Anti-BCMA single-domain antibody, and preparation method therefor and use thereof

Abstract

Provided are an anti-BCMA single-domain antibody, and a preparation method therefor and the use thereof. Specifically, provided is a single-domain antibody having an amino acid sequence of SEQ ID No. 1. The single-domain antibody has high affinity, can thoroughly specifically target BCMA-positive cells, and can be applied to the detection of BCMA expression in bone marrow cells of MM patients. The single-domain antibody can be prepared into a specific antibody drug clinically used for preventing and treating BCMA-target-related diseases (such as multiple myeloma, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma); or a BCMA protein detection kit, etc. The single-domain antibody has a stable structure, a small molecular size, is easily recombinantly expressed and has a low production cost, can be used alone or as a drug delivery system to carry relevant drugs, and has very wide prospects and important significance in fields such as drug application and clinical diagnosis.

Description

Anti-BCMA single-domain antibody and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202311320141.7 filed with the China Patent Office on October 12, 2023, entitled “Anti-BCMA Single Domain Antibodies and Preparation Methods and Applications Thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of biomedicine, and specifically relates to anti-BCMA single-domain antibodies and their preparation methods and applications. Background Art

[0004] Multiple myeloma (MM) is a malignant disease characterized by the abnormal proliferation of monoclonal plasma cells and is a common hematologic malignancy. Common symptoms of MM include myeloma-related organ dysfunction and secondary amyloidosis. The Chinese Guidelines for the Diagnosis and Treatment of Multiple Myeloma (revised in 2022) recommend autologous hematopoietic stem cell transplantation (ASCT) as the preferred treatment for patients with multiple myeloma who have a good overall performance status. Allogeneic hematopoietic stem cell transplantation (Allo-SCT) can be considered for younger patients with high-risk prognostic factors and a suitable donor. Alternatively, chimeric antigen receptor T cells (CAR-T) (clinical trials, investigator-initiated studies) are also available for relapsed patients. CAR-T therapy targeting BCMA (B-cell maturation antigen) has demonstrated clinical efficacy in multiple myeloma. BCMA (CD269, TNFRSF17) is a type III transmembrane glycoprotein that belongs to the human tumor necrosis factor receptor (TNFR) superfamily and plays a major role in B cell maturation and differentiation into plasma cells. BAFF and APRIL are two B cell activation and expansion-inducing ligands that bind to BCMA and jointly regulate B cell function. BCMA is primarily expressed in mature B cells, where its expression gradually increases with B cell maturation. It is selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival, and the development of drug resistance. BCMA is highly expressed on various tumor cells, but is absent or expressed at low levels in other normal tissues, making it an ideal target for targeted cancer therapy.

[0005] Heavy-chain antibodies (HcAbs) are a naturally occurring type of antibody, unique to camelids and cartilaginous fish. Their antibody domains naturally lack light chains and consist solely of two heavy chains. The antigen recognition function of heavy-chain antibodies is primarily determined by the variable region (VHH) of the heavy chain. Because the VHH alone can recognize antigens, they are also called single-domain antibodies (sdAbs). Single-domain antibodies lack light chains and consist only of the heavy chain variable region. Due to their small molecular weight, they are also called nanobodies (Nb). With a molecular weight of only approximately 13-15 kDa, a diameter of approximately 2.5 nm, and a length of 4 nm, they are the smallest antigen-binding antibody fragments to date. Their antigen-binding ability and stability are essentially the same as those of full antibodies, or they possess higher specific antigen affinity. Compared to traditional antibodies, single-domain antibodies also possess many unique properties, such as improved tissue penetration, greater thermal and chemical stability, the ability to reach specific antigenic epitopes, the flexibility to form arbitrary building blocks, and low production costs. Conventional methods for obtaining single-domain antibodies (SDAs) involve numerous steps, including multiple immunizations of camelids, isolation of B lymphocytes, amplification of the VHH domain, construction and screening of display libraries. With the advancement of synthetic biology, it has become possible to construct high-quality, randomized, and large-capacity SDAs libraries based on total synthesis. Currently, VHH SDAs are widely used in miniaturized genetically engineered antibody research, new drug development, and disease diagnosis and treatment due to their advantages, including stable structure, small size, good solubility, excellent tissue penetration, ease of recombinant expression, high homology with human antibodies, ease of humanization, and low immunogenicity. SDAs hold broad application prospects in biomedical research and therapeutics.

[0006] SUMMARY OF THE INVENTION

[0007] One of the purposes of this application is to provide a single-domain antibody targeting BCMA antigen and its application in targeted tumor therapy. This application first uses BCMA recombinant protein to immunize alpacas to obtain single-domain antibody genes, and constructs a single-domain antibody expression library, and then screens high-affinity anti-BCMA single-domain antibodies (BCMA-VHH-54) from the single-domain antibody expression library through phage display screening technology. After sequencing the antibody gene sequence, BCMA-VHH-54 was prepared by genetic engineering methods. Functional experiments such as affinity and specificity were further performed on the obtained antibodies. The results showed that the single-domain antibody of the present application has high affinity, can specifically target BCMA-positive cells (cells expressing BCMA protein), and can be successfully used to detect the expression of BCMA in bone marrow cells of MM patients. The single-domain antibodies of the present application can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells, and any recombinant system, and can be made into specific antibody drugs for clinical prevention and treatment of BCMA target-related diseases (multiple myeloma, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma), or BCMA protein detection kits, etc. The single-domain antibody drugs of the present application have a stable structure, a small molecule, are easy to express recombinantly, and have low production costs. They can be used alone or as a drug delivery system to carry related drugs, and have very broad prospects and important significance in the fields of drug application and clinical diagnosis. Technical issues

[0008] One of the purposes of the present application is to provide a single-domain antibody targeting BCMA antigen and its application in tumor targeted therapy. Technical Solutions

[0009] To achieve the above objectives, the present application first provides a single-domain antibody named BCMA-VHH-54, which may be composed of a heavy chain variable region (VHH), and the heavy chain variable region may include the complementary determining region CDR1 of positions 26-33 of SEQ ID No.1, the complementary determining region CDR2 of positions 51-58 of SEQ ID No.1, and the complementary determining region CDR3 of positions 97-106 of SEQ ID No.1.

[0010] The single-domain antibody (anti-BCMA single-domain antibody) may be a single-domain antibody that specifically binds to the BCMA protein.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region may be SEQ ID No. 1 or an amino acid sequence that is at least 80% identical to SEQ ID No. 1 and has the same function.

[0012] The single-domain antibody BCMA-VHH-54 only has the variable region (VHH) of the heavy chain antibody, which is composed of the framework region FR1, the complementary determining region CDR1, the framework region FR2, the complementary determining region CDR2, the framework region FR3, the complementary determining region CDR3 and the framework region FR4.

[0013] The amino acid sequence of the single-domain antibody BCMA-VHH-54 may be SEQ ID No. 1. Positions 1-25 of SEQ ID No. 1 are the framework region FR1, positions 26-33 are the complementarity determining region CDR1, positions 34-50 are the framework region FR2, positions 51-58 are the complementarity determining region CDR2, positions 59-96 are the framework region FR3, positions 97-106 are the complementarity determining region CDR3, and positions 107-117 are the framework region FR4. The nucleotide sequence of the nucleic acid molecule encoding the single-domain antibody BCMA-VHH-54 (BCMA-VHH-54 gene) is shown in SEQ ID No. 2.

[0014] The sequences of the complementarity determining regions are defined according to the Kabat numbering system.

[0015] Other variants of the single domain antibody sequences of the present application with improved affinity and / or titer can be obtained by using methods known in the art and are included in the scope of protection of the present application. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can also be used to improve translation efficiency in expression systems for producing antibodies. In addition, polynucleotides comprising sequences that optimize antibody specificity by applying directed evolution methods to any nucleic acid sequence of the present application also fall within the scope of the present application.

[0016] In certain embodiments, substitutions, insertions, or deletions may occur within one or more complementary determining regions or framework regions of the single-domain antibodies described herein, as long as such changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, where conservative substitutions of amino acids do not alter the properties and functions of the protein) may be made to the complementary determining regions and / or framework regions without substantially reducing binding affinity. For example, such changes may be outside the antigen contact residues in the complementary determining regions.

[0017] The present application also provides a biomaterial related to the single-domain antibody BCMA-VHH-54, which may be any of the following:

[0018] C1) a nucleic acid molecule encoding the heavy chain variable region of the single-domain antibody BCMA-VHH-54;

[0019] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0020] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0021] C4) A recombinant host cell containing the nucleic acid molecule of C1), or a recombinant host cell containing the expression cassette of C2), or a recombinant host cell containing the recombinant vector of C3).

[0022] Among them, the recombinant host cell described in C4) can express the single-domain antibody BCMA-VHH-54.

[0023] In the above-mentioned biological material, the nucleic acid molecule may be any of the following:

[0024] D1) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2;

[0025] D2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in D1) and having the same function.

[0026] The DNA molecule shown in SEQ ID No. 2 encodes the single-domain antibody BCMA-VHH-54 with the amino acid sequence of SEQ ID No. 1.

[0027] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0028] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0029] Host cell (also referred to as recipient cell) described herein refers to any type of cell that can be used for importing vector.Host cell can be eukaryotic cell (such as HEK293, CHO and myeloma cell etc.), or can be prokaryotic cell (such as Escherichia coli etc.).Described host cell can be understood as referring to not only specific recipient cell, but also to the offspring of such cell, and due to natural, accidental or intentional mutation and / or change, this progeny may not necessarily be completely consistent with original parental cell, but is still included in the scope of host cell.In one or more embodiments of the application, described host cell is Escherichia coli TG1 and / or HEK293 cell.

[0030] The recombinant vector described herein refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. The recombinant vector can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into recipient cells and provide the exogenous target gene with the ability to replicate, integrate, amplify, and / or express in the recipient cells. In one or more embodiments of the present application, the recombinant vector is pcDNA3.1-BCMA-VHH-54-hFc.

[0031] The recombinant vector pcDNA3.1-BCMA-VHH-54-hFc is designed to facilitate purification by adding a human IgG1 Fc gene (hFc gene, nucleotide sequence SEQ ID No. 3) to the 3' end of the gene encoding the single-domain antibody BCMA-VHH-54 (SEQ ID No. 2) to obtain a BCMA-VHH-54-hFc fusion gene. A BamHI restriction site is then added before the 5' start codon and an EcoRI restriction site is added after the 3' stop codon of the fusion gene. The fusion gene is then cloned between the BamHI and EcoRI recognition sites of the eukaryotic expression vector pcDNA3.1 to obtain the recombinant eukaryotic expression vector pcDNA3.1-BCMA-VHH-54-hFc expressing the single-domain antibody BCMA-VHH-54 with a C-terminal fusion hFc gene. The recombinant eukaryotic expression vector pcDNA3.1-BCMA-VHH-54-hFc contains the gene encoding the single-domain antibody BCMA-VHH-54 shown in SEQ ID No. 2.

[0032] The nucleotide sequence of the human IgG1Fc gene (696 bp) is shown in SEQ ID No. 3, and the amino acid sequence it encodes is shown in SEQ ID No. 4.

[0033] The present application also provides any of the following applications of the single-domain antibody BCMA-VHH-54 and / or the biomaterial:

[0034] E1) Use in the preparation of a medicament for preventing or treating tumors, or use in preventing or treating tumors;

[0035] E2) Use in the preparation of a medicament for preventing or treating a BCMA target-related disease, or use in preventing or treating a BCMA target-related disease;

[0036] E3) Use in the preparation of products for screening, diagnosis, or auxiliary diagnosis of BCMA target-related diseases, or use in screening, diagnosis, or auxiliary diagnosis of BCMA target-related diseases;

[0037] E4) Use in detecting BCMA protein or preparing a product for detecting BCMA protein;

[0038] E5) Use in the preparation of a product for binding BCMA protein;

[0039] E6) Use in mediating drug-specific recognition of tumors expressing BCMA antigens or in preparing products for mediating drug-specific recognition of tumors expressing BCMA antigens;

[0040] E7) Use in in vivo imaging of BCMA protein or in the preparation of a product for in vivo imaging of BCMA protein;

[0041] E8) in the preparation of CAR cells targeting BCMA.

[0042] Herein, the BCMA target-related disease may be BCMA-positive cancer (such as multiple myeloma).

[0043] Herein, the BCMA-positive cancer may include multiple myeloma (MM), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma, but is not limited thereto.

[0044] The product described in E6) that mediates the drug to specifically recognize tumors expressing the BCMA antigen may be a drug delivery system that specifically targets the BCMA protein, and the drug delivery system contains the single-domain antibody BCMA-VHH-54.

[0045] The drug delivery system can be a liposome drug delivery system, a polymer micelle drug delivery system, a polymer disc drug delivery system or a nanoparticle drug delivery system.

[0046] The drug-carrying system can be used for targeted transport and / or site-specific release of drugs.

[0047] The present application also provides a kit, which contains the single-domain antibody BCMA-VHH-54, and which can have any of the following uses:

[0048] F1) Detection of BCMA protein;

[0049] F2) Screening, diagnosis, or auxiliary diagnosis of BCMA target-related diseases;

[0050] F3) is used for in vivo imaging of BCMA protein.

[0051] The kit may be a chemiluminescence immunoassay kit, an enzyme-linked immunosorbent assay kit, a colloidal gold immunoassay kit, or a fluorescence immunoassay kit, but is not limited thereto.

[0052] Furthermore, the kit may also include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic particles, blocking solution, diluent, washing solution, color development solution, stop solution, etc., but is not limited thereto.

[0053] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0054] The components of the kit can be provided in the form of solutions, such as aqueous solutions. When provided in aqueous solutions, the concentrations or amounts of these components can be readily determined by those skilled in the art based on individual needs. For example, for storage purposes, the components can be provided in a higher concentration. When in working condition or use, the higher concentration solution can be diluted to reduce the concentration to the working concentration.

[0055] The kit may also contain, for example, a buffer, a preservative, or a protein stabilizer. The kit may also contain components necessary for detecting the detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples that can be assayed and compared to the test sample. The kit may include a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.

[0056] The detection of BCMA protein described herein may be detecting whether the sample to be tested contains BCMA protein and / or detecting the content of BCMA protein in the sample to be tested.

[0057] The sample to be tested can be a cell or tissue sample.

[0058] The products described herein for detecting BCMA protein include products that detect antigen-antibody binding using enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination assay, or immunoturbidimetry.

[0059] The product described herein may be a reagent, a kit, a chip or a test paper.

[0060] The present application also provides a pharmaceutical composition, which may contain the single-domain antibody BCMA-VHH-54.

[0061] Furthermore, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier.

[0062] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

[0063] The pharmaceutical composition may have at least one of the following uses:

[0064] M1) For the prevention or treatment of tumors;

[0065] M2) for preventing or treating BCMA target-related diseases;

[0066] M3) is used to mediate drug-specific recognition of tumors expressing BCMA antigen.

[0067] The dosage form of the pharmaceutical composition may include, but is not limited to, an inhalation preparation, an oral preparation, and an injection preparation. The inhalation preparation may be an inhalation aerosol, an inhalation powder inhalation, an inhalation spray, an inhalation liquid preparation, an inhalation atomizer, a lyophilized powder injection for inhalation, or a nasal spray. The oral preparation may be a tablet, powder, capsule, granule, pill, powder, ointment, solid beverage, or oral liquid. The injection preparation may be an injection solution or a powder injection for injection.

[0068] In order to prepare the pharmaceutical composition into an injectable preparation, such as a solution, emulsion, lyophilized powder injection, and suspension, all diluents (carriers) commonly used in the art can be used, for example, water, saline, phosphate-buffered saline, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose, or glycerol can be added to the injectable preparation. In addition, conventional carriers such as cosolvents, buffers, and pH adjusters can also be added.

[0069] The pharmaceutical composition can be prepared according to methods well known and commonly practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Loyd V. Allen et al., eds., Pharmaceutical Press (2012). The pharmaceutical composition is preferably manufactured under GMP or cGMP conditions.

[0070] The present application also provides a method for preparing the single-domain antibody BCMA-VHH-54, which may include: constructing a recombinant expression vector containing the nucleic acid molecule described herein (a nucleic acid molecule encoding the heavy chain variable region of the single-domain antibody BCMA-VHH-54); introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the single-domain antibody; culturing the recombinant cell, and obtaining the single-domain antibody through separation and purification.

[0071] Furthermore, the nucleic acid molecule encoding the heavy chain variable region of the single-domain antibody BCMA-VHH-54 may be any of the following:

[0072] D1) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2;

[0073] D2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in D1) and having the same function.

[0074] Furthermore, the host cell may be a HEK293 cell.

[0075] Furthermore, the introduction can be carried out by transforming the vector carrying the single domain antibody gene of the present application into the host cell through any known transfection method such as calcium phosphate co-precipitation method, liposome-mediated method, electroporation method or viral vector method.

[0076] The present application also provides a method for diagnosing or assisting in the diagnosis of a BCMA target-related disease, which may include: isolating a sample from a subject, then using the single-domain antibody BCMA-VHH-54 to detect the content of BCMA protein in the sample, and performing diagnosis or assisting in the diagnosis of a BCMA target-related disease based on the content of the BCMA protein.

[0077] Furthermore, the method may also include the step of comparing the BCMA protein content in the test result with a reference value (the BCMA protein content of the disease diagnostic standard). The reference value may be the level of BCMA protein in a sample from a subject (e.g., a healthy control) known not to have a disease associated with the BCMA target (also referred to as a "negative reference value"). The result of the comparison may indicate whether the subject has a disease associated with the BCMA target or is at risk of having a disease associated with the BCMA target, or indicate the patient's disease progression and / or recovery after treatment.

[0078] The present application also provides a method for screening for BCMA target-related diseases, which may include: isolating a sample from a subject, then using the single-domain antibody BCMA-VHH-54 to detect the content of BCMA protein in the sample, and screening for BCMA target-related diseases based on the content of the BCMA protein (compared with the content of BCMA protein in the disease screening standard).

[0079] Furthermore, the method may also include the step of comparing the BCMA protein content in the test result with a reference value (the BCMA protein content of the disease screening standard). The reference value can be the level of BCMA protein in a sample from a subject known not to have a disease associated with the BCMA target (e.g., a healthy control) (also referred to as a "negative reference value"). The result of the comparison can indicate a wide range of people suitable for the therapeutic product of the present application.

[0080] The present application also provides a method for preventing or treating a BCMA target-related disease, which may include administering the single-domain antibody BCMA-VHH-54 or the pharmaceutical composition described herein to a subject suffering from a BCMA target-related disease.

[0081] In the above method, the BCMA target-related disease may be a BCMA target-related tumor.

[0082] In the above method, the BCMA target-related tumor may be a BCMA-positive cancer.

[0083] In the above method, the BCMA-positive cancer may include multiple myeloma, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma but is not limited thereto.

[0084] Furthermore, the amount administered may be a therapeutically effective amount. The therapeutically effective amount may refer to an amount of the drug that (i) treats or prevents a specific disease, condition, or disorder; (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder; or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. A therapeutically effective amount can be determined by testing in known in vitro or in vivo (e.g., animal models) systems.

[0085] Furthermore, the administration includes but is not limited to intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumor injection, mucosal administration, oral administration, oral and nasal spraying, aerosol inhalation, in vivo implantation and in vitro device administration.

[0086] The preferred mode of administration (route of administration) is parenteral (such as intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular) administration. The skilled person will appreciate that the route of administration will vary according to the intended purpose. In certain embodiments, the nanometer antibodies, bispecific antibodies or multispecific antibodies of the present application, isolated nucleic acid molecules, recombinant vectors, recombinant host cells, antibody conjugates or pharmaceutical compositions can be administered by injection or continuous infusion (including but not limited to intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular and intraportal vein).

[0087] The present application also provides a method for detecting BCMA protein in vitro, which may include using the single-domain antibody BCMA-VHH-54 or the kit described herein to detect BCMA protein.

[0088] The present application also provides an antibody-drug conjugate, comprising an antibody portion and a conjugated portion, wherein the antibody portion may comprise the single-domain antibody BCMA-VHH-54.

[0089] Furthermore, the conjugated moiety may include a detectable label, a chemical drug or a cytotoxin.

[0090] The antibody moiety and the conjugated moiety may be covalently linked directly or through a linker.

[0091] The detectable label may be selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

[0092] The chemical drug can be a drug that can kill tumor cells.

[0093] The cytotoxin may be a tubulin inhibitor (such as maytansine, auristatin), a DNA synthesis inhibitor (such as calicheamicin) or an RNA synthesis inhibitor (such as amatoxin).

[0094] The purpose of the applications and methods described in this application may be disease diagnosis, disease prognosis and / or disease treatment, or their purpose may be non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0095] The subject described herein can be a human or non-human animal. In certain embodiments, the subject suffers from a BCMA target-related disease.

[0096] The non-human animal described herein may be a non-human mammal.

[0097] The non-human mammals mentioned herein may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but are not limited thereto.

[0098] BCMA (CD269, TNFRSF17), described herein, is a type III transmembrane glycoprotein that belongs to the human tumor necrosis factor receptor (TNFR) superfamily and plays a major role in B cell maturation and differentiation into plasma cells. BAFF and APRIL are two B cell activation and expansion-inducing ligands that bind to BCMA and jointly regulate B cell function. BCMA is primarily expressed in mature B cells, where its expression gradually increases with B cell maturation. It is selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival, and the development of drug resistance. BCMA is expressed on tumor cells such as multiple myeloma, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma. Beneficial effects

[0099] The single-domain antibody BCMA-VHH-54 of the present application has high affinity, can specifically target BCMA-positive cells (cells expressing BCMA protein), and can be successfully used to detect the expression of BCMA in bone marrow cells of MM patients. The single-domain antibody of the present application can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system, and can be made into specific antibody drugs for clinical prevention and treatment of BCMA target-related diseases (multiple myeloma, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma), or BCMA protein detection kits, etc. The single-domain antibody drug of the present application has a stable structure, small molecules, easy recombinant expression, low production cost, can be used alone or as a drug delivery system to carry related drugs, and has very broad prospects and important significance in the fields of drug application and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is a research flow chart of this application.

[0101] Figure 2 shows the results of detecting BCMA expression on different tumor cell lines using the FITC-labeled BCMA-VHH-54 antibody. Isotype represents the control group, and BCMA represents the FITC-BCMA-VHH-54-hFc antibody.

[0102] Figure 3 shows the results of detecting BCMA expression on bone marrow cells from MM patients using FITC-labeled BCMA-VHH-54 antibody. FMOC represents the control group, and BCMA-FITC represents FITC-labeled BCMA-VHH-54 antibody. Modes for Carrying Out the Invention

[0103] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0104] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0105] The pComb3XSS plasmid in the following examples is a product of Beijing Zhuangmeng International Biogene Technology Co., Ltd., product number ZK129.

[0106] The helper phage M13K07 in the following examples is a product of Chengdu Renyu Biotechnology Co., Ltd., product number A001-1.

[0107] The K562 cells (BCMA-negative cells) in the following examples are products of Nanjing Kebai Biotechnology Co., Ltd., with the product number CBP60529; KG-1a cells (BCMA-negative cells) are products of Nanjing Kebai Biotechnology Co., Ltd., with the product number CBP61455; RPMI-8226 (BCMA-positive cells) cells are products of Beijing Kebai Biotechnology Co., Ltd., with the product number CBP60244; Daudi (BCMA-positive cells) are products of Nanjing Kebai Biotechnology Co., Ltd., with the product number CBP60262; MM1.S cells (BCMA-positive cells) are products of Beijing Biobo Biotechnology Co., Ltd., with the product number bio-129535.

[0108] In the following examples, the bone marrow cells of MM patients were obtained from Zhejiang Provincial People's Hospital.

[0109] In the following examples, the method for preparing BCMA-positive cells K562-BCMA is to insert BCMA between the BamHI and EcoRI sites of pcDNA3.1, and transfect K562 cells with the prepared recombinant vector to obtain BCMA-positive cells K562-BCMA.

[0110] The vector pcDNA3.1 in the following examples is a product of Changsha Abiwei Biotechnology Co., Ltd., product number: HG-VPI0001.

[0111] The research flow chart of this application is shown in Figure 1.

[0112] Example 1. Screening and preparation of anti-BCMA single-domain antibodies

[0113] In this example, alpacas were immunized with BCMA-Fc recombinant protein to obtain single-domain antibody genes, and a single-domain antibody expression library was constructed. Then, high-affinity anti-BCMA single-domain antibodies were screened from the single-domain antibody expression library using phage display screening technology. The specific steps are as follows:

[0114] 1. Animal immunization

[0115] 1.1. Acquisition of antigens

[0116] The BCMA recombinant protein used as the antigen is shown below:

[0117] BCMA immune antigen: Human BCMA Llama IgG2b FC (BCA-H5259) protein is a product of Shanghai Baiying Biotechnology Co., Ltd.

[0118] BCMA screening antigen: Human BCMA-6×His (BCA-H522Y) protein is a product of Shanghai Baiying Biotechnology Co., Ltd.

[0119] 1.2. Immune Alpaca

[0120] Alpacas (2-3 years old, weighing approximately 100 catties) were immunized three times with the BCMA antigen Human BCMA Llama IgG2b FC. Each subcutaneous injection of 0.5 mg of Human BCMA Llama IgG2b FC was administered two weeks apart. Before the next immunization, 5 ml of blood was drawn, and the plasma was separated and assayed for antibody titers following the previous immunization. The titers after the three immunizations were then measured one week after each immunization.

[0121] The titer of antibodies in serum was detected by indirect ELISA method. The steps are as follows:

[0122] A. Dilute the antigen to 5 μg / mL in PBS and coat at 100 μL / well at 4°C overnight.

[0123] B. Discard the coating solution, wash three times with PBST, add 300 μL of 5% skim milk to each well, and block at 37°C for 1 hour.

[0124] C. Wash three times with PBST, add 100 μL / well of serum dilution buffer (starting from 1:2000 dilution), and incubate at 37°C for 45 min.

[0125] D. Wash five times with PBST, add horseradish peroxidase-conjugated goat anti-Alpaca secondary antibody (diluted 1:1 in PBS) at 100 μL / well, and incubate at 37°C for 45 min.

[0126] E. Wash the plate five times with PBST. Add TMB colorimetric solution (100 μL / well) for development at 37°C for 5 min. Terminate the reaction by adding stop solution (50 μL / well) and measure the optical density at 450 nm. Positive wells are identified when the OD450 reading of the immune serum sample is greater than that of the non-immune serum sample and is greater than 0.5.

[0127] 1.3 Immunization Results

[0128] The results of serum titer test are shown in Table 1. The ones in bold are positive wells. After three immunizations, the serum titer in alpaca B reached 64K, that is, the antibody level reached 64K.

[0129] Table 1. Serum titer test results

[0130] As shown in Table 1, after the third immunization, the antibody level in the alpaca reached 64K. The Ag coating was the antigen Human BCMA Protein His tag.

[0131] 2. Phage library construction

[0132] After successful immunization, the above-mentioned B alpaca peripheral blood mononuclear cells (PBMC) were collected to construct a phage library: namely, an anti-BCMA VHH phage antibody library.

[0133] 2.1 RNA extraction and reverse transcription

[0134] Alpaca peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stored in Trizol. Total RNA was extracted from the PBMCs and reverse transcribed into cDNA using the Takara reverse transcription kit. The specific steps are as follows:

[0135] A: Peripheral blood lymphocyte isolation

[0136] After three immunizations, 50 mL of peripheral blood was collected from alpaca B, and PBMCs were isolated according to the instructions of the lymphocyte separation medium.

[0137] B: RNA extraction

[0138] Total RNA was extracted using RNAiso Plus reagent.

[0139] C: Gel electrophoresis to detect RNA purity

[0140] 1 μg RNA was electrophoresed to detect RNA purity, and the results showed that the RNA purity was good.

[0141] D: Reverse transcription

[0142] See PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A) instructions, a total of 5 μg RNA was transcribed.

[0143] 2.2. PCR amplification of the VHH target fragment of BCMA antibody

[0144] The obtained cDNA stock solutions were mixed in equal proportions and diluted 5-fold. 5.0 μL was selected as the optimal amplification concentration. After the first round of nested PCR, a VHH fragment of approximately 750 bp was recovered by gel tapping. After the second round of nested PCR, the VHH fragment was purified using a DNA product purification kit.

[0145] 2.3 Enzyme Digestion and Ligation

[0146] 1) Using pComb3XSS as a phage plasmid vector, the pComb3XSS vector and the VHH PCR product (i.e., the VHH target fragment) were digested with the restriction endonuclease SfiI, respectively, and incubated at 50°C overnight.

[0147] 2) Use a DNA recovery and purification kit to purify the pComb3XSS vector and VHH PCR product after enzyme digestion to obtain a total of 3.1 μg of ligation product, which was stored at 4°C.

[0148] 3) The digested pComb3XSS vector and the VHH target fragment were ligated and recovered to obtain a total of 3.1 μg of ligation product with a molar ratio of Vector:VHH=1:3.

[0149] 2.4 Bacterial library construction

[0150] A total of 10 electroporation transformations were performed. Immediately after electroporation, 1 mL of 2YT medium (preheated at 37°C) was added to the electroporation cup for recovery. The electroporation product was aspirated and the electroporation cup was washed with 2YT medium. A total of 100 mL of recovery product was obtained. The product was recovered at 37°C, 180 rpm for 45 min, and 100 μL was taken for gradient dilution to 10 -3 and 10 -4 The number of transformants in the library was determined and plated onto 90 mm plates. The remaining liquid was centrifuged, resuspended in 8 mL of 2YT, and plated onto eight 200 mm plates. The next day, 105 clones were found on the plate used to determine the number of transformants in the library. Forty-eight clones were randomly selected from the plate used to determine the number of transformants in the library and were identified. The results showed an insertion rate of 100%. Based on the number of transformants in the library, the insertion rate, and the results of the diversity sequencing analysis, the library capacity was set at 1.05 × 10 9 (105×1000×10 4 ×1×1). The library has good capacity and diversity and can be used for the next step of screening.

[0151] 2.5. Phage library preparation

[0152] (1) The bacterial library was inoculated into 2×300 mL 2YT+A+G (Amp: 100 μg / ml, Glu: 1%) medium to an initial OD600 of 0.1-0.2, and cultured at 37°C and 230 rpm until the OD600 reached above 0.8.

[0153] (2) Add helper phage M13KO7 according to the OD600 value (helper phage: bacteria = 20:1).

[0154] (3) After adding helper phage M13KO7, mix well and let stand at 37°C for 30 min.

[0155] (4) Shake slowly at 180 rpm at 37°C for 30 min.

[0156] (5) Centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend the pellet with an equal volume of 2YT+A+K (Amp: 100 μg / ml, Kan: 50 μg / ml) culture medium and incubate at 30°C, 220 rpm overnight.

[0157] (6) Centrifuge the overnight culture at 4°C, 10,000 rpm for 20 min, collect the supernatant, and discard the precipitate. Replace the centrifuge tube and centrifuge at 4°C, 10,000 rpm for 20 min, and collect the supernatant.

[0158] (7) Add PEG8000 / NaCl at 1 / 5 of the supernatant volume, mix well, and precipitate on ice for more than 2 hours.

[0159] (8) Centrifuge at 10,000 rpm for 20 min at 4°C. Discard the supernatant and evacuate once to remove all the supernatant. Resuspend the pellet in 1 mL of 1× PBS and add 1 / 5 volume of PEG8000 / NaCl for a second precipitation for 1 h.

[0160] (9) Centrifuge at 4°C, 12,000 rpm for 10 min, discard the supernatant, and evacuate once to remove all the supernatant. Depending on the amount of precipitate, add 1× PBS to resuspend the precipitate.

[0161] (10) Add 100% glycerol to a final concentration of 50%, mix well, and dispense into 1.5 mL EP tubes and store at -80°C.

[0162] (11) Take 10uL of library phage and dilute it with 2YT gradient, from 10 -8 and 10 -9 Take 10 μl of the culture medium from the tube and add it to 90 μL of TG1 bacterial solution. Mix gently. Incubate at 37°C for 15 minutes. Spread on Amp-resistant plates and culture overnight.

[0163] (12) The next day, at 10 -9 There were 540 clones on the titer plate, and the titer of the phage library was 5.40×10 13 cfu / mL(540×10 9 ×100).

[0164] 3. Affinity screening

[0165] 3.1. Two rounds of affinity screening of the phage library were performed using the screening antigen (BCMA-6×His), including titer detection, amplification of phage eluates, and purification of phages.

[0166] 1) Dilute the target molecule BCMA-6×His antigen to a final concentration of 5 μg / mL in carbonate buffer (pH 9.6) and add 100 μL / well to the enzyme-labeled wells. Coat 8 wells per target molecule (4 wells for the second screening round, 2 wells each for the third and fourth screening rounds) at 4°C overnight.

[0167] 2) Discard the coating solution, wash with PBS three times, add 300 μL of 3% OVA-PBS blocking solution to each well, and block at 37°C for 1 hour;

[0168] 3) Wash with PBS three times, add 100 μL of phage library, and incubate at 37°C for 1 h;

[0169] 4) Aspirate unbound phages, wash six times with PBST, and then wash twice with PBS;

[0170] 5) Add 100 μL of Gly-HCl eluent and incubate at 37°C for 8 min to elute specifically bound phage; transfer the eluate to a 1.5 mL sterile centrifuge tube and quickly neutralize with 10 μL of Tris-HCl neutralization buffer;

[0171] 6) Take 10 μL for serial dilution, determine the titer, calculate the panning recovery rate, and mix the remaining eluate for amplification and purification for the next round of affinity panning, changing the panning conditions.

[0172] 3.2. Amplification of the library after panning

[0173] 1) The panning eluate was mixed with 20 mL of E. coli TG1 culture in the early logarithmic growth phase, incubated at 37°C for 30 min, and then 1 mL of 20% glucose was added. The culture was shaken at 220 rpm for 30 min. Then, M13K07 phage and 4 μL of Amp+ were added at a cell:phage ratio of 1:20. The culture was incubated at 37°C for 30 min, and then 2 mL of 2YT liquid medium was added. The culture was shaken at 220 rpm for 30 min.

[0174] 2) The culture was divided into centrifuge tubes and incubated at 4°C, 5000 rpm for 10 min. The cell pellet was resuspended in 50 mL of 2×YT-AK liquid medium and cultured with shaking at 30°C, 250 rpm overnight.

[0175] 3) Centrifuge the overnight culture at 4°C, 10,000 rpm for 20 min. Transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG-NaCl, mix well, and incubate at 4°C for at least 2 h.

[0176] 4) 4°C, 10,000 rpm, 20 min. Remove the supernatant, resuspend the pellet in 1 mL of PBS, add 1 / 5 volume of PEG / NaCl, mix well, and incubate at 4°C for at least 1 h.

[0177] 5) Incubate at 4°C, 12,000 rpm for 2 min. Remove the supernatant and suspend the precipitate in 200 μL PBS. This is the amplified product. Measure the titer for use in the next round of panning or analysis.

[0178] 3.3 Identification and Analysis of Specific Phage Clones

[0179] 1) From the plate used for panning eluate titer, randomly pick 480 clones from the second round titer determination plate using a sterile toothpick, inoculate them into 300 μl of 2×YT-A medium, and culture at 37°C, 230 rpm, and shake for 8 h.

[0180] 2) Take 100 μL of the above culture and add M13K07 phage at a ratio of cell:phage = 1:20. Incubate at 37°C for 15 min and shake at 220 rpm for 45 min.

[0181] 3) Add 300 μL of 2×YT-AK and culture at 30°C with vigorous shaking overnight.

[0182] 4) The next day, centrifuge at 12,000 rpm for 2 minutes and collect the supernatant for monoclonal ELISA identification.

[0183] 3.4 Identification of positive phage clones

[0184] 1) Dilute the target molecule hBCMA-his antigen with carbonate buffer (pH 9.6) to a final concentration of 2 μg / mL. Add 100 μL / well to the enzyme-labeled wells and coat overnight at 4°C.

[0185] 2) Discard the coating solution, wash three times with PBST, add 300 μL of 5% skim milk to each well, and block at 37°C for 1 h;

[0186] 3) Wash once with PBST, add 50 μL of phage culture supernatant and 50 μL of 5% skim milk to each well, and incubate at 37°C for 1 h;

[0187] 4) Wash with PBST five times, add horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10,000 in PBS) at 100 μL / well and incubate at 37°C for 1 h.

[0188] 5) Wash the plate six times with PBST. Add TMB colorimetric solution (100 μL / well) for color development at 37°C for 7 min. Add stop solution (50 μL / well) to terminate the reaction and measure the optical density at 450 nm.

[0189] 6) hBCMA antigen affinity panning: Gly-HCl acid elution was used for screening. The enrichment during the screening process is shown in the table below.

[0190] Table 2. Recovery of hBCMA antigen by acid elution screening

[0191] 4. BCMA VHH monoclonal ELISA identification

[0192] Using hBCMA antigen as the target molecule, specific phages were eluted by Gly-HCl acid elution method. 480 clones (numbered NB405-ANTI-Human-1-96 and NB405-ANTI-HBCMA-97-480, 1-480 being a second-round monoclonal clone) were randomly selected from the second-round HBCMA titer determination plate using a sterile toothpick. Positive clones were screened by indirect ELISA, and the optical density was measured at 450 nm.

[0193] After two rounds of screening, a total of 480 clones were identified on the second round of titer plate testing, and 317 positive clones were obtained. Of these, clones 48, 342, and 423 were non-monoclonal. Clones 146, 150, 155, 321, 414, and 445 were retested as negative clones. Sequencing and data analysis of these positive clones revealed 41 unique sequences based on CDR3 sequence differences. Combined with the results of ELISA screening of the positive clones, clone 54 was selected for expression of VHH-hIgG1Fc in HEK293 cells.

[0194] 5. Preparation of anti-BCMA single domain antibodies

[0195] 5.1 Structure and Sequence of Anti-BCMA Single-Domain Antibodies

[0196] The positive monoclonal antibodies in step 4 were selected and sequenced to obtain the antibody gene sequence. Anti-BCMA single-domain antibodies (BCMA-VHH) were prepared using genetic engineering methods, and one of the antibodies was named BCMA-VHH-54.

[0197] Single-domain antibodies only have the variable region (VHH) of the heavy chain antibody, which is composed of the framework region FR1, the complementary determining region CDR1, the framework region FR2, the complementary determining region CDR2, the framework region FR3, the complementary determining region CDR3 and the framework region FR4. The specific sequence information is as follows:

[0198] The amino acid sequence of the single-domain antibody BCMA-VHH-54 is shown in SEQ ID No. 1. Positions 1-25 of SEQ ID No. 1 are the framework region FR1, positions 26-33 are the complementarity determining region CDR1, positions 34-50 are the framework region FR2, positions 51-58 are the complementarity determining region CDR2, positions 59-96 are the framework region FR3, positions 97-106 are the complementarity determining region CDR3, and positions 107-117 are the framework region FR4. The nucleotide sequence of the nucleic acid molecule encoding the single-domain antibody BCMA-VHH-54 (BCMA-VHH-54 gene) is shown in SEQ ID No. 2.

[0199] The amino acid sequence of the single-domain antibody BCMA-VHH-54 is: 5'-QLQLVESGGGLVQPGGSLKLSCAASGFTFKDYAMNWYRQAPGKEREWVALISSDGGTTEYEQSVKGRFTISRDNAKNTVYLQMNSVKPEDTAVYRCNKFGAGSYNYWGQGTLVTVSS-3' (SEQ ID No. 1). The three underlined parts are CDR1, CDR2, and CDR3, respectively.

[0200] Nucleotide sequence of BCMA-VHH-54 gene: 5'-CAGTTGCAGCTCGTGGAGTCA GGTGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTTCAAAGACTATGCCATGAACTGGTACCGTCAGGCTCCAGGGAAGGAGCGCGAGTGGGTCGCACTTATTAGTAGTGATGGTGGTACCACAGAGTATGAGCAGTCC GTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGTATCTGCAAATGAACAGCGTGAAACCTGAGGACACGGCCGTGTATCGCTGTAATAAGTTCGGAGCAGGATCCTATAACTACTGGGGCCAGGGGGACCCTGGTCACCGTCTCCAGC-3'(SEQ ID No.2).

[0201] The sequences of the complementarity determining regions are defined according to the Kabat numbering system.

[0202] The above-mentioned single-domain antibody is a single-domain antibody that specifically binds to the BCMA protein.

[0203] 5.2 Expression and Purification of Anti-BCMA Single Domain Antibodies

[0204] The eukaryotic expression vector pcDNA3.1 was used to express the anti-BCMA single-domain antibody (BCMA-VHH-54).

[0205] 5.2.1 Construction of recombinant vector

[0206] To facilitate purification, a human IgG1 Fc gene (hFc gene, nucleotide sequence SEQ ID No. 3, encoded amino acid sequence SEQ ID No. 4) was added to the 3' end of the gene encoding the single-domain antibody BCMA-VHH-54 (SEQ ID No. 2) to obtain the BCMA-VHH-54-hFc fusion gene. A BamHI restriction site was then added before the 5' start codon and an EcoRI restriction site after the 3' stop codon of the fusion gene. The gene was then cloned into the eukaryotic expression vector pcDNA3.1 between the BamHI and EcoRI recognition sites to obtain the recombinant eukaryotic expression vector pcDNA3.1-BCMA-VHH-54-hFc expressing the single-domain antibody BCMA-VHH-54 with the C-terminal hFc gene fused thereto.

[0207] 5.2.2 Expression of anti-BCMA single-domain antibodies

[0208] The eukaryotic expression vector pcDNA3.1-BCMA-VHH-54-hFc obtained in step 5.2.1 was transfected into HEK293 cells by conventional electroporation for expression.

[0209] 5.2.3 Purification of Anti-BCMA Single Domain Antibodies

[0210] Protein A has the activity of binding to the Fc fragment of IgG, so the anti-BCMA single domain antibody expressed in HEK293 cells was purified using a Protein A column (conventional purification step).

[0211] Finally, a purified anti-BCMA single-domain antibody fused with the hFc gene (BCMA-VHH-54-hFc) was prepared.

[0212] Example 2: Functional identification of anti-BCMA single domain antibodies

[0213] 1. Antigen-antibody affinity determination

[0214] Test antibody: the anti-BCMA single-domain antibody fused with the hFc gene (BCMA-VHH-54-hFc) prepared in step 5.2.3.

[0215] (1) The test antibody BCMA-VHH-54-hFc was injected into the Pierce TMThe FITC-labeled antibody FITC-BCMA-VHH-54-hFc was obtained by labeling with the NHS-Fluorescein Antibody Labeling Kit for flow cytometry detection;

[0216] (2) Resuspend BCMA-positive K562-BCMA cells to a density of 2×10 6 cells / mL, 100 μL was placed in 96-well V-bottom plates, centrifuged, and the supernatant was discarded to collect the cells (K562 and KG-1a cells were used as BCMA-negative cell controls);

[0217] (3) 40 μL of FITC-BCMA-VHH-54-hFc diluted in different gradients (see Table 3 for specific groups) was mixed with the above cells and incubated in the dark at room temperature for 10 min;

[0218] Table 3. Grouping of FITC-BCMA-VHH-54-hFc Antibody Affinity Test

[0219] (4) Wash twice by centrifugation with 200 μL FACS buffer, resuspend in 200 μL FACS buffer, and immediately perform flow cytometry to measure the mean fluorescence intensity (MFI);

[0220] (5) The affinity is determined by calculating the equilibrium dissociation constant (KD) of the BCMA VHH antibody binding to the target cell according to the formula: 1 / (MFI-Con) = 1 / Fmax + (KD / Fmax)(1 / [BCMA IgG Fc]). The smaller the KD value, the stronger the affinity.

[0221] Among them, MFI-Con represents the relative mean fluorescence intensity, which is the value of the mean fluorescence intensity of the experimental group minus the mean fluorescence intensity of the background; BCMA IgG Fc represents the antibody dosage (unit: nM).

[0222] Plot a standard curve using the reciprocal of the antibody dosage as the abscissa and the reciprocal of the relative mean fluorescence intensity as the ordinate to generate a linear regression equation. Calculate the KD using the linear regression equation, where the intersection of the standard curve and the ordinate is 1 / Fmax. The KD is the slope of the line multiplied by Fmax.

[0223] The calculated equilibrium dissociation constants are shown in Table 4:

[0224] Table 4. Equilibrium dissociation constant (KD) of BCMA-VHH-54-hFc and K562-BCMA cells

[0225] 2. Specificity detection of the single-domain antibody BCMA-VHH-54

[0226] FITC-labeled BCMA-VHH-54 antibody (FITC-BCMA-VHH-54-hFc) was used to detect the expression of BCMA on different tumor cells.

[0227] (1) BCMA-negative cell lines (control group): K562 cells and KG-1a cells; BCMA-positive cell lines (experimental group): RPMI-8226, Daudi, MM1.S cells and the BCMA-overexpressing cell line K562-BCMA were all revived at 37°C and added to cell culture flasks, cultured at 37°C, 5% CO2, and passaged twice before detection of anti-BCMA single-domain antibody (FITC-BCMA-VHH-54-hFc).

[0228] (2) Adjust the cell concentration to 2×10 6 pcs / mL, 200 μL / well was added to a 96-well plate and centrifuged at 1500 rpm for 5 min;

[0229] (3) Wash with FACS buffer and centrifuge;

[0230] (4) Add 20 μL of diluted FITC-BCMA-VHH-54-hFc antibody (1:400), mix well, and incubate at room temperature for 10 min;

[0231] (5) Add FACS buffer and centrifuge to wash away the antibodies, then add buffer to resuspend and detect using flow cytometry.

[0232] Flow cytometry results showed that the FITC-BCMA-VHH-54-hFc antibody could specifically bind to the BCMA antigen molecules expressed on tumor cells (Figure 2).

[0233] 3. Detection of BCMA expression in bone marrow cells of MM patients

[0234] FITC-labeled BCMA-VHH-54 antibody (FITC-BCMA-VHH-54-hFc) was used to detect bone marrow cells from two multiple myeloma (MM) patients. The flow cytometry analysis steps were as follows:

[0235] (1) Cryopreserved bone marrow cells from two multiple myeloma (MM) patients were revived at 37°C.

[0236] (2) Adjust the cell density to 2×10 6 pcs / ml, 200 μL / well was added to a 96-well plate and centrifuged at 1500 rpm for 5 min;

[0237] (3) Wash with FACS buffer and centrifuge;

[0238] (4) Add 20 μL of diluted FITC-BCMA-VHH-54-hFc antibody (1:400), mix well, and incubate at room temperature for 10 min;

[0239] (5) Add FACS buffer and centrifuge to wash away the antibodies, then add buffer to resuspend and detect using flow cytometry.

[0240] The results showed that the single-domain antibody BCMA-VHH-54 of the present application can specifically recognize BCMA expressed by MM tumor cells ( Figure 3 ).

[0241] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art. Industrial Applicability

[0242] The single-domain antibody (BCMA-VHH-54) of the present application has high affinity, can specifically target BCMA-positive cells (cells expressing BCMA protein), and can be successfully used to detect the expression of BCMA in bone marrow cells of MM patients. The single-domain antibody of the present application can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system, and can be made into specific antibody drugs for clinical prevention and treatment of BCMA target-related diseases (multiple myeloma, B-cell acute lymphoblastic leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma), or BCMA protein detection kits, etc. The single-domain antibody drug of the present application has a stable structure, small molecules, easy recombinant expression, low production cost, can be used alone or as a drug delivery system to carry related drugs, and has very broad prospects and important significance in the fields of drug application and clinical diagnosis.

Claims

1. A single domain antibody, characterized in that The single-domain antibody is composed of a heavy chain variable region, which includes a complementary determining region CDR1 with amino acid sequences at positions 26-33 of SEQ ID No.1, a complementary determining region CDR2 with amino acid sequences at positions 51-58 of SEQ ID No.1, and a complementary determining region CDR3 with amino acid sequences at positions 97-106 of SEQ ID No.

1.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is SEQ ID No. 1 or an amino acid sequence that has at least 80% identity with SEQ ID No. 1 and has the same function.

3. A biological material related to the single domain antibody according to claim 1 or 2, characterized in that: The biological material is any of the following: C1) a nucleic acid molecule encoding the heavy chain variable region of the single-domain antibody according to claim 1 or 2; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant host cell containing the nucleic acid molecule described in C1), or a recombinant host cell containing the expression cassette described in C2), or a recombinant host cell containing the recombinant vector described in C3).

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecule is any of the following: D1) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2; D2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in D1) and has the same function.

5. Any of the following uses of the single domain antibody according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4: E1) Use in the preparation of a medicament for preventing or treating a tumor, or use in preventing or treating a tumor; E2) Use in the preparation of a drug for preventing or treating a BCMA target-related disease, or use in the prevention or treatment of a BCMA target-related disease; E3) Use in the preparation of products for screening, diagnosis or auxiliary diagnosis of BCMA target-related diseases, or use in screening, diagnosis or auxiliary diagnosis of BCMA target-related diseases; E4) Use in detecting BCMA protein or preparing a product for detecting BCMA protein; E5) Use in the preparation of a product for binding BCMA protein; E6) Use in mediating drug-specific recognition of tumors expressing BCMA antigen or in preparing a product for mediating drug-specific recognition of tumors expressing BCMA antigen; E7) Use in the in vivo imaging of BCMA protein or in the preparation of a product for the in vivo imaging of BCMA protein; E8) is used in the preparation of CAR cells targeting BCMA.

6. The use according to claim 5, characterized in that: The BCMA target-related disease is BCMA-positive cancer.

7. The use according to claim 6, characterized in that: The BCMA-positive cancers include multiple myeloma, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkin lymphoma and Hodgkin lymphoma.

8. A kit, characterized in that The kit contains the single domain antibody according to claim 1 or 2, and the kit has any of the following uses: F1) Detection of BCMA protein; F2) Screening, diagnosis or auxiliary diagnosis of BCMA target related diseases; F3) is used for in vivo imaging of BCMA protein.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the single domain antibody according to claim 1 or 2.

10. The method for preparing a single domain antibody according to claim 1 or 2, characterized in that: The method comprises: constructing a recombinant expression vector containing the nucleic acid molecule of claim 3 or 4; introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the single-domain antibody; culturing the recombinant cell to obtain the single-domain antibody by separation and purification.

11. A method for diagnosing or assisting in the diagnosis of a BCMA target-related disease, characterized in that: The method comprises: obtaining a sample by separation from a subject, then detecting the content of BCMA protein in the sample using the single domain antibody according to claim 1 or 2, and performing diagnosis or auxiliary diagnosis of a BCMA target-related disease based on the content of the BCMA protein.

12. A method for screening BCMA target-related diseases, characterized in that: The method comprises: obtaining a sample by separation from a subject, then detecting the content of BCMA protein in the sample using the single domain antibody according to claim 1 or 2, and screening for BCMA target-related diseases based on the content of BCMA protein.

13. A method for preventing or treating a BCMA target-related disease, characterized in that: The method comprises administering the single domain antibody of claim 1 or 2 or the pharmaceutical composition of claim 9 to a subject suffering from a BCMA target-related disease.

14. The method according to any one of claims 11 to 13, characterized in that: The BCMA target-related disease is a BCMA target-related tumor.

15. The method according to claim 14, characterized in that The BCMA target-related tumor is a BCMA-positive cancer.

16. The method according to claim 15, characterized in that The BCMA-positive cancers include multiple myeloma, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkin lymphoma and Hodgkin lymphoma.

17. A method for detecting BCMA protein in vitro, characterized in that: The method comprises detecting BCMA protein using the single domain antibody of claim 1 or 2 or the kit of claim 8.

18. An antibody-drug conjugate, comprising an antibody portion and a conjugate portion, characterized in that: The antibody portion comprises the single domain antibody of claim 1 or 2.

19. The antibody-drug conjugate according to claim 18, characterized in that: The conjugated moieties include detectable labels, chemical drugs or cytotoxins.

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