:12) and DD003-5 (SEQ ID NO:13) targeting BCMA antigen and T cell marker CD3, and the positive control molecule DD003-BM (SEQ ID NO:27), are respectively composed of human interleukin-10 (IL-10)-SP signal peptide (SEQ ID NO:14), the VHH sequence of humanized BCMA single-domain antibody CBCHC-25 (SEQ ID NO:28), or the VHH double tandem (CBCHC-25 - G4S - CBCHC-25) sequence of humanized BCMA single-domain antibody CBCHC-25 (SEQ ID NO:15) (SEQ ID NO:15) from the N-terminus to the C-terminus, or the BCMA targeting sequence, G4S-linker sequence (SEQ ID NO:16) of B38MCAR molecule from US10934363B2 patent, targeting CD3ε (CD3 The monoclonal antibody clone SP34, consisting of the scFv sequence (SEQ ID NO:17) and the 6xHis sequence (SEQ ID NO:18) of the epsilon chain, is shown in Table 15.

[0180] Table 15 BCMA TCE and its component sequence

[0181]

[0182]

[0183]

[0184] Example 13: BCMA / CD3 TCE Expression

[0185] This embodiment aims to characterize the physicochemical properties of candidate bispecific TCE proteins DD003-3 and DD003-5 produced by a mammalian cell transient expression system, in order to confirm their structural integrity and process scalability, and to meet the quality requirements for therapeutic proteins in preclinical studies.

[0186] The samples used were DD003-3 and DD003-5 proteins purified by affinity chromatography. The buffer was replaced with PBS (pH 7.4), and the concentration was determined using the BCA method before being used for various assays. SDS-PAGE gel electrophoresis results showed that under non-reducing conditions, both DD003-3 and DD003-5 exhibited a single main band, with migration positions corresponding to approximately 35–55 kDa, consistent with the expected monomer molecular weight range, and without obvious high-molecular-weight aggregates or low-molecular-weight degradation bands. Under reducing conditions, the bands did not change significantly (see [link to reducing gel electrophoresis]). Figure 7 Size exclusion high-performance liquid chromatography (SEC-HPLC) results showed that the retention time of the main peak of DD003-3 was 8.279 min, with a peak area accounting for 99.71%, while the retention time of the main peak of DD003-5 was 7.811 min, with a peak area accounting for 98.39%. Both samples showed only a single main peak, with no visible aggregate peaks (>10% high molecular weight peaks were not detected), indicating that the products were highly monomeric and without significant aggregation. The comprehensive physicochemical parameters showed that DD003-3 and DD003-5 had good expression yields, high purity, and no endotoxin risk (see Table 16).

[0187] Table 16 BCMA / CD3 TCE Expression Parameters and Physicochemical Properties

[0188]

[0189] Example 14: In vitro functional study of BCMA / CD3 TCE

[0190] This embodiment aims to evaluate the targeted killing ability of two novel BCMA / CD3 bispecific TCE proteins (DD003-3 and DD003-5) designed in this invention against autologous B cells and BCMA-positive target cells in the presence of healthy donor PBMCs, and to conduct parallel comparisons with the positive control molecule DD003-BM and the CD19 / CD3 positive control BT003-BM1 (i.e., Blinatumomab, synthesized by Baiying Biotechnology) with the same target.

[0191] The experimental method is as follows: 1) Healthy donor W059K119-derived PBMCs were seeded at 1e5 cells per well in a 96-well round-bottom plate, and 1e4 BCMA-positive target cells H929-GFP were added to each well to achieve an effector-to-target ratio of 10:1. Control groups were set up with either PBMCs alone or H929-GFP alone; 2) TCE molecules were added in a 10-fold serially diluted form, and the cells were incubated at 37°C in a 5% CO2 incubator for 48 hours; 3) Cells were collected and incubated for 15 minutes in staining buffer containing anti-CD20-APC (Biolegend, catalog number 302310), anti-Live / Dead fluorescent labeling antibody (Zombie Aqua™ Fixable Viability Kit, Biolegend, catalog number 423102), and CountingBeads (Thermo Fisher CountBright™ Absolute Counting Beads, catalog number C36950); 4) After washing twice with PBS, the cells were resuspended in 200 μL of PBS. In PBS, at least 3000 Counting Beads events were collected using flow cytometry, and the absolute number of live CD20-positive B cells or H929-GFP cells in each well was calculated using the following formula:

[0192] Absolute number of live CD20-positive B cells = (CD20-positive live cell events / Counting Beads events) × Total number of Counting Beads;

[0193] Absolute number of live H929 cells = (GFP-positive live cell events / Counting Beads events) × Total number of Counting Beads;

[0194] 5) Using PBMCs without TCE or H929-GFP alone as negative controls, the killing rates against CD20-positive B cells and BCMA-positive target cells were calculated respectively:

[0195] Kill rate (%) = [1 – (absolute number of viable cells in the experimental group / absolute number of viable cells in the negative control group)] × 100%;

[0196] 6) The kill curve was fitted using GraphPad Prism 10.1.2 software, and the maximum effect value (Emax, %) and half-maximal effective concentration (EC50, pM) were calculated.

[0197] Experimental results showed that: 1) In the co-culture system of healthy donor PBMCs and BCMA target cells, both DD003-3 and DD003-5 could efficiently induce T cell-mediated BCMA+ target cell killing in a significant dose-dependent manner. Compared with the BT003-BM1 control, the Emax killing rate of TCE in the DD003 group reached approximately 90%. The EC50 levels of DD003-3 and DD003-5 were several orders of magnitude lower than those of the positive control group (see...). Figure 8 (A) 2) In the killing of B cells, DD003-3 and DD003-5 showed a certain ability to induce TDCC, and their Emax was lower than that of the positive control BT003-BM1, but significantly higher than that of the DD003-BM control (see A). Figure 8 B).

[0198] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-BCMA antibody or its active variant or its antigen-binding fragment, characterized in that, The heavy chain variable region of the anti-BCMA antibody contains three complementarity-determining regions (CDRs), wherein the CDRs include CDR1, CDR2, and / or CDR3; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

2. The anti-BCMA antibody or its active variant or its antigen-binding fragment as described in claim 1, characterized in that, The framework region FR of the anti-BCMA antibody includes FR1, FR2, FR3 and / or FR4; the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively; or, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 29, SEQ ID NO: 20, SEQ ID NO: 30 and SEQ ID NO: 31, respectively. Preferably, the amino acid sequence of the heavy chain variable region of the anti-BCMA antibody is as shown in SEQ ID NO: 1 or SEQ ID NO: 28; More preferably, the anti-BCMA antibody is a VHH or a nanobody.

3. The anti-BCMA antibody or its active variant or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The anti-BCMA antibody is a nanobody and also contains Fc; Preferably, the Fc is the Fc of human or mouse IgG1, IgG2, IgG3 or IgG4 or a variant thereof; the variant has 80%-99.9% sequence identity with the sequence from which it originates; More preferably, the active variant has 80%-99.9% identity with the amino acid sequence of the anti-BCMA antibody; for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity.

4. A chimeric antigen receptor, characterized in that, The extracellular recognition domain of the chimeric antigen receptor comprises the anti-BCMA antibody as described in any one of claims 1-3, or an active variant thereof, or an antigen-binding fragment thereof; Preferably, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the anti-BCMA antibody, a hinge region and a transmembrane sequence, and an intracellular signal sequence; optionally, it also includes a signal peptide.

5. The chimeric antigen receptor as described in claim 4, characterized in that, The chimeric antigen receptor is selected from one or more of the following: The hinge region and transmembrane sequence are the hinge region and transmembrane sequence of CD8 or CD28; their amino acid sequences are, for example, as shown in SEQ ID NO: 8 or SEQ ID NO: 9; The intracellular signal sequence is a CD3ζ or FcεR1γ intracellular signal sequence; its amino acid sequence is, for example, shown in SEQ ID NO:10 or SEQ ID NO:

11. The signal peptide may be optionally the CSF2Rα-SP signal peptide or the IL-10 signal peptide; its amino acid sequence is shown, for example, as SEQ ID NO:7 or SEQ ID NO:14; Preferably, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO:

33.

6. A bispecific or multispecific antibody, characterized in that, It comprises the anti-BCMA antibody as described in any one of claims 1-3, or an active variant thereof, or an antigen-binding fragment thereof; Preferably, the bispecific or multispecific antibody is a bispecific T-cell conjugate; More preferably, the bispecific T-cell connector further comprises an antibody targeting a T-cell marker, such as CD3, CD2, CD4, or CD8.

7. The bispecific or multispecific antibody as described in claim 6, characterized in that, The bispecific T cell conjugate comprises, from N-terminus to C-terminus, the anti-BCMA antibody or its dual tandem antibody, a G4S-linker sequence, and an antibody targeting CD3ε. Preferably, the amino acid sequence of the G4S-linker sequence is shown in SEQ ID NO:16; The antibody targeting CD3ε is the scFv sequence of the monoclonal antibody clone SP34 that targets the CD3ε chain; its amino acid sequence is shown, for example, as SEQ ID NO:

17. More preferably, the amino acid sequence of the dual-specific T cell conjugate is shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:25 or SEQ ID NO:

26.

8. A polynucleotide, characterized in that, The polynucleotide is selected from any one of the following groups: (1) Encoding the anti-BCMA antibody as described in any one of claims 1-3, or its active variant or antigen-binding fragment thereof, or the bispecific or multispecific antibody as described in claim 6 or 7; (2) Encoding the chimeric antigen receptor as described in claim 4 or 5.

9. A recombinant expression vector, characterized in that, The recombinant expression vector is selected from any one of the following groups: (i) comprising (1) of the polynucleotide as described in claim 8, or expressing the anti-BCMA antibody as described in any one of claims 1-3 or its active variant or its antigen-binding fragment or the bispecific or multispecific antibody as described in claim 6 or 7; (ii) comprising (2) of the polynucleotide as described in claim 8, or expressing the chimeric antigen receptor as described in claim 4 or 5; Preferably, the backbone plasmid of the recombinant expression vector is pFcIG or GS-CMV-100A-BspQI.

10. A recombinant host cell, characterized in that, The recombinant host cell expresses the anti-BCMA antibody of any one of claims 1-3 or its active variant or its antigen-binding fragment, or the bispecific or multispecific antibody of claim 6 or 7; for example, the recombinant host cell contains (i) of the polynucleotide of claim 8, or contains (i) of the recombinant expression vector of claim 9. Preferably, the original cell of the recombinant host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the original cells are mammalian cells, Escherichia coli or yeast cells, such as Escherichia coli trans5α, Expi293F cells, CHO cells, HEK293F cells, HEK293T cells.

11. A recombinant immune cell comprising or expressing the chimeric antigen receptor as described in claim 4 or 5; for example, the recombinant immune cell comprising (2) of the polynucleotide as described in claim 8, or comprising (ii) of the recombinant expression vector as described in claim 9. Preferably, the recombinant immune cells originate from any one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, NKT cells, and iPSCs; More preferably, the macrophages are human primary macrophages; the T cells are regulatory T cells, helper T cells, cytotoxic T cells, or γδ T cells; and the monocytes are human monocytes, such as THP-1 cells.

12. A method for preparing the anti-BCMA antibody or its active variant or its antigen-binding fragment as described in any one of claims 1-3, or the bispecific or multispecific antibody as described in claim 6 or 7, characterized in that, The method includes: culturing the recombinant host cells as described in claim 10 to obtain the BCMA antibody or its active variant or its antigen-binding fragment or bi- or multi-specific antibody; Preferably, the method includes the following steps: inserting a polynucleotide encoding an anti-BCMA antibody or its active variant or its antigen-binding fragment or a bispecific or multispecific antibody into an expression vector to obtain the recombinant expression vector; The recombinant expression vector was transferred into host cells to obtain the recombinant host cells; The recombinant host cells were cultured to obtain a culture. Optionally, the process also includes purifying the culture to obtain the anti-BCMA antibody or its active variant or its antigen-binding fragment or bi- or multi-specific antibody.

13. An antibody-drug conjugate or a pharmaceutically usable salt thereof, comprising an anti-BCMA antibody as described in any one of claims 1-3 or an active variant thereof or an antigen-binding fragment thereof, or a bispecific or multispecific antibody as described in claim 6 or 7.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: an anti-BCMA antibody as described in any one of claims 1-3 or an active variant thereof or an antigen-binding fragment thereof, a bispecific or multispecific antibody as described in claim 6 or 7, a recombinant immune cell as described in claim 11 or an antibody-drug conjugate as described in claim 13 or a pharmaceutically acceptable salt thereof; and pharmaceutically acceptable excipients.

15. A reagent kit, characterized in that, The kit comprises an anti-BCMA antibody or its active variant or its antigen-binding fragment as described in any one of claims 1-3, a bispecific or multispecific antibody as described in claim 6 or 7, a polynucleotide as described in claim 8, a recombinant expression vector as described in claim 9, a recombinant host cell as described in claim 10, a recombinant immune cell as described in claim 11, an antibody-drug conjugate or its pharmaceutically usable salt as described in claim 13, and / or a pharmaceutical composition as described in claim 14; and a detection-acceptable reagent.

16. A method for detecting BCMA, characterized in that, The method includes contacting the anti-BCMA antibody or its active variant or its antigen-binding fragment as described in any one of claims 1-3, or the bi- or multi-specific antibody as described in claim 6 or 7, with the sample to be tested. Preferably, the method is for non-diagnostic and / or therapeutic purposes.

17. The use of the anti-BCMA antibody or its active variant or antigen-binding fragment as described in any one of claims 1-3, the chimeric antigen receptor as described in claim 4 or 5, the bispecific or multispecific antibody as described in claim 6 or 7, the polynucleotide as described in claim 8, the recombinant expression vector as described in claim 9, the recombinant host cell as described in claim 10, the recombinant immune cell as described in claim 11, the antibody-drug conjugate as described in claim 13 or its pharmaceutically usable salt, or the pharmaceutical composition as described in claim 14, in the preparation of a medicament for the prevention and / or treatment of cancer or a kit for the detection of BCMA protein; Preferably, the cancer is a BCMA-positive cancer or an autoimmune disease containing BCMA-positive autoreactive B cells or plasma cells; More preferably, the cancer is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasmama, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, plasmacytoma, and melanoma; the autoimmune disease is selected from one or more of the following: systemic lupus erythematosus, multiple sclerosis, inflammatory myositis, antineutrophil cytoplasmic antibody vasculitis, rheumatoid arthritis, Sjögren's syndrome, antiphospholipid syndrome, myasthenia gravis, autoimmune thyroid disease, pemphigus / pemphigoid, and type 1 diabetes; More preferably, the myeloma is multiple myeloma or plasma cell myeloma; the leukemia is chronic lymphocytic leukemia or acute B-lymphocytic leukemia; the malignant lymphoma is non-Hodgkin lymphoma or Hodgkin lymphoma; the plasma cell tumor is solitary plasma cell tumor; and the autoimmune thyroid disease is Graves' disease or Hashimoto's thyroiditis.

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