Human bcma antibodies and uses thereof

Abstract

The present disclosure relates to the field of biological medicine, and provides a human BCMA antibody and application thereof. The anti-BCMA monoclonal antibody 1F5 provided by the present disclosure has strong affinity to BCMA antigen, and the Kd value is 0.752×10 ‑9 M; the light chain is a kappa chain; and the monoclonal antibody 1F5 can bind to the BCMA positive cell line H 929 and RPMI 8226 with high affinity, and can also bind to the CD138 positive bone marrow mononuclear cells derived from multiple myeloma patients with high affinity. In view of these characteristics, the monoclonal antibody 1F5 can be used for detecting cells expressing human BCMA, and can be used in tumor immunotherapy targeting human BCMA alone or in combination with other methods, and has diagnostic and therapeutic values.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, and in particular, to an anti-human BCMA antibody or antigen-binding moiety and its applications. Background Technology

[0002] BCMA, also known as TNFRSF17 or CD269, is a member of the tumor necrosis factor receptor superfamily and is a type III transmembrane glycoprotein. BCMA ligands include B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Because BCMA is highly expressed only in malignant plasma cells, it is less commonly found in normal tissues (except plasma cells and some mature B cells) and CD34. + BCMA is not expressed on stem / group cells, which makes it an important biomarker for the diagnosis of diseases such as multiple myeloma and a highly promising target for immunotherapy.

[0003] Currently, treatments and drugs for human BCMA, such as bispecific antibodies, CAR-T therapy, and antibody-drug conjugates, are either on the market or undergoing clinical trials. Therefore, developing new BCMA antibodies will bring more hope for overcoming related diseases. Summary of the Invention

[0004] Technical problems to be solved:

[0005] One aspect of this disclosure is to provide an anti-human BCMA antibody, addressing the problem of the lack of an ideal molecule that specifically binds to human BCMA in the prior art.

[0006] Technical solution:

[0007] An isolated antibody or antigen-binding moiety that specifically binds to human BCMA protein, the antibody or antigen-binding moiety comprising:

[0008] (1) Heavy chain variable region CDRH1 as shown in SEQ ID No. 1, heavy chain variable region CDRH2 as shown in SEQ ID No. 2, and heavy chain variable region CDRH3 as shown in SEQ ID No. 3; and

[0009] (2) Light chain variable region CDRL1 as shown in SEQ ID No.4, light chain variable region CDRL2 as shown in SEQ ID No.5 and light chain variable region CDRL3 as shown in SEQ ID No.6.

[0010] In some embodiments, the antibody or antigen-binding portion may include:

[0011] (1) Heavy chain variable region CDRH1 as shown in SEQ ID No. 1, heavy chain variable region CDRH2 as shown in SEQ ID No. 2, and heavy chain variable region CDRH3 as shown in SEQ ID No. 3; or

[0012] (2) Light chain variable region CDRL1 as shown in SEQ ID No.4, light chain variable region CDRL2 as shown in SEQ ID No.5 and light chain variable region CDRL3 as shown in SEQ ID No.6.

[0013] Furthermore, in some embodiments, the antibody or antigen-binding portion comprises: a heavy chain variable region as shown in SEQ ID No. 7 and a light chain variable region as shown in SEQ ID No. 8, or an amino acid sequence having more than 90% identity with the heavy chain variable region or the light chain variable region.

[0014] In some embodiments, the antibody may be a mammalian-derived antibody, such as that from mice, rabbits, sheep, horses, monkeys, pigs, camels, sharks, chickens, etc. In other embodiments, the antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody.

[0015] In some embodiments, the antibody may be IgG, IgA, IgM, IgD, or IgE. Preferably, in some embodiments, the antibody may be IgG. Further, in some embodiments, the antibody may be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody may be IgG1.

[0016] In some embodiments, the antibody is a monoclonal antibody.

[0017] In some embodiments, the antibody or antigen-binding portion is modified, including N-glycosylation, O-glycosylation, phosphorylation, methylation, acetylation, or labeling.

[0018] In some embodiments, the antibody includes an Fc moiety. Preferably, in some embodiments, the Fc moiety of the antibody is modified or altered to enhance its ADCC, CDC, or ADCP activity.

[0019] In some embodiments, the antigen-binding moiety is Fab, Fab', F(ab')2, Fd, FCL, dAb, or a single-chain antibody scFv. Preferably, in some embodiments, the antigen-binding moiety is a single-chain antibody scFv.

[0020] Another aspect of this disclosure is to provide a multivalent antibody comprising the antibody or antigen-binding portion described above.

[0021] The multivalent antibody can be, for example, bivalent, trivalent, tetravalent, hexavalent, nonavalent, etc. The multivalent antibody can be prepared using suitable methods in the prior art. Preferably, in some embodiments, the multivalent antibody is a bispecific antibody or a trispecific antibody.

[0022] Another aspect of this disclosure is to provide a multispecific antibody that selectively binds at least to human BCMA, the multispecific antibody comprising the antibody or antigen-binding portion described above; the multispecific antibody is a monovalent antibody or a multivalent antibody.

[0023] Another aspect of this disclosure is to provide an isolated polynucleotide that encodes the aforementioned antibody or antigen-binding moiety, or encodes the aforementioned multivalent antibody.

[0024] Another aspect of this disclosure is to provide a vector comprising the aforementioned polynucleotide.

[0025] Another aspect of this disclosure is to provide a cell comprising the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, or the aforementioned carrier. In some embodiments, the cell may be any suitable host cell used as a tool for producing the target protein. For example, SP2 / 0, YB2 / 0, IR983F, human myeloma Namalwa, PERC6 or CHO cell lines, insect cells, or Escherichia coli cells.

[0026] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier or the aforementioned cell, and a pharmaceutically acceptable carrier. In some embodiments, to achieve better therapeutic effects, the pharmaceutical composition may further comprise other therapeutic agents.

[0027] Another aspect of this disclosure is to provide an immunoconjugate comprising:

[0028] a) The antibody or antigen-binding moiety described above, or the multivalent antibody described above; and

[0029] b) Therapeutic agents or detectable markers; and

[0030] c) The connecting body between parts a) and b) above;

[0031] The therapeutic agents include drugs, enzymes, toxins, cytokines, or radionuclides.

[0032] Another aspect of this disclosure is the use of the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier, the aforementioned cell, the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate in the preparation of a medicament for treating B-cell-related tumors, said B-cell-related tumors including multiple myeloma, plasma cell leukemia, diffuse large B-cell lymphoma, plasmablastoma, or light chain amyloidosis.

[0033] Another aspect of this disclosure is the use of the aforementioned antibody or antigen-binding moiety or the aforementioned multivalent antibody in the preparation of products for detecting the presence or level of human BCMA molecules in a sample.

[0034] Beneficial effects:

[0035] The anti-BCMA monoclonal antibody 1F5 disclosed herein exhibits a strong affinity for the BCMA antigen, with a Kd value of 0.752 × 10⁻⁶. -9 M; is a murine IgG1 subtype with a κ light chain; it can bind with high affinity to BCMA-positive cell lines H929 and RPMI 8226, and also with high affinity to CD138-positive bone marrow mononuclear cells derived from multiple myeloma patients. Given these properties, monoclonal antibody 1F5 can be used to detect cells expressing human BCMA, and can also be used alone or in combination with other methods in tumor immunotherapy targeting human BCMA, thus possessing diagnostic and therapeutic value. Attached Figure Description

[0036] Figure 1 This is a diagram illustrating the identification of the 1F5 antibody subtype in an embodiment of this disclosure.

[0037] Figure 2 The image shown is an SDS-PAGE image of the 1F5 pure antibody in the embodiments of this disclosure, where NR represents electrophoresis under non-reducing conditions and R represents electrophoresis under reducing conditions.

[0038] Figure 3 This is a graph showing the affinity constant analysis between the 1F5 antibody and the multiple myeloma cell line H929 in the embodiments of this disclosure;

[0039] Figure 4A This is a graph showing the results of FACS detection of the CON group in the binding of antibody 1F5 to human BCMA-positive cell line H929 in the embodiments of this disclosure;

[0040] Figure 4B This is a graph showing the results of FACS detection of the IF5 group in the binding of antibody 1F5 to human BCMA-positive cell line H929 in the embodiments of this disclosure;

[0041] Figure 5AThis is a graph showing the results of the CON group in the FACS method for detecting the binding of antibody 1F5 to the human BCMA-positive cell line RPMI 8226 in the embodiments of this disclosure;

[0042] Figure 5B This is a graph showing the results of FACS detection of the IF5 group in the binding of antibody 1F5 to the human BCMA-positive cell line RPMI 8226 in this embodiment of the present disclosure.

[0043] Figure 6A In this embodiment of the invention, the FACS method is used to detect antibody 1F5 and CD138 derived from bone marrow specimens of multiple myeloma patients. + Image showing the results of patients in group 1-CON in the binding of mononuclear cells;

[0044] Figure 6B In this embodiment of the invention, the FACS method is used to detect antibody 1F5 and CD138 derived from bone marrow specimens of multiple myeloma patients. + Graph showing the results of patients in the 1-IF5 group in the binding of mononuclear cells;

[0045] Figure 6C In this embodiment of the invention, the FACS method is used to detect antibody 1F5 and CD138 derived from bone marrow specimens of multiple myeloma patients. + Image showing the results of patients in group 2-CON in the binding of mononuclear cells;

[0046] Figure 6D In this embodiment of the invention, the FACS method is used to detect antibody 1F5 and CD138 derived from bone marrow specimens of multiple myeloma patients. + A graph showing the results of patients in the 2-IF5 group in the binding of mononuclear cells. Detailed Implementation

[0047] This disclosure provides a human BCMA antibody and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0048] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" means two or more. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0049] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0050] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0051] In this disclosure, terms such as "one embodiment," "an example," "some embodiments," "a particular embodiment," "related embodiment," "a certain embodiment," "some embodiments," "additional embodiment," or "further embodiment," "further implementation," or "another embodiment," "some other embodiments," mean that at least one feature or characteristic description is included in relation to the embodiment. Therefore, throughout this disclosure, the above phrases do not necessarily refer to the same embodiment. Furthermore, specific features may be combined in any suitable manner in one or more embodiments.

[0052] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, David L. Nelson, Michael M. Cox, WHFreeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Jones & Bartlett Learning.

[0053] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0054] definition:

[0055] The term "separated" in this disclosure refers to a substance or entity that has been removed from its natural environment or the environment in which it existed before separation and is separate from other components. For example, a separated protein substantially does not originate from cellular material or other proteins derived from the cell or tissue from which it originates. The separation ratio may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Separated substances may have different purity levels relative to the substances before their separation.

[0056] The term "antibody" in this disclosure refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody-binding sites. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be different isotypes of antibody, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibody. In some embodiments, the antibody can be IgG, IgA, IgM, IgD, or IgE. Preferably, in some embodiments, the antibody type can be IgG. Further, in some embodiments, the antibody can be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody can be IgG1.

[0057] Antibody preparation:

[0058] In some implementations, the antibodies are generated using mammalian cells. For example, monoclonal antibodies are generated in mammalian cells using hybridoma technology. Mice or other suitable host animals are first immunized with an immunogen (with adjuvants added if necessary).

[0059] Immunogens or adjuvants are typically administered via subcutaneous multi-point injection or intraperitoneal injection. Adjuvants can include Freund's adjuvant (complete or incomplete) or MPL-TDM. After immunization, animals produce lymphocytes that secrete antibodies specifically binding to the immunogen. These target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (such as PEG4000) to obtain hybridoma cells.

[0060] The hybridoma cells prepared above are seeded into a suitable culture medium for growth, the culture medium containing one or more substances that can inhibit the growth of unfused maternal myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine-guanine phosphotransferase (HGPRT or HPRT), the addition of substances such as hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium can inhibit the growth of HGPRT-deficient cells.

[0061] Preferred myeloma cells should possess high fusion rates, stable antibody secretion capabilities, and sensitivity to HAT culture medium. Among these, murine myeloma cells are preferred, such as the MOP-21 and MC-11 mouse tumor-derived lines (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Cell Collection, Rockville, Md. USA). Additionally, human myeloma and human-mouse heterologous myeloma cell lines can be used to prepare human monoclonal antibodies.

[0062] The culture medium used to grow hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined using the following methods: immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).

[0063] After determining the specificity, affinity, and reactivity of the antibodies produced by hybridomas, the target cell line can be subcloned using limiting dilution. Suitable culture media include DMEM or RPMI-1640. Additionally, hybridoma cells can also grow in animals in the form of ascites tumors.

[0064] Traditional immunoglobulin purification methods, such as protein A agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, can be used to separate monoclonal antibodies secreted by subclonal cells from cell culture medium, ascites fluid, or serum, thereby obtaining the monoclonal antibodies.

[0065] In other embodiments, antibodies against human BCMA can also be generated by known recombinant methods, such as selecting a recombinant antibody library in a phage or similar vector.

[0066] Antibody modification and alteration:

[0067] In some embodiments, the isolated antibody may be a humanized antibody. Antibody humanization may improve antibody affinity or other characteristics.

[0068] In some embodiments, the antibody Fc (crystallizable region fragment, Fc) is modified to enhance its effector functions triggered by binding to Fc receptors or complement. These functions may include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The modifications may include: 1) modifying glycosylation, for example, modifying the aspartic acid at position 297 (N297) in the Fc region with N-acetylglucosamine. Mutations of N297 to alanine (A), glutamine (Q), or glycine (G) all inhibit antibody glycosylation, thereby reducing Fc-mediated effector functions. Antibody deglycosylation reduces its ability to induce ADCC or CDC activity; sialic acid modification reduces its binding affinity to FcγRIIIa, thus leading to a decrease in CDC and ADCC activity.

[0069] In addition to the functions mentioned above, glycosylation modification of antibodies can also affect their conformation and stability. For example, the glycans in glycosylation can maintain the antibody's conformation, preventing aggregation or unfolding. For instance, the sugars on the a1-3 arm do not contact the antibody surface but are embedded deep within the space formed by the Fc segments of the two heavy chains. The mannose on the a1-3 arms of the two glycans interacts with each other, which is crucial for maintaining the antibody's conformation. Without the presence of glycans, the CH2 domain of the Fc segment would be slightly enlarged, leading to earlier elution times in size exclusion chromatography and increased sensitivity and aggregation in thermally accelerated stability experiments. Simultaneously, glycosylation modification can also affect the binding of antibodies to receptors on cell membranes, forming complexes that play an important role in signal transduction. This regulation of signal transduction is crucial for physiological processes such as cell proliferation, differentiation, and apoptosis.

[0070] The above modifications may also include: 2) point mutations, for example, LALA mutations (L234A / L235A) can lead to changes in the antibody's affinity for FcγR (eliminating binding to low-affinity FcγR and reducing binding to FcγRI), thereby significantly reducing its ADCC and CDC activities. Additionally, combining trans-subtype antibodies can also modulate the antibody's effector function.

[0071] In some embodiments, the point mutation results in the substitution of some conserved amino acids, thereby obtaining a “conservative amino acid substitution variant”. The change results in some amino acids being substituted with others that are chemically and / or functionally similar. Providing conserved substitutions of amino acids with similar chemical properties and / or functions is well known in the art. Typical examples of mutually conserved substitutions include, for example, (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine ​​(C), methionine (M).

[0072] The above modifications can also include: 3) Phosphorylation modification. Phosphorylation modification refers to the process of adding phosphate groups to amino acids of proteins within cells. Phosphorylation antibodies can specifically recognize specific phosphorylation sites, thereby detecting the increase or decrease in the phosphorylation level of proteins when cells are stimulated. These antibodies play an important role in life science research fields such as cell signaling, apoptosis, and cancer. 4) Methylation modification: Methylation modification is an important dynamic modification and biological phenomenon catalyzed by methyltransferases acting on specific residues of proteins. Methylation antibodies can specifically recognize specific methylated amino acid sites, used to distinguish between methylated and unmethylated forms of proteins. They have wide applications in research fields such as epigenetics, cancer, Alzheimer's disease, and aging. 5) Acetylation modification. Acetylation is one of the most common types of acylation modification. Acetylation antibodies can specifically recognize the acetylated form of target proteins and specific acetylated amino acid sites, detecting the activity level of the protein. These antibodies are widely used in research on cell cycle regulation, signal transduction, neurodegenerative diseases, metabolic diseases, and the occurrence and development of cancer.

[0073] The above modifications can also include: 6) Labeling. Antibodies can be cross-linked with different chemical reagents to attach to substances such as enzymes, fluorescent dyes, biotin, or colloidal gold, thereby altering their detection or analytical performance. For example, enzyme labeling: Antibodies can be cross-linked to enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase. This is commonly used in immunohistochemistry, ELISA, and other experiments, where a color reaction is produced through enzyme catalysis, thus detecting the presence of the antibody. For example, HRP-labeled antibodies can produce a color precipitate after binding to an antigen by adding a substrate, facilitating observation and quantification. Fluorescent dye labeling: Antibodies can also be bound to fluorescent dyes (such as FTC, PE, APC, etc.) for detection methods such as flow cytometry and fluorescence microscopy. Fluorescently labeled antibodies can locate specific antigens in cells or tissue sections, and the intensity of the fluorescence signal can be used to determine the antigen expression level. Biotin labeling: Biotin is a small molecule compound that can bind to antibodies without affecting their antigen-binding ability. Biotin-labeled antibodies can amplify and detect signals by binding to avidin (such as streptavidin). It is commonly used in multiplex immunolabeling assays to detect multiple antigens simultaneously.

[0074] In this disclosure, modifications and alterations to the antibody typically occur in the Fc region and the framework regions (FRs) of the antibody variable region, but not in the complementarity-determining regions (CDRs) of the antibody variable region. The framework regions of the antibody variable region have relatively conserved amino acid sequences, providing stable support for the hypervariable structure and participating in maintaining the three-dimensional conformation of the antigen-binding groove. Therefore, modifications and alterations to these regions do not affect the antibody's binding ability. In this disclosure, the term "having more than 90% identity" means that, under the aforementioned modifications or alterations, it has approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identity with the corresponding sequence.

[0075] Separated antigen-binding portion:

[0076] The term "antigen-binding moiety" in this disclosure refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding fragment." Antigen-binding fragments of antibodies can be generated through recombinant DNA technology or through enzymatic or chemical fragmentation of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, etc.

[0077] The term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL, and CH1 domains; the term "F(ab′)2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains; and the term "Fv fragment" refers to an antibody fragment composed of the VL and VH domains of a single arm of the antibody.

[0078] In some embodiments, the antigen-binding moiety is prepared using protease digestion, such as papain or pepsin. In other embodiments, the antigen-binding moiety is prepared using chemical reagent treatment. In still other embodiments, the antigen-binding moiety is prepared using genetic engineering methods. That is, a fragment containing all or part of the gene sequence of the antigen-binding moiety is ligated into a suitable vector and expressed. Examples of the expression vector include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0079] Multivalent and multispecific antibodies:

[0080] In some embodiments, the multivalent antibody comprises at least two antibody or antigen-binding moieties as described in this disclosure, capable of competitively binding to human BCMA molecules and producing effects different from those of monovalent antibodies. For example, by increasing the number of antigen-binding sites, a tighter antigen-antibody complex can be formed, thereby enhancing binding affinity and stability. This enhanced binding affinity helps improve the affinity of anti-human BCMA antibodies for antigens and enhances the interaction between antigens and cell surface receptors or other molecules. The multivalent antibody can be obtained, for example, by protein fusion, the addition of linkers, covalent bonds, or non-covalent bonds.

[0081] In some embodiments, the multispecific antibody, such as a bispecific antibody or a trispecific antibody, includes the antibody or antigen-binding portion described in this disclosure, which, in addition to competitively binding to human BCMA molecules, can also bind to at least one other different site or target molecule. Non-limiting examples include CD33, CD123, CLEC12A, CD47, FLT-3, PDGFR, VEGFR, KIT, IDH1, IDH2, SMO, BCL-2, ALT, c-KIT, CD70, CD45, PD-1 / PD-L1, CTLA-4, TIM-3, TLR-2, CD3, CD4, IL-2, CD20, BAFF, TLR-4, TLR-7, TLR-8, TNF-α, IL-6, IL-6R, IL-17A, IL-17RA, IL-12, IL-23, IL-4, α4 integrin, cell adhesion molecules, complement factor D, JAK1, JAK2, JAK3, TYK2, IL-5, URAT1, TSLP, MASP-2, CSF1R, and ROCK2. These multispecific antibodies can be linked together directly or via linkers. The multispecific antibody can be expressed through recombination. To achieve better therapeutic effects, the multispecific antibody can be monovalent or multivalent.

[0082] The above-mentioned multivalent antibodies and multispecific antibodies can be prepared using conventional techniques in this field.

[0083] The term "polynucleotide" in this disclosure may also be used interchangeably with "nucleic acid," referring to a chain of nucleotides of any length, including DNA or RNA. It may include any known nucleotide analogs or modified nucleotides or bases.

[0084] The term "vehicle" as used in this disclosure refers to a polynucleotide molecule capable of delivering and / or expressing one or more target genes. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as generating cells.

[0085] Example:

[0086] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0087] Example 1: Screening of monoclonal antibodies for mouse hybridomas.

[0088] After lentiviral transfection, human BCMA protein was stably expressed on the cell membrane of 3T3 cells. Following multiple positive sorting processes, a 3T3-BCMA cell line stably expressing BCMA protein was established. The lentiviral expression vector used was constructed by inserting the gene encoding the extracellular segment of human BCMA into its multiple cloning site using pCDH-CMV-MCS-EF1-copGFP as the empty vector.

[0089] Female Balb / c mice aged 4–5 weeks were immunized with 3T3-BCMA as the immunogen via intraperitoneal injection at a dose of 1×10⁻⁶. 7 3T3-BCMA cells / 500 µL PBS / mouse were administered as booster immunizations at weeks 3 and 5 following the initial immunization. On day 8 post-boost immunization, 10 µL of tail blood was collected from mice and placed in 90 µL PBS. The mixture was incubated at room temperature for 1 hour, then centrifuged at 12000 rpm for 10 minutes at 4°C. Serum was collected and diluted with PBS to different concentrations: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600. 3T3 and 3T3-BCMA cells were collected separately, washed once with PBS, and cell counts were performed. 100 μL of each serum dilution was added to a 1×10⁻⁶ cell line. 6 In the test cells, serum from unimmunized mice was used as a negative control. Cells were incubated at 37°C for 30 minutes and washed twice with PBS. 0.2 μL of APC-labeled anti-mouse IgG secondary antibody was added to each cell, and the cells were incubated at 37°C in the dark for 30 minutes. Cells were resuspended in 200 μL PBS buffer, and the percentage of antibody binding to cells and fluorescence intensity in the serum were detected by flow cytometry. A dilution with an average fluorescence intensity of 3T3-BCMA cells at least twice that of 3T3 cells was considered an effective titer. Fusion was only performed when the titer was higher than 6400. Three days prior to fusion, immunized mice were given a pulse immunization via tail vein injection of the immunogen.

[0090] Spleen cells from successfully immunized mice were isolated under sterile conditions. After washing twice with serum-free 1640 medium at 1200 rpm for 6 min, the cells were fused with SP2 / 0 myeloma cells in logarithmic growth phase (at a ratio of 10:1). The fusion process was performed in a 37°C water bath. 1 mL of preheated 50% PEG was added dropwise over 1 min to the mixed spleen and myeloma cell clusters (after discarding the supernatant). Then, 10 mL of preheated serum-free 1640 medium was slowly added over 5 min. The cell suspension was centrifuged twice at 800 rpm for 6 min, the supernatant was discarded, and the cells were resuspended in 1640 medium containing HAT and seeded into 96-well plates (2.5 × 10⁻⁶). 7 Cells / plate). Cells were cultured at 37°C and 5% CO2.

[0091] When the cell clones are large enough under a microscope, 100 μL of the supernatant from the corresponding well is taken. 3T3 and 3T3-BCMA cells are mixed at a 1:1 ratio as the test cells. The supernatant and test cells are co-incubated for detection, using the same method as for titer detection. When the average immunofluorescence intensity of 3T3-BCMA is higher than that of 3T3, the well is considered a positive well for further expansion culture. Positive hybridoma clones are expanded from 96-well plates to 24-well plates and cultured for 3–5 days. The culture supernatant is screened again. A small portion of cells from clones that are still positive are used for further subcloning, and the remaining cells are cryopreserved. Hybridoma cells are collected from the 24-well plates, counted, and the cell density is adjusted to 10 cells / mL. 100 μL of the mixture is seeded into each well of a 96-well plate and cultured at 37°C and 5% CO2 for approximately 10 days. Cloning is observed. Only wells with single clones are selected, and the culture supernatant is used for detection, using the same method as before. Clones with positive test results were further cultured in 24-well plates. After a second test of the supernatant, clones still showing positive results underwent a second round of subclonal culture. This process was generally repeated multiple times until all test wells were positive, thus obtaining a stable hybridoma cell line. The positive hybridoma culture supernatant was selected, and antibody subtype detection strips were used to determine the antibody subtype. In this example, the monoclonal antibody was designated 1F5, representing the mouse IgG1 subtype with a κ light chain (subtype identification results are shown in...). Figure 1 ).

[0092] Example 2: Preparation and purification of ascites fluid.

[0093] Hybridoma cells were washed with sterile PBS solution at 5 × 10⁻⁶ ppm. 6 / 0.5 mL PBS / cell was intraperitoneally injected into Balb / c mice pre-sensitized with liquid paraffin. Ascites fluid was collected 7 to 10 days later, centrifuged at 3000 rpm for 10 min at room temperature, and the intermediate layer was collected. The antibody was crudely purified using 45% saturated ammonium sulfate. The method was as follows: one part ascites fluid was added to one part PBS, and 1.64 parts saturated ammonium sulfate was added dropwise while stirring. The mixture was incubated overnight at 4°C, centrifuged at 10000 rpm for 10 min to remove the supernatant, and the precipitate was dissolved with a small amount of PBS. Dialysis with PBS at 4°C for 24 h was performed, changing the medium three times during dialysis. The crudely purified antibody was further purified according to the purification manual provided by GE using an AKTA protein purification system with a 1 mL ProteinG pre-packed column. The obtained purified antibody was analyzed by SDS-PAGE (results are shown in [link to results]). Figure 2 ).

[0094] Example 3: Monoclonal antibody titer detection.

[0095] 1F5 antibody was administered at final concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.2 nM, 1.6 nM, 0.8 nM, 0.4 nM, 0.2 nM, and 0.1 nM, respectively, in combination with 2 × 10⁻⁶ antibodies. 5 Incubate H929 at room temperature for 30 min, avoiding light. Centrifuge at 1800 rpm for 10 min, discard the supernatant, wash twice with PBS; resuspend cells in 100 μL, add 0.2 μL of APC-labeled anti-mouse IgG secondary antibody, incubate at room temperature in the dark for 30 min, wash three times with PBS, resuspend cells in 200 μL of PBS, measure fluorescence intensity using FACS, and calculate the mean value. Calculate the Kd value of the antibody using GraphPad Prism 7 data analysis software. The Kd value of the 1F5 antibody is 0.752 × 10⁻⁶. -9 M. (See results) Figure 3 )

[0096] Example 4: Cloning of the Ig variable region gene by RT-PCR.

[0097] Total RNA was extracted, and a cDNA library was synthesized. The steps are as follows:

[0098] Total RNA was extracted from 1F5 hybridoma cells using Trizol reagent (Invitrogen), and the total RNA was reverse-transcribed into a cDNA library using M-MLV reverse transcriptase (Invitrogen). The variable regions of the heavy and light chains of the anti-human BCMA antibody gene fragment were amplified by RT-PCR. Primers used are shown in SEQ ID Nos. 9, 10, 11, and 12. The PCR reaction mixture (50 μL) was prepared as follows:

[0099] cDNA: 2 μL; upstream primer (10 μM): 2 μL; downstream primer (10 μM): 2 μL; dNTP mixture: 2 μL; pfu DNA polymerase (5 U / μL): 1 μL; 10 × pfu Buffer II: 5 μL; ddH2O: bring to 50 μL. Reaction conditions: 95℃ pre-denaturation for 5 min; repeat the following cycle 35 times: 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min; finally, 72℃ extension for 10 min. VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated with the pMD19-T(simple) vector (Takara) using T4 ligase (Takara). The ligation system was as follows: 70 ng each of VL and VH PCR products, 1 μL of pMD19-T(simple) vector, 5 μL of Solution I ligation reaction solution, and ddH2O to a final volume of 10 μL. Ligation was performed overnight at 4°C. The ligation products were transformed into E. coli DH5α competent cells and cultured overnight at 37°C. Single colonies were picked, shaken at 37°C for 2 hours, and then subjected to colony PCR for identification. The cDNA of the corresponding antibody was used as a positive control. The reaction mixture (25 μL) was prepared as follows: bacterial culture: 1 μL; upstream primer (10 μM): 1 μL; downstream primer (10 μM): 1 μL; dNTP Mixture (2.5 Mm each): 2 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; 10×Taq Buffer (Mg2+) 2+ (Plus): 2.5 μL; add water to 25 μL. Reaction conditions are the same as before. Select PCR-positive clones for amplification culture, extract plasmids from positive clones using a plasmid extraction kit (Takara), and send for sequencing. At least 5 clone samples for each chain of each antibody should be sent for testing, and sequencing results should be obtained from at least three samples. Successful cloning yielded the heavy and light chain variable region sequences of 1F5, which conform to the characteristics of typical antibody variable region sequences.

[0100] Example 5: High affinity binding with H929 cells that highly express BCMA.

[0101] FACS detection of antibody 1F5 binding to BCMA protein on the surface of H929: 1 µL antibody (200 µg / mL) and 1×10 6H929 cells were incubated at room temperature for 30 minutes, followed by two washes with PBS. Cells were resuspended in 100 μL of the solution, and 0.2 μL of APC-labeled anti-mouse IgG secondary antibody was added. Cells were incubated at room temperature in the dark for 30 minutes, followed by three washes with PBS. Cells were then resuspended in 200 μL of PBS buffer and analyzed by FACS. The results showed that 1F5 effectively bound to H929 cells, exhibiting strong affinity (see results below). Figure 4A , Figure 4B ).

[0102] Example 6: High affinity binding with RPMI 8226 cells that highly express BCMA.

[0103] FACS detection of antibody 1F5 binding to BCMA protein on the surface of RPMI 8226: 1 µL antibody (200 µg / mL) and 1×10 6 RPMI 8226 cells were incubated at room temperature for 30 minutes, followed by two washes with PBS. Cells were resuspended in 100 μL of the solution, and 0.2 μL of APC-labeled anti-mouse IgG secondary antibody was added. Cells were incubated at room temperature in the dark for 30 minutes, followed by three washes with PBS. Cells were then resuspended in 200 μL of PBS buffer and analyzed by FACS. The results showed that 1F5 cells effectively bound to RPMI 8226, exhibiting strong affinity (see results below). Figure 5A , Figure 5B ).

[0104] Example 7: Antibody 1F5 and CD138 derived from bone marrow specimens of multiple myeloma patients + High affinity binding to mononuclear cells.

[0105] With the patients' consent, bone marrow cell samples were obtained from two patients with multiple myeloma. Bone marrow cell samples were subjected to density gradient centrifugation to obtain bone marrow mononuclear cells (BMMCs). BMMCs were then analyzed using CD138... + CD138 was obtained through positive sorting of magnetic beads. + BMMC.

[0106] 2 cases of CD138 + BMMCs were incubated with 1 μL of 1F5 antibody (200 µg / mL) at room temperature in the dark for 30 minutes, followed by washing twice with PBS. Cells were resuspended in 100 μL of the solution, and 0.2 μL of APC-labeled anti-mouse IgG secondary antibody was added. The cells were incubated at room temperature in the dark for 30 minutes, followed by washing three times with PBS. Cells were then resuspended in 200 μL of PBS buffer and analyzed by FACS. The results showed that 1F5 effectively bound to bone marrow samples from multiple myeloma patients, exhibiting strong affinity (see results below). Figure 6A , Figure 6B , Figure 6C , Figure 6D ).

[0107] Citation notes.

[0108] The sequence information disclosed herein has been submitted in accordance with the ST.26 standard and is hereby incorporated herein by reference.

[0109] 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. A separated antibody or antigen-binding moiety, characterized in that, The antibody or antigen-binding moiety specifically binds to human BCMA protein, and the antibody or antigen-binding moiety comprises: (1) Heavy chain variable region CDRH1 as shown in SEQ ID No. 1, heavy chain variable region CDRH2 as shown in SEQ ID No. 2, and heavy chain variable region CDRH3 as shown in SEQ ID No. 3; and (2) Light chain variable region CDRL1 as shown in SEQ ID No.4, light chain variable region CDRL2 as shown in SEQ ID No.5 and light chain variable region CDRL3 as shown in SEQ ID No.

6.

2. The isolated antibody or antigen-binding moiety according to claim 1, characterized in that, The antibody or antigen-binding region comprises a heavy chain variable region as shown in SEQ ID No. 7 and a light chain variable region as shown in SEQ ID No.

8.

3. The isolated antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

4. The isolated antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antibody is IgG1.

5. The isolated antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antibody or antigen-binding portion is modified. The modifications include N-glycosylation, O-glycosylation, phosphorylation, methylation, acetylation, or labeling.

6. The isolated antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antigen-binding portion is Fab, Fab', F(ab')2 or a single-chain antibody scFv.

7. An isolated polynucleotide, characterized in that, The polynucleotide encodes the antibody or antigen-binding portion as described in any one of claims 1 to 6.

8. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 7.

9. A cell characterized by, The cell includes an antibody or antigen-binding portion as described in any one of claims 1 to 6, a polynucleotide as described in claim 7, or a carrier as described in claim 8.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody or antigen-binding moiety as described in any one of claims 1 to 6, a polynucleotide as described in claim 7, a carrier as described in claim 8 or a cell as described in claim 9, and a pharmaceutically acceptable carrier.

11. The use of the antibody or antigen-binding moiety as described in any one of claims 1 to 6, the polynucleotide as described in claim 7, the carrier as described in claim 8, the cell as described in claim 9, or the pharmaceutical composition as described in claim 10 in the preparation of a medicament for treating B-cell-related tumors, characterized in that, The B-cell-related tumors include multiple myeloma, plasma cell leukemia, diffuse large B-cell lymphoma, or plasmablastoma.

12. The use of the antibody or antigen-binding moiety as described in any one of claims 1 to 6 in the preparation of a product for detecting the presence or level of human BCMA molecules in a sample.

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