Anti-cd40 monoclonal antibodies and uses thereof

Abstract

The application provides an anti-CD40 monoclonal antibody and application thereof. The anti-CD40 monoclonal antibody comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 1-3, and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 4, YAS and SEQ ID NO: 5. The antibody has binding activity to in vitro CD40 and cell surface CD40, can competitively bind CD40 with CD40L, blocks the combination of the two to inhibit CD40 / CD40L signal transduction, inhibits B cell proliferation and activation, and can further inhibit immune hyperactivity and rejection. Therefore, when the antibody is applied to preparation of a drug for treating an immune disease, the antibody has advantages of high target specificity, high affinity, few side effects and the like, and has a very good application prospect.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to an anti-CD40 monoclonal antibody and its applications. Background Technology

[0002] CD40 is a type I transmembrane glycoprotein. Its extramembrane amino acid residues are highly homologous to nerve growth factor receptor (NGFR), tumor necrosis factor receptor (TNFR), Fas antigen (CD95), and CD27 of T lymphocytes. These molecules constitute the TNFR superfamily or NGFR superfamily.

[0003] CD40 is widely expressed on the surface of immune cells, including B cells, monocytes, macrophages, and dendritic cells. It is also expressed on the surface of non-immune cells such as epithelial cells, endothelial cells, adipocytes, stromal cells (fibroblasts, smooth muscle cells, synovial cells, stellate cells, etc.), and platelets. In addition, CD40 is also expressed on leukemia B cells, and is also expressed on the surface of syndactyly cells, dendritic cells, thymic epithelial cells, and some epithelial cancer cells.

[0004] CD40 plays a crucial role in antigen presentation within the immune system. Activation of dendritic cells (DCs) and macrophages via CD40 enhances their antigen-presenting capacity and promotes T-cell activation. CD40 also plays a significant role in autoimmune diseases and immune rejection. A brief summary follows:

[0005] The CD40-CD40L co-stimulatory pathway plays a crucial role in thymus-dependent humoral immune responses, being essential for B cell activation, differentiation, and memory development. It is also vital for activating antigen-presenting cells (APCs) and is key to T cell activation. The CD40-CD40L co-stimulatory pathway influences various diseases, and studies on these diseases have shown that this molecule enhances the body's protective capabilities. Pathogens can interfere with the CD40-CD40L co-stimulatory pathway, thereby disrupting CD40 signaling. The CD40-CD40L co-stimulatory pathway plays a vital role in immune regulation and homeostasis. In inflammatory responses, CD40 and CD40L are transiently expressed in T cells and other non-immune cells to regulate numerous cellular and molecular processes, including the development and progression of cellular and humoral adaptive immunity. Therefore, CD40 and CD40L are involved in the pathological processes of many inflammatory diseases, including autoimmune diseases, atherosclerotic thrombosis, tumors, and respiratory diseases.

[0006] Autoimmune diseases associated with CD40 include the following: rheumatoid arthritis (CD40 is not only involved in the production of autoantibodies in RA, but is also directly expressed by synovial fibroblasts. Contact with CD40L leads to the production of inflammatory mediators such as TNF, IL-1β, and MMP, resulting in further inflammation and joint destruction); autoimmune nephritis; inflammatory bowel disease (CD40L and CD40 are increased in circulation and intestinal mucosa in IBD. CD40 is strongly overexpressed in endothelial and submucosal cells, and CD40 expression is correlated with the clinical activity of UC); autoimmune skin diseases, multiple sclerosis, etc.

[0007] Currently, the most commonly used drugs for treating autoimmune diseases and suppressing immune rejection are chemical drugs or small molecule drugs, while antibody drugs are less frequently used. Furthermore, existing drugs have drawbacks such as unsatisfactory efficacy and significant toxic side effects. Summary of the Invention

[0008] The main purpose of this application is to provide an anti-CD40 monoclonal antibody and its application, in order to solve the problem of high toxicity and side effects of drugs in the prior art.

[0009] To achieve the above objectives, according to one aspect of this application, an anti-CD40 monoclonal antibody is provided, comprising three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1: GYAFTTTG; the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2: INTHSGVP; the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3: VRSGNGNYDLAYFAY; the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4: QSISDY; the amino acid sequence of LCDR2 is: YAS; and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 5: QHGHSFPNT.

[0010] Furthermore, the anti-CD40 monoclonal antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7;

[0011] SEQ ID NO: 6:

[0012] QIQLVSGPELKKPGETVRISCKASGYAFTTGMQWVQEMPGKGLKWIGWINTHSGVPKYVEDFKGRFAFSLETSANTAYLISNLKNEDTATYFCVRSGNGNYDLAYFAYWGQGTLVTVSA;

[0013] SEQ ID NO: 7:

[0014] DIVLTSPATLSVIPGDRVSISCRASQSISDYLHWYQKSHESPRLLIKYASHSISGIPSRFSGSGSGSDFTLSINSVEPEDVGIYYCQHGHSFPNTFGGGTKLEIKRTVA.

[0015] Furthermore, the anti-CD40 monoclonal antibody also includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is a constant region of immunoglobulins from humans, mice, monkeys, rabbits, or sheep, preferably a constant region of any of the following mouse types: IgG, IgA, IgM, IgE, or IgD, more preferably a constant region of any of the following mouse IgG types: IgG1, IgG2a, IgG2b, or IgG3; the light chain constant region is a constant region of a mouse κ type.

[0016] Further, the anti-CD40 monoclonal antibody is any one of the following: murine antibody molecule, humanized antibody molecule or chimeric antibody; preferably, the anti-CD40 monoclonal antibody is a full-length antibody or antibody fragment, preferably, the antibody fragment is selected from any one of the following: Fab, F(ab)2, Fv or ScFv.

[0017] According to a second aspect of this application, a medicament is provided comprising any of the aforementioned anti-CD40 monoclonal antibodies.

[0018] According to a third aspect of this application, a nucleic acid molecule is provided that encodes any of the aforementioned anti-CD40 monoclonal antibodies.

[0019] According to a fourth aspect of this application, a recombinant expression vector is provided, which comprises any of the above-described nucleic acid molecules.

[0020] According to a fifth aspect of this application, a host cell is provided, which is transfected with the above-described recombinant expression vector; preferably, the host cell is selected from prokaryotic cells, yeast cells, insect cells or mammalian cells; preferably, the mammalian cell is HEK293 cell, CHO cell or NSO cell.

[0021] According to the fifth aspect of this application, the use of any of the above-mentioned anti-CD40 monoclonal antibodies, nucleic acid molecules, group expression vectors or host cells in the preparation of medicaments for the treatment and / or prevention of diseases related to CD40 activation is provided.

[0022] Furthermore, diseases associated with CD40 activation include autoimmune diseases or immune rejection.

[0023] Furthermore, autoimmune diseases include one or more of the following: connective tissue diseases, neuromuscular diseases, endocrine diseases, digestive system diseases, urinary system diseases, and hematological diseases.

[0024] Further, connective tissue diseases include any one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, or scleroderma; preferably, neuromuscular diseases include any one or more of the following: multiple sclerosis, myasthenia gravis, or demyelinating diseases; preferably, endocrine diseases include any one or more of the following: primary adrenal atrophy, chronic thyroiditis, or hyperthyroidism; preferably, digestive system diseases include any one or more of the following: ulcerative colitis, primary biliary cirrhosis, or autoimmune hepatitis; preferably, urinary system diseases include autoimmune glomerulonephritis or pulmonary-renal hemorrhagic syndrome; preferably, hematologic diseases include autoimmune hemolysis, idiopathic thrombocytopenic purpura, or idiopathic leukopenia.

[0025] Furthermore, immune rejection includes immune rejection after organ transplantation, and more preferably includes immune rejection after allogeneic organ transplantation or xenogeneic organ transplantation.

[0026] Applying the technical solution of this application, a novel anti-CD40 antibody is provided. This antibody exhibits binding activity to both in vitro and cell surface CD40, competitively binding to CD40L and blocking the binding of CD40 to its ligand CD40L, thereby inhibiting CD40 / CD40L signaling transduction, suppressing B cell proliferation and activation, and ultimately inhibiting immune overreaction and rejection. Therefore, when this antibody is used to prepare drugs for treating immune diseases, it has advantages such as strong targeting specificity, high affinity, and few side effects, making it highly promising for application. Attached Figure Description

[0027] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0028] Figure 1The diagram shows the results of detecting the binding activity of antibody R5C7 to hCD40 protein by ELISA in Example 2 of this application;

[0029] Figure 2 The diagram shows the results of detecting the binding activity of antibody R5C7 to cell surface hCD40 by flow cytometry in Example 3 of this application;

[0030] Figure 3 The diagram shows the results of detecting the in vitro competitive activity of antibody R5C7 by flow cytometry in Example 4 of this application;

[0031] Figure 4 The figure shows the results of detecting the inhibitory effect of antibody R5C7 on CD40 signal transduction activity by luciferase in Example 5 of this application;

[0032] Figure 5 The antagonistic effect of antibody R5C7 on CD40L-induced B cell activation was detected by flow cytometry in Example 6 of this application.

[0033] Figure 6 The invention illustrates the inhibitory effect of antibody R5C7 on CD40L-induced B cell proliferation as detected by flow cytometry in Example 7 of this application. Detailed Implementation

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to the embodiments.

[0035] Terminology Explanation:

[0036] Autoimmune diseases: Disease states caused by the body's immune system's immune response to its own components. While an immune response to foreign antigens usually results in the clearance of those antigens, an immune response to antigens affecting one's own cells or tissues is not easily and completely cleared by the immune system's effector cells; instead, the cells or tissues are continuously attacked, leading to a disease state.

[0037] Immune rejection: Immune rejection is the process by which the body destroys a graft (allogeneic cells, tissues, or organs) through a specific immune response. Generally, it refers to the process after transplantation where the recipient can recognize graft antigens and generate a response, and the immune cells in the graft can also recognize the recipient's antigenic tissues and generate a response.

[0038] Antibody: A protein that binds to a specific antigen, broadly referring to all proteins and protein fragments containing a complementarity-determining region (CDR). "Antibody" specifically refers to full-length antibodies. Full-length antibodies include polyclonal and monoclonal antibodies. When an antibody is a protein fragment containing a CDR, it can be a substance containing part or all of the antibody's CDR, lacking at least some amino acids present in a full-length antibody but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. In some embodiments, antibodies in this fragment form have the function of specifically recognizing and binding to CD40. In some embodiments, antibodies in this fragment form are fragments that block the binding of CD40 to its CD40L. In some specific embodiments, such fragments are selected from Fab (composed of the complete light chain and Fd), Fv (composed of VH and VL), ScFv (a single-chain antibody with a linker peptide connecting VH and VL), or single-domain antibodies (composed only of VH). Antibodies in this fragment form can be produced using recombinant nucleic acid technology, or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0039] "Complementarity-determining region" (CDR): This refers to a highly variable region of the heavy and light chains of an immunoglobulin. There are three heavy-chain CDRs and three light-chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of antibodies to the antigens or epitopes they recognize. In another specific embodiment, CDR refers to the highly variable region of the heavy and light chains of an immunoglobulin as defined by IMGT.

[0040] In this application, the complementarity-determining region (CDR) of the heavy chain is denoted by HCDR, and the complementarity-determining region of the light chain is denoted by LCDR. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. In their compilation of "Sequences of Proteins of Immunological Interest," the amino acid sequences of the light chain (λ, κ) variable regions and antibody heavy chains, as well as the variable regions (α, β, γ, δ) of T cell receptors, are aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This application uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of protection of this application.

[0041] In this application, the binding ability of an antibody to a predetermined epitope on an antigen can be measured using affinity K. D To represent. Typically, antibodies are expressed in terms of approximately less than 10. -6 M, for example, approximately less than 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D It binds to the target antigen.

[0042] In this application, the antibody can specifically recognize CD40 from various genera, such as humans, mice, monkeys (e.g., cynomolgus monkeys), rabbits, or sheep.

[0043] As mentioned in the background section, the most commonly used drugs for treating autoimmune diseases and suppressing immune rejection are chemical drugs or small molecule drugs, with antibody drugs being less frequently used. Furthermore, existing drugs suffer from drawbacks such as unsatisfactory efficacy and significant toxic side effects. To improve this situation, in this application, the inventors, based on the important role of CD40 in autoimmune diseases and immune rejection, attempted to use CD40 as a therapeutic target, preparing an anti-CD40 monoclonal antibody that specifically recognizes CD40. A series of experiments demonstrated that this antibody competitively blocks the binding of CD40L to CD40 and inhibits B cell proliferation and activation, thereby suppressing immune overreaction and rejection. Based on this, the applicant proposes a series of protective measures in this application.

[0044] In a first typical embodiment of this application, an anti-CD40 monoclonal antibody is provided, comprising three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and three light chain variable regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1: GYAFTTTG; the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2: INTHSGVP; the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3: VRSGNGNYDLAYFAY; the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4: QSISDY; the amino acid sequence of LCDR2 is: YAS; and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 5: QHGHSFPNT.

[0045] In some embodiments, the CD40 monoclonal antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO: 7.

[0046] SEQ ID NO: 6:

[0047]

[0048] SEQ ID NO: 7:

[0049]

[0050] It should be noted that proteins or polypeptides having 95% to 99.9% homology (e.g., 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) to SEQ ID NOs: 1-7, and having the same or similar CD40 binding activity as SEQ ID NOs: 1-7, are also within the scope of this application.

[0051] In this application, homology refers to the “sequence identity” between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, it can be determined using the BLAST program of the NCBI database. For information on the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991.

[0052] Amino acid residues can be represented by a three-letter or one-letter amino acid code according to standards known and agreed upon in the art. Conservative amino acid substitutions or replacements are known in the art. For example, a conserved amino acid substitution is preferably one amino acid residue from the following groups (1)-(5) replaced by another amino acid from the same group: (1) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (3) positively charged polar residues: His, Arg, and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by lysine; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; and Val is replaced by Ile or Leu.

[0053] Therefore, the modified sequences of SEQ ID NOs: 1-7 obtained by replacing the conventionally conserved amino acids are also within the scope of protection of this application.

[0054] In some embodiments, the anti-CD40 monoclonal antibody further includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is a constant region of immunoglobulins from humans, mice, monkeys, rabbits, or sheep, preferably a constant region of any of the following mice: IgG, IgA, IgM, IgE, or IgD, more preferably a constant region of any of the following mouse IgGs: IgG1, IgG2a, IgG2b, or IgG3; the light chain constant region is a constant region of a mouse κ type.

[0055] In some embodiments, the anti-CD40 monoclonal antibody is any one of the following: murine antibody molecule, humanized antibody molecule, or chimeric antibody; preferably, the anti-CD40 monoclonal antibody is a full-length antibody or antibody fragment, and preferably, the antibody fragment is selected from any one of the following: Fab, F(ab)2, Fv, or ScFv.

[0056] In a second typical embodiment of this application, a medicament is provided comprising any of the aforementioned anti-CD40 monoclonal antibodies. The medicament herein can be a pharmaceutical composition containing excipients with an anti-CD40 monoclonal antibody as a single active ingredient, or it can be a pharmaceutical composition comprising two or more active ingredients, including an anti-CD40 monoclonal antibody.

[0057] In some embodiments, excipients include pharmaceutically acceptable carriers. “Pharmaceutically acceptable carriers” can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc., used to extend the shelf life or potency of antibodies.

[0058] In a third typical embodiment of this application, a nucleic acid molecule is provided that encodes the aforementioned anti-CD40 monoclonal antibody.

[0059] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA nucleic acids are preferred when ligated into a vector.

[0060] In a third typical embodiment of this application, a recombinant expression vector is provided, which contains the aforementioned nucleic acid molecules.

[0061] The term "recombinant expression vector," also simply called a vector, refers to a nucleic acid delivery vehicle into which nucleic acid molecules can be inserted. When a vector enables the expression of the protein encoded by the inserted nucleic acid molecule, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; or animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). In some embodiments, the vectors in this application contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0062] In a fourth typical embodiment of this application, a host cell is provided, which is transfected with the aforementioned recombinant expression vector. Preferably, the host cell is selected from prokaryotic cells, yeast cells, insect cells, or mammalian cells; preferably, the mammalian cell is HEK293 cell, CHO cell, or NSO cell.

[0063] In a fifth typical embodiment of this application, the use of the above-described anti-CD40 monoclonal antibody in the preparation of medicaments for the treatment and / or prevention of diseases related to CD40 activation is provided.

[0064] In this application, the aforementioned diseases related to CD40 activation include autoimmune diseases or immune rejection; preferably, autoimmune diseases include one or more of connective tissue diseases, neuromuscular diseases, endocrine diseases, digestive system diseases, urinary system diseases, and hematological diseases; preferably, connective tissue diseases include any one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, or scleroderma; preferably, neuromuscular diseases include any one or more of the following: multiple sclerosis, myasthenia gravis, or demyelinating diseases; preferably, endocrine diseases include any one or more of the following The following are possible conditions: primary adrenal cortical atrophy, chronic thyroiditis, or hyperthyroidism; preferably, digestive system diseases include any one or more of the following: ulcerative colitis, primary biliary cirrhosis, or autoimmune hepatitis; preferably, urinary system diseases include autoimmune glomerulonephritis or pulmonary-renal hemorrhagic syndrome; preferably, hematologic diseases include autoimmune hemolysis, idiopathic thrombocytopenic purpura, or idiopathic leukopenia; preferably, immune rejection includes immune rejection after organ transplantation, more preferably including immune rejection after allogeneic organ transplantation or immune rejection after xenotransplantation.

[0065] In some specific embodiments, immune diseases include any of the following: Addison's disease, allergic reactions, anaphylactic reactions, ankylosing spondylitis, asthma, atherosclerosis, atopic dermatitis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune mumps, bronchial asthma, coronary heart disease, Crohn's disease, diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drugs (e.g., factor VII in hemophilia), systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthritis, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.

[0066] It should be noted that the use of the anti-CD40 antibody or its antigen-binding fragment, nucleic acid molecule, recombinant expression vector, and host cell in the preparation of drugs for the treatment and / or prevention of immune diseases should also be within the scope of protection of this application.

[0067] This application also provides a kit for diagnosing CD40-related diseases, the kit containing the aforementioned anti-CD40 antibody or its antigen-binding fragment.

[0068] The beneficial effects of this application will be further explained in detail below with reference to specific embodiments. It should be noted that the isotype control antibodies used in the following embodiments are the same, all being mouse monoclonal antibodies (catalog number: ab99757) with clone number JDC-10 manufactured by Abcam. Unless otherwise specified, the reagents, methods, and equipment used in the following embodiments are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0069] Example 1 Antibody Preparation

[0070] The CD40 monoclonal antibody and its sequence in this application were obtained through hybridoma technology, as detailed below:

[0071] I. Animal Immunization

[0072] The extracellular segment sequence of hCD40 (NP_001241.1) and the mouse Fc fragment were linked to form a recombinant protein, which was used as an antigen to immunize female Balb / c mice.

[0073] For the first immunization, mice were subcutaneously injected with purified antigen and complete Freund's adjuvant after thorough emulsification. For the second, third, and fourth immunizations, the antigen and incomplete adjuvant were emulsified and then injected subcutaneously. On day 7 after the fourth immunization, blood was collected from the tail vein of the mice for titer analysis. Mice with the highest titers were selected for booster immunization via intraperitoneal injection of antibodies.

[0074] II. Cell Fusion

[0075] Three days later, mouse spleens were harvested under sterile conditions. The spleen tissue was separated, minced, and prepared into a single-cell suspension. The two cell lines were mixed at a ratio of spleen cells to myeloma cells (SP2 / 0) of 5:1, and the collected spleen cells were washed twice with IMEM medium. After centrifugation of the mixed cells, the supernatant was removed, and PEG1500 was slowly added to the cell clumps. The mixture was gently shaken and incubated for 2 minutes. Then, 2 ml of IMEM was added to terminate the cell fusion reaction, followed by the slow addition of 10 ml of IMEM. The mixture was centrifuged at 800 rpm, and the supernatant was discarded.

[0076] III. Screening of hybridoma-positive clones and cell cloning

[0077] Then, add sufficient IMEM (1*HAT) containing penicillin antibiotics and 15% FBS to fully resuspend the cells. Add 300 μl of cell suspension to each well of a 96-well cell culture plate. Incubate the 96-well plate at 37°C. Hybridoma clones can be obtained after 10 days, and single-cell positive clones can be obtained by limiting dilution.

[0078] IV. Antibody Preparation

[0079] Positive clones were expanded and cultured, and the cell culture supernatant was harvested. The cell supernatant was purified using a Protein A / G affinity chromatography column to obtain the anti-human CD40 mouse antibody R5C7.

[0080] V. Sequencing of the variable region of monoclonal antibodies

[0081] The amino acid sequence of the anti-human CD40 murine antibody R5C7 monoclonal antibody was determined by proteomic analysis.

[0082] The heavy chain variable region contains 120 amino acids, and the amino acid sequence is shown in SEQ ID NO:6. The underlined parts are HCDR1, HCDR2 and HCDR3, respectively.

[0083]

[0084] The light chain variable region contains 109 amino acids, and the amino acid sequence is shown in SEQ ID NO:7. The underlined parts are LCDR1, LCDR2 and LCDR3, respectively.

[0085]

[0086] Example 2: Detection of in vitro binding activity of antibody R5C7 to CD40

[0087] The binding activity of antibody R5C7 was detected using an ELISA method. The specific steps are as follows:

[0088] The hCD40-his protein (self-constructed) was diluted to 1 μg / mL with CBS buffer and coated into microplates at a rate of 50 μL / well. The plates were then incubated overnight at 4°C.

[0089] Wash the plate three times with 200 μL / well of PBST buffer. Then add 1% BSA dissolved in PBS and incubate at 37°C for 1 hour to block the plate.

[0090] After incubation, wash the plate three times with PBST, 200 μL / well. Dilute hCD40 antibody R5C7 to 30 μg / mL with PBST, perform serial dilutions using the 3-fold dilution method, and transfer 100 μL to the microplate using a multi-channel pipette. Incubate at 37°C for 1 h.

[0091] After incubation, wash the plate three times with PBST, 200 μL / well, and blot dry. Dilute the HRP-labeled anti-mouse IgG antibody (1:5000 dilution) with 1% BSA dissolved in PBS, 100 μL / well. Incubate at 37°C for 1 h; after incubation, wash the plate three times with PBST, 200 μL / well, and blot dry.

[0092] Add 50 μL of TMB chromogenic solution to each well and allow the reaction to proceed for 5 min. Then, add 50 μL of hydrochloric acid per well to terminate the reaction. Measure the absorbance at 450 nm (OD450) using an ELISA reader in absorbance mode. Results are as follows: Figure 1 As shown, antibody R5C7 has a strong binding ability to hCD40-His protein.

[0093] Example 3: Detection of the binding activity of antibody R5C7 to CD40 on the cell surface

[0094] The binding activity of antibody R5C7 to cell surface hCD40 was detected by flow cytometry. The specific steps are as follows:

[0095] The binding activity of antibody R5C7 to hCD40 on the cell surface was detected using a self-constructed 293F-hCD40 cell line. Cells from the 293F-hCD40 line were counted, and 2 × 10⁶ cells were collected. 5 The cells were added to a 1.5 mL EP tube.

[0096] Add the prepared antibody R5C7 or isotype control antibody. Dilute antibody R5C7 and isotype control antibody (Abcam, clone number JDC-10) to an initial concentration of 30 μg / mL, then perform a 1:3 serial dilution to seven concentrations, adding 150 μL / tube to the corresponding tube. For the blank control, add 150 μL of PBS, gently pipette to mix, and incubate on ice for 1 hour.

[0097] After incubation, add 500 μL of PBS and wash once by centrifugation, then centrifuge at 2000 rpm for 5 min and discard the supernatant. Add goat anti-mouse fluorescent secondary antibody diluted with PBS (1:1000, 200 μL / tube to the corresponding tube), gently pipette to mix, and incubate on ice in the dark for 30 min.

[0098] After 30 minutes of incubation, flow cytometry (FCM) was used to detect the binding activity of antibody R5C7 and isotype control antibody on hCD40 surface cells of 293F-hCD40 cells. EC50 was then calculated. 50 .

[0099] like Figure 2 As shown, the murine hCD40 antibody R5C7 has a strong binding affinity to hCD40 on the cell surface, EC5C6. 50 It was 1.26 ± 0.15 μg / mL.

[0100] Example 4: In vitro competitive activity of antibody R5C7

[0101] 293F-hCD40 cells were collected and counted, and the cell density was adjusted to 4 × 10⁻⁶. 6Count per mL. Take 2 × 10⁻⁶ cells / mL at a rate of 50 μL / tube. 5 The cells were added to a 1.5 mL EP tube.

[0102] The hCD40 murine antibody R5C7 or isotype control antibody was diluted to an initial concentration of 30 μg / mL, and five concentrations were obtained by serial dilution at a ratio of 1:3. 50 μL of each concentration was added to the corresponding tube. 50 μL of PBS was added, and the mixture was gently pipetted to mix. The tubes were then incubated on ice for 15 min.

[0103] After 15 minutes, add the prepared hCD40L-hFc protein (self-constructed) (6 μg / mL), 100 μL / tube, gently pipette to mix, and incubate on ice for another 1 hour.

[0104] After incubation, each tube was washed once with 500 μL of PBS by centrifugation at 2000 rpm for 5 min, and the supernatant was discarded. The Alexa 488 fluorescently labeled anti-mouse antibody (Abcam, catalog number ab150113) was diluted 1:1000 with PBS, and 200 μL was added to each tube. The tubes were gently mixed by pipetting and incubated on ice in the dark for 30 min. After 30 min of incubation, the signal was detected using flow cytometry.

[0105] like Figure 3 As shown, antibody R5C7 exhibits significant antagonistic activity against the binding of CD40L to CD40.

[0106] Example 5: Detection of antibody R5C7's inhibitory activity on CD40 / CD40L signal transduction.

[0107] We introduced the NFκB-Luciferase reporter gene vector (self-constructed) into the 293T-hCD40 cells we constructed earlier to obtain the hCD40-NFκB-Luciferase cell line. The activation ability of the hCD40 antibody on the hCD40 protein-related signaling pathway in 293T-hCD40 cells was detected using a Luciferase assay. This experiment tests the activity of antibodies antagonizing the CD40 protein; therefore, the weaker the detected fluorescence intensity, the stronger the inhibitory activity of the hCD40 murine antibody on the signaling pathway, which represents the stronger the antibody's antagonistic activity against CD40L.

[0108] Collect 293T-hCD40 cells at a concentration of 2.0 × 10⁻⁶. 4 Cells / wells were added to 6-well plates. Prepared CD40 antibody R5C7 and isotype control antibody were added, with three concentration gradients of 1 μg / mL, 5 μg / mL, and 15 μg / mL for each antibody. The dilution buffer was DMEM complete medium (containing 10% FBS).

[0109] Add 2 mL of reagent to each well, with three replicates per sample. Incubate the 6-well plate at 37°C in a 5% CO2 cell culture incubator for 6 hours. After incubation, remove the 6-well plate and add 50 μL of luciferase assay reagent (equilibrated to room temperature) per well. Incubate at room temperature for approximately 3 minutes. Detect the chemiluminescence signal using the luminescence mode of a multi-mode microplate reader.

[0110] Experimental results are as follows Figure 4 The results showed that antibody R5C7 had a significant inhibitory effect on the hCD40 protein-related signaling pathway, exhibiting a concentration-dependent relationship.

[0111] Example 6: Antibody R5C7 Inhibits B Cell Activation

[0112] When B cells in human blood are activated, various proteins can be elevated. Among them, the upregulation of CD95 protein expression in B cells after stimulation by various cytokines is one of the important markers of B cell activation. This embodiment uses flow cytometry (FCM) to detect the ability of hCD40 antibody to induce CD95 expression in Ramos cells (tumor cells derived from B cells), thereby determining the inhibitory effect of the test antibody on B cell activation. The experimental method is briefly described below:

[0113] Ramos cells in good growth condition were digested, terminated, centrifuged, resuspended, and counted at a concentration of 1.0 × 10⁻⁶. 5 Add cells / well to a 6-well plate. Add prepared antibody R5C7 and isotype control antibody, with three concentration gradients of 1 μg / mL, 5 μg / mL, and 15 μg / mL for each antibody. The dilution buffer is 1640 complete medium (containing 10% FBS). Add 1640 complete medium to the control group. Add 100 μL / well to the corresponding well. Each well contains P / S double antibody (1:100).

[0114] The 24-well plates were cultured at 37°C in a 5% CO2 incubator for 16 hours. After culture, cells were collected into corresponding 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. PE-Cy5-labeled CD95 antibody (diluted 1:50) was added to each tube at 50 μL / tube, and the tubes were incubated at 4°C in the dark for approximately 40 min. After incubation, 200 μL of PBS was added to each tube, and signal detection was performed using flow cytometry. Data were processed using FlowJo and GraphPad Prism 5 software, and bar charts were generated.

[0115] Experimental results are as follows Figure 5 The results showed that antibody R5C7 had a good antagonistic effect on CD40L-induced B cell activation, and significantly reduced CD95 expression levels in a dose-dependent manner.

[0116] Example 7: Antibody R5C7 inhibits B cell proliferation

[0117] B cells possess several important membrane proteins on their surface, which perform crucial physiological functions. CD19 is one of the more specific biomarkers on the B cell membrane, expressed in precursor B cells and absent until differentiation into plasma cells. The density of CD19 on the B cell membrane is highly regulated during B cell development and maturation, with its expression level in mature B cells being three times higher than in immature B cells. Activation of antigen-presenting cells such as B cells can further promote the activation of anti-tumor T cells, thereby enhancing the body's ability to eliminate tumor cells.

[0118] This embodiment uses flow cytometry (FCM) to detect the expression level of CD19 on B cells in vitro to reflect the ability of candidate hCD40 antibodies to antagonize B cell proliferation. The experimental method is briefly described below:

[0119] First, peripheral blood mononuclear cells (PBMCs) were collected, and B cells were purified using the Dynabeads Untouched Human B Cells Kit according to the manufacturer's instructions. The B cells were then resuspended in 1640 complete culture medium (containing 10% FBS) and counted. The cells were then purified at a concentration of 2.0 × 10⁶ cells / mL. 5 Cells were seeded into 6-well plates. Prepared R5C7 antibody and isotype control antibody were added. The antibody was prepared at three concentration gradients: 1 μg / mL, 5 μg / mL, and 15 μg / mL, using 1640 complete culture medium as the dilution buffer. The 6-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 48 hours.

[0120] After culture, cells were collected into corresponding numbered 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 50 μL of CD19-APC (1:50) solution was added to each tube (except for the CD19- group) to resuspend the cells and mix well. The cells were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed with 1 mL PBS. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were then resuspended in 150 μL PBS and analyzed using flow cytometry. Samples were analyzed using flow cytometry. Data processing was performed using FlowJo software.

[0121] The results are as follows Figure 6 The results showed that, compared with the control group, the R5C7 antibody significantly inhibited CD40L-induced B cell proliferation, and the inhibitory effect was concentration-dependent.

[0122] As can be seen from the above description, the embodiments of this application achieve the following technical effects: The antibody R5C7 targeting CD40 provided in this application can competitively bind to CD40 with CD40L, blocking the binding of CD40 to its ligand CD40L, thereby inhibiting CD40 / CD40L signal transduction, inhibiting B cell proliferation and activation (i.e., inhibiting the maturation and development of memory B cells, T cell-dependent immunoglobulin type switching, etc.), and thus inhibiting immune overreaction and rejection. Therefore, applying this antibody to the preparation of drugs for treating autoimmune diseases has advantages such as high specificity and few side effects.

[0123] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An anti-CD40 monoclonal antibody, characterized in that, It includes three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, among which... The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1: GYAFTTTG; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 2: INTHSGVP; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 3: VRSGNGNYDLAYFAY; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4: QSISDY; The amino acid sequence of LCDR2 is: YAS; The amino acid sequence of LCDR3 is shown in SEQ ID NO: 5: QHGHSFPNT.

2. The anti-CD40 monoclonal antibody according to claim 1, characterized in that, The anti-CD40 monoclonal antibody includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7. SEQ ID NO: 6: QIQLVSGPELKKPGETVRISCKAS GYAFTTTG MQWVQEMPGKGLKWIGW INTHSGVP KYVEDFKGRFAFSLETSANTAYLISNLKNEDTATYFC VRSGNGNYDLAYFAY WGQGTLVTVSA; SEQ ID NO: 7: DIVLTSPATLSVIPGDRVSISCRAS QSISDY LHWYQKSHESPRLLIK YAS HSISGIPSRFSGSGSGSDFTLSINSVEPEDVGIYYC QHGHSFPNT FGGGTKLEIKRTVA。 3. The anti-CD40 monoclonal antibody according to claim 2, characterized in that, The anti-CD40 monoclonal antibody further includes a heavy chain constant region and / or a light chain constant region; The heavy chain constant region is the constant region of immunoglobulins from humans, mice, monkeys, rabbits, or sheep; The light chain constant region is a murine κ-type constant region.

4. The anti-CD40 monoclonal antibody according to claim 3, characterized in that, The heavy chain constant region is a constant region of any of the following in mice: IgG, IgA, IgM, IgE, or IgD.

5. The anti-CD40 monoclonal antibody according to claim 4, characterized in that, The heavy chain constant region is a constant region of any of the following mouse IgGs: IgG1, IgG2a, IgG2b, or IgG3.

6. The anti-CD40 monoclonal antibody according to any one of claims 1-5, characterized in that, The anti-CD40 monoclonal antibody is any one of the following: murine antibody molecules, humanized antibody molecules, or chimeric antibodies.

7. The anti-CD40 monoclonal antibody according to claim 6, characterized in that, The anti-CD40 monoclonal antibody is a full-length antibody.

8. A drug, characterized in that, It comprises the anti-CD40 monoclonal antibody according to any one of claims 1-7.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD40 monoclonal antibody according to any one of claims 1-7.

10. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 9.

11. A host cell, characterized in that, The host cell is transfected with the recombinant expression vector of claim 10.

12. The host cell according to claim 11, characterized in that, The host cell is selected from prokaryotic cells, yeast cells, insect cells, or mammalian cells.

13. The host cell according to claim 12, characterized in that, The mammalian cells are HEK293 cells, CHO cells, or NSO cells.

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