Anti-CD40 monoclonal antibody and application thereof

Abstract

The invention provides an anti-CD40 monoclonal antibody and application of the anti-CD40 monoclonal antibody. The anti-CD40 monoclonal antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) as shown in SEQ ID NOs: 1-3, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) as shown in SEQ ID NO: 4, YAS and SEQ ID NO: 5. The antibody has binding activity with in-vitro CD40 and CD40 on the cell surface, and can be competitively bound with CD40L to block binding of the antibody and CD40L so as to inhibit CD40 / CD40L signal transduction and inhibit B cell proliferation and activation, so that immune overexpression and rejection can be inhibited. Therefore, when the antibody is applied to preparation of drugs for treating immune diseases, the antibody has the advantages of high targeting specificity, high affinity, few side effects and the like, and has a great application prospect.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular, to an anti-CD40 monoclonal antibody and its application. Background Art

[0002] CD40 is a type I transmembrane glycoprotein. The amino acid residues outside its envelope 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, and is also expressed on the surface of non-immune cells such as epithelial cells, endothelial cells, adipocytes, interstitial cells (fibroblasts, smooth muscle cells, synoviocytes, astrocytes, etc.), and platelets. In addition, CD40 is also expressed on leukemia B cells, and on the surface of synaptic cells, dendritic cells, thymic epithelial cells and some epithelial cancer cells.

[0004] CD40 plays an important role in antigen presentation of the immune system. After DC cells and macrophages are activated by CD40, they enhance antigen presentation ability and promote T cell activation. CD40 plays an important role in autoimmune diseases and immune rejection. A brief description is as follows:

[0005] The CD40-CD40L co-stimulatory pathway plays an important role in the thymus-dependent humoral immune response. It is crucial for B cell activation, differentiation and memory generation. It is also very important for the activation of antigen presenting cells (APCs) and is the key to T cell activation. The CD40-CD40L co-stimulatory pathway has an impact on a variety of diseases. Through the study of these diseases, it was found that this pair of molecules can enhance the body's protective ability. Pathogens can interfere with the CD40-CD40L co-stimulatory pathway and thus destroy the CD40 signal. The CD40-CD40L co-stimulatory pathway plays an important 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 many cellular and molecular processes, including the occurrence and development 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] Among them, the autoimmune diseases associated with CD40 include the following: rheumatoid arthritis (CD40 is not only related to the production of autoantibodies in RA, but is also directly expressed by synovial fibroblasts, which contact with CD40L, leading to the production of inflammatory mediators such as TNF, IL-1b and MMP, leading to further inflammation and joint destruction); autoimmune nephritis; inflammatory bowel disease (CD40L and CD40 in IBD are increased in both the circulation and intestinal mucosa. CD40 is strongly overexpressed in endothelial and mesenchymal cells in the mucosa and submucosa, and the expression of CD40 is associated with the clinical activity of UC); autoimmune skin diseases, multiple sclerosis, etc.

[0007] At present, the most commonly used drugs for treating autoimmune diseases and suppressing immune rejection are chemical drugs or small molecule drugs, while antibody drugs are rarely used. In addition, existing drugs have defects such as poor effects and large toxic side effects. Summary of the invention

[0008] The main purpose of the present application is to provide an anti-CD40 monoclonal antibody and its application to solve the problem of large toxic and side effects of drugs in the prior art.

[0009] To achieve the above-mentioned purpose, according to one aspect of the present application, an anti-CD40 monoclonal antibody is provided, comprising three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and three light chain complementary 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; the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 5: QHGHSFPNT.

[0010] Further, the anti-CD40 monoclonal antibody comprises 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 an immunoglobulin of human, mouse, monkey, rabbit or sheep, preferably any one of the following mouse constant regions: IgG, IgA, IgM, IgE or IgD, more preferably any one of the following mouse IgG constant regions: IgG1, IgG2a, IgG2b or IgG3; the light chain constant region is a mouse κ-type constant region.

[0016] Furthermore, the anti-CD40 monoclonal antibody is any one of the following: a murinized antibody molecule, a humanized antibody molecule or a chimeric antibody; preferably, the anti-CD40 monoclonal antibody is a full-length antibody or an 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 the present application, a drug is provided, comprising any one of the above-mentioned anti-CD40 monoclonal antibodies.

[0018] According to the third aspect of the present application, a nucleic acid molecule is provided, which encodes any one of the above-mentioned anti-CD40 monoclonal antibodies.

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

[0020] According to the fifth aspect of the present application, a host cell is provided, which is transfected with the above-mentioned recombinant expression vector; preferably, the host cell is selected from prokaryotic cells, yeast cells, insect cells or mammalian cells; preferably, the mammalian cells are HEK293 cells, CHO cells or NS0 cells.

[0021] According to a fifth aspect of the present application, there is provided a use of any of the above anti-CD40 monoclonal antibodies, nucleic acid molecules, expression vectors or host cells in the preparation of a drug for treating and / or preventing diseases associated with CD40 activation.

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

[0023] Furthermore, the autoimmune diseases include one or more of connective tissue diseases, neuromuscular diseases, endocrine diseases, digestive system diseases, urinary system diseases and blood system 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 disease; preferably, endocrine diseases include any one or more of the following: 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, blood system 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 immune rejection after xenogeneic organ transplantation.

[0026] By applying the technical solution of the present application, the present application provides a new anti-CD40 antibody, which has binding activity with both in vitro CD40 and CD40 on the cell surface, and can competitively bind to CD40 with CD40L, blocking the binding of CD40 with its ligand CD40L, thereby inhibiting CD40 / CD40L signal transduction, inhibiting B cell proliferation and activation, and further inhibiting immune excitation and rejection. Therefore, when the antibody is used to prepare drugs for treating immune diseases, it has the advantages of strong targeting specificity, high affinity, and few side effects, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings constituting part of the present application are used to provide a further understanding of the present application. The exemplary embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0028] Figure 1The figure shows the result of detecting the binding activity of antibody R5C7 and hCD40 protein by ELISA method in Example 2 of the present application;

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

[0030] Figure 3 The figure shows the result of detecting the in vitro competitive activity of antibody R5C7 by flow cytometry in Example 4 of the present application;

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

[0032] Figure 5 It is shown that the antagonistic effect of antibody R5C7 on CD40L-induced B cell activation was detected by flow cytometry in Example 6 of the present application;

[0033] Figure 6 The inhibitory effect of antibody R5C7 on CD40L-induced B cell proliferation detected by flow cytometry in Example 7 of the present application is shown. DETAILED DESCRIPTION

[0034] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below in conjunction with the embodiments.

[0035] Terminology explanation:

[0036] Autoimmune disease: A disease state caused by the body's immune system's immune response to its own components. The result of the body's immune response to foreign antigens is usually the elimination of the antigens, but when the body's immune response occurs to its own cell or tissue antigens, the body's own cells or tissues are not easily completely eliminated by the immune system's effector cells but are constantly attacked, resulting in the body entering a disease state.

[0037] Immune rejection: Immune rejection is the process by which the body destroys the transplant (allogeneic cells, tissues or organs) through a specific immune response. Generally speaking, after transplantation, the recipient can recognize the transplant antigens and respond, and the immune cells in the transplant can also recognize the recipient antigen tissue and respond.

[0038] Antibody: refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments that contain a complementary determining region (CDR region). "Antibody" refers in particular to a full-length antibody. "Full-length antibody" includes polyclonal antibodies and monoclonal antibodies. When an antibody is a protein fragment that includes CDR, it may be a substance that includes part or all of the antibody CDR, which lacks at least some of the amino acids present in the full-length antibody but is still able to specifically bind to the 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 the form of such fragments have the function of specifically recognizing and binding to CD40. In some embodiments, antibodies in the form of such fragments are fragments that have the ability to block the binding of CD40 to its CD40L. In some specific embodiments, such fragments are selected from Fab (composed of a complete light chain and Fd), Fv (composed of VH and VL), ScFv (single-chain antibody, VH and VL are connected by a connecting peptide) or single-domain antibodies (consisting only of VH). Such fragment forms of antibodies can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

[0039] "Complementarity determining region" (i.e., CDR): refers to the highly variable regions of the heavy and light chains of immunoglobulins. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the circumstances, 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 an antibody to its recognized antigen or epitope. In another specific embodiment, CDR refers to the highly variable regions of the heavy and light chains of immunoglobulins as defined by IMGT.

[0040] In the present application, the complementarity determining region of the heavy chain is represented by HCDR, and the complementarity determining region of the light chain is represented by LCDR. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. 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 light chain (λ, κ) variable regions and antibody heavy chains, as well as the variable regions of T cell receptors (α, β, γ, δ) are aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. In the present application, the Kabat annotation standard is used to label the CDR region, but CDR regions labeled by other methods also belong to the scope of protection of the present application.

[0041] In the present application, the ability of an antibody to bind to an epitope on a predetermined antigen can be expressed as affinity K. D Typically, antibodies are expressed as less than 10 -6 M, for example, less than about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (K D ) binds to the target antigen.

[0042] In the present application, the antibody provided can specifically recognize CD40 of various genera, such as human, mouse, monkey (such as cynomolgus monkey), rabbit or sheep.

[0043] As mentioned in the background technology, the most commonly used drugs for treating autoimmune diseases and inhibiting immune rejection are chemical drugs or small molecule drugs, and antibody drugs are less used. In addition, existing drugs have defects such as poor effects and large toxic and side effects. In order to improve this situation, in this application, the inventors tried to use CD40 as a therapeutic target based on the important role played by CD40 in autoimmune diseases and immune rejection, and prepared anti-CD40 monoclonal antibodies that specifically recognize CD40. After a series of experiments, it was proved that the antibody has the activity of competitively blocking the binding of CD40L to CD40, and can inhibit B cell proliferation and activation, thereby inhibiting immune hyperactivity and rejection. On this basis, the applicant proposed a series of protection schemes for this application.

[0044] In a first typical embodiment of the present application, an anti-CD40 monoclonal antibody is provided, which includes three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region includes three light chain complementary 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; the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 5: QHGHSFPNT.

[0045] In some embodiments, the CD40 monoclonal antibody comprises 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.

[0046] SEQ ID NO: 6:

[0047]

[0048] SEQ ID NO: 7:

[0049]

[0050] It should be noted that proteins or polypeptides having an amino acid sequence with 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 the present application.

[0051] Homology in this application refers to the "sequence identity" between two amino acid sequences, i.e., the percentage of identical amino acids between the sequences. Methods for evaluating 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 usually measured using sequence analysis software. For example, it can be determined by the BLAST program of the NCBI database. For 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., M Stockton Press, New York, 1991.

[0052] Amino acid residues can be represented by the standard three-letter or one-letter amino acid code known and agreed upon in the art. Conservative amino acid substitutions or replacements are well known in the art. For example, conservative amino acid substitutions preferably replace one amino acid residue in the following groups (1)-(5) with another amino acid in the same group: (1) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) polar positively charged residues: His, Arg and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (5) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; 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 variant sequences of the above SEQ ID NOs: 1-7 obtained by replacing the above conventional conservative amino acids are also within the protection scope of the present application.

[0054] In some embodiments, the anti-CD40 monoclonal antibody further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a constant region of an immunoglobulin of human, mouse, monkey, rabbit or sheep, preferably any one of the following mouse constant regions: IgG, IgA, IgM, IgE or IgD, more preferably any one of the following mouse IgG constant regions: IgG1, IgG2a, IgG2b or IgG3; the light chain constant region is a mouse κ-type constant region.

[0055] In some embodiments, the anti-CD40 monoclonal antibody is any one of the following: a murinized antibody molecule, a humanized antibody molecule or a chimeric antibody; preferably, the anti-CD40 monoclonal antibody is a full-length antibody or an antibody fragment, 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 the present application, a drug is provided, which comprises any of the above-mentioned anti-CD40 monoclonal antibodies. The drug herein can be a pharmaceutical composition containing excipients and having the anti-CD40 monoclonal antibody as a single active ingredient, or a pharmaceutical composition comprising two or more active ingredients including the anti-CD40 monoclonal antibody.

[0057] In some embodiments, the excipient includes a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible to extend the shelf life or efficacy of the antibody.

[0058] In a third typical embodiment of the present application, a nucleic acid molecule is provided, which 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 are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is 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 operably linked to the coding sequence. DNA nucleic acids are preferably used when they are linked to a vector.

[0060] In a third typical embodiment of the present application, a recombinant expression vector is provided, which comprises the aforementioned nucleic acid molecule.

[0061] The term "recombinant expression vector (also referred to as vector)" refers to a nucleic acid carrier into which a nucleic acid molecule can be inserted. When a vector can express the protein encoded by the inserted nucleic acid molecule, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage or animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40). In some embodiments, the vector in the present application contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0062] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell is transfected with the aforementioned recombinant expression vector. Preferably, the host cell is selected from a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell; preferably, the mammalian cell is a HEK293 cell, a CHO cell or a NS0 cell.

[0063] In a fifth typical embodiment of the present application, there is provided use of the above anti-CD40 monoclonal antibody in the preparation of a drug for treating and / or preventing diseases associated with CD40 activation.

[0064] In the present application, the above-mentioned diseases associated with CD40 activation include autoimmune diseases or immune rejection; preferably, the autoimmune diseases include one or more of connective tissue diseases, neuromuscular diseases, endocrine diseases, digestive system diseases, urinary system diseases and blood system 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 disease; preferably, endocrine diseases include any one or more of the following: Preferably, the digestive system diseases include any one or more of the following: ulcerative colitis, primary biliary cirrhosis or autoimmune hepatitis; preferably, the urinary system diseases include autoimmune glomerulonephritis or pulmonary-renal hemorrhagic syndrome; preferably, the blood system diseases include autoimmune hemolysis, idiopathic thrombocytopenic purpura or idiopathic leukopenia; preferably, the immune rejection includes immune rejection after organ transplantation, more preferably includes immune rejection after allogeneic organ transplantation or immune rejection after xenogeneic organ transplantation.

[0065] In some specific embodiments, the immune disease includes any one of the following: Addison's disease, allergy, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune ear diseases, autoimmune eye diseases, autoimmune hepatitis, autoimmune parotitis, 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 of the present application in the preparation of drugs for treating and / or preventing immune diseases should also be within the scope of protection of the present application.

[0067] The present application also provides a kit for diagnosing CD40-related diseases, wherein the kit contains the aforementioned anti-CD40 antibody or antigen-binding fragment thereof.

[0068] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments. It should be noted that the isotype control antibodies used in the following embodiments are the same, all of which are mouse monoclonal antibodies with clone number JDC-10 produced by abcam (article number: ab99757). Unless otherwise specified, the reagents, methods and equipment used in the following embodiments are conventional reagents, methods and equipment in the art. 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 of the present application are obtained by hybridoma technology, as follows:

[0071] 1. Animal Immunization

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

[0073] For the first immunization of mice, purified antigen and complete Freund's adjuvant were fully emulsified and then injected subcutaneously; for the second, third and fourth immunizations, antigen and incomplete adjuvant were fully emulsified and then injected subcutaneously. On the 7th day after the fourth immunization, blood was collected from the tail vein of the mice for titer detection. The mice with the highest titer were selected for booster immunization, and the booster immunization was performed by intraperitoneal injection of antibodies.

[0074] 2. Cell Fusion

[0075] After 3 days, the spleen of the mouse was taken under a sterile environment. The spleen of the mouse was separated, minced, and made into a single cell suspension. The two cells were mixed at a ratio of spleen cells: myeloma cells (SP2 / 0) = 5:1, and the collected spleen cells were washed twice with IMEM medium. After the mixed cells were centrifuged, the supernatant was removed, PEG1500 was slowly added to the cell mass, and it was gently shaken. After incubation for 2 minutes, 2ml of IMEM was added to terminate the cell fusion reaction, and then 10ml of IMEM was slowly added and centrifuged at 800rpm to discard the supernatant.

[0076] 3. Screening of hybridoma positive clones and cell cloning

[0077] Then add enough IMEM (1*HAT) containing dual antibodies and 15% FBS to fully resuspend the cells. Add 300μl of cell suspension per well to a 96-well cell culture plate. Place the 96-well plate in a 37°C cell culture incubator and culture. After 10 days, hybridoma clones can be obtained, and single-cell positive clones can be obtained by limiting dilution method.

[0078] IV. Antibody Preparation

[0079] The 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] 5. Monoclonal antibody variable region sequence determination

[0081] The anti-human CD40 mouse antibody R5C7 monoclonal antibody was subjected to protein spectrum detection and its amino acid sequence was determined.

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

[0083]

[0084] The light chain variable region comprises 109 amino acids, the amino acid sequence of which is shown in SEQ ID NO:7, and the underlined portions are LCDR1, LCDR2, and LCDR3;

[0085]

[0086] Example 2 In vitro binding activity detection of antibody R5C7 and CD40

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

[0088] hCD40-his protein (self-constructed) was diluted to 1 μg / mL with CBS buffer, coated on an ELISA plate at 50 μL / well, and placed at 4°C overnight.

[0089] The plate was washed three times with 200 μL / well of PBST buffer, and then 1% BSA dissolved in PBS was added at 200 μL / well and incubated at 37°C for 1 hour for blocking.

[0090] After incubation, wash the plate 3 times with PBST, 200 μL / well. Dilute hCD40 antibody R5C7 to 30 μg / mL with PBST, perform gradient dilution by 3-fold dilution method, transfer 100 μL to the ELISA plate with a pipette, and incubate at 37°C for 1 hour.

[0091] After incubation, wash the plate 3 times with PBST, 200 μL / well, and pat dry. Dilute HRP-labeled anti-mouse IgG antibody (1:5000 dilution) with 1% BSA dissolved in PBS, 100 μL / well. Incubate at 37°C for 1 hour; after incubation, wash the plate 3 times with PBST, 200 μL / well, and pat dry.

[0092] Add 50 μL TMB colorimetric solution to each well for 5 min, then add 50 μL / well hydrochloric acid to terminate the reaction. Use the absorbance mode of the microplate reader to detect the absorbance value at 450 nm (OD450). The 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] Flow cytometry was used to detect the binding activity of antibody R5C7 to hCD40 on the cell surface. The specific steps are as follows:

[0095] The self-constructed 293F-hCD40 cell detection antibody R5C7 was used to detect the binding activity of hCD40 on the cell surface. 293F-hCD40 cell lines were counted and 2×10 5 The cells were added to a 1.5 mL EP tube.

[0096] Add the prepared antibody R5C7 or isotype control antibody. Antibody R5C7 and isotype control antibody (abcam, clone number JDC-10) were diluted to a starting concentration of 30 μg / mL, 1:3 gradient dilution to 7 concentrations, and 150 μL / tube was added to the corresponding tube. Add 150 μL PBS to the blank group as a blank control, gently pipet and mix, and incubate on ice for 1 hour.

[0097] After incubation, add 500 μL PBS and wash once by centrifugation at 2000 rpm for 5 min, discard the supernatant. Add goat anti-mouse fluorescent secondary antibody diluted with PBS (diluted at 1:1000), add 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 to the surface hCD40 of 293F-hCD40 cells, and the EC 50 .

[0099] like Figure 2 As shown, hCD40 mouse antibody R5C7 has a strong binding ability with hCD40 on the cell surface. 50 It is 1.26±0.15μg / mL.

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

[0101] The 293F-hCD40 cell line was collected and counted, and the cell density was adjusted to 4×10 6 / mL. Press 50μL / tube, take 2×10 5 The cells were added to a 1.5 mL EP tube.

[0102] hCD40 mouse antibody R5C7 or isotype control antibody was diluted to a starting concentration of 30 μg / mL, and 5 concentrations were obtained by 1:3 gradient dilution method, and 50 μL / tube was added to the corresponding tubes. 50 μL PBS was added, and the mixture was gently pipetted to mix, and then placed on ice for incubation 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, add 500 μL PBS and wash once by centrifugation at 2000 rpm for 5 min, and discard the supernatant. Dilute Alexa 488 fluorescently labeled anti-mouse antibody (abcam, catalog number ab150113) with PBS (all diluted at 1:1000), add 200 μL / tube to the corresponding tube, gently blow and mix, and incubate on ice in the dark for 30 min. After incubation for 30 min, use flow cytometer to detect the signal.

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

[0106] Example 5 Detection of Antibody R5C7 Inhibiting CD40 / CD40L Signal Transduction Activity

[0107] The NFκB-Luciferase reporter gene vector (self-constructed) was introduced into the 293T-hCD40 cells we constructed above to obtain the hCD40-NFκB-Luciferase cell line. The Luciferase assay was used to detect the activation ability of hCD40 antibodies on the hCD40 protein-related signaling pathways in 293T-hCD40 cells. This experiment is to detect the activity of antagonistic antibodies against CD40 protein. Therefore, the weaker the detected fluorescence intensity, the stronger the inhibitory activity of the corresponding hCD40 mouse antibody on the signaling pathway, which also means that the antibody has a stronger activity of antagonizing CD40L.

[0108] 293T-hCD40 cells were collected and counted at 2.0×10 4 Cells / well were added to a 6-well plate. Prepared CD40 antibody R5C7 and isotype control antibody were added, and the antibodies were set at three concentration gradients of 1 μg / mL, 5 μg / mL and 15 μg / mL. The diluent was DMEM complete medium (containing 10% FBS).

[0109] Add 2 mL / well to the corresponding wells, with 3 replicates for each sample. Place the 6-well plate in a 37°C, 5% CO2 cell culture incubator for 6 hours. After the culture is completed, remove the 6-well plate and add 50 μL / well of luciferase detection reagent that has been equilibrated to room temperature. Place at room temperature for about 3 minutes. Detect the chemiluminescent signal using the luminescence mode (Luminescence) of the multifunctional microplate reader.

[0110] The 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, showing a concentration-dependent relationship.

[0111] Example 6 Antibody R5C7 inhibits B cell activation

[0112] When B cells in human blood are activated, multiple proteins may be elevated. Among them, after being stimulated by multiple cytokines, B cells upregulate the expression of CD95 protein, which is one of the important signs of B cell activation. In this example, the ability of hCD40 antibody to induce Ramos cells (tumor cells derived from B cells) to express CD95 is detected by flow cytometry (FCM), thereby determining the inhibitory ability of the tested antibody on B cell activation. The experimental method is briefly described as follows:

[0113] The Ramos cells with good growth status were digested, terminated, centrifuged, resuspended, and counted. 5 Add cells / well to a 6-well plate. Add the prepared antibody R5C7 and isotype control antibody, and set the antibody concentration gradient to 1μg / mL, 5μg / mL and 15μg / mL. The diluent is 1640 complete medium (containing 10% FBS). Add 1640 complete medium to the blank group. Add 100μL / well to the corresponding well. Each well contains P / S dual antibody (1:100).

[0114] The 24-well plate was placed in a 37°C, 5% CO2 cell culture incubator for 16 hours. After the culture, the cells were collected in the corresponding 1.5mL EP tubes, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. 50μL / tube of PE-Cy5-labeled CD95 antibody (diluted at 1:50) was added to each tube and incubated at 4°C in the dark for about 40 minutes. After the incubation, 200μL PBS was added to each tube, and the signal was detected by flow cytometry. FlowJo and GraphPad Prism 5 data processing software were used for data processing and bar graphs were made.

[0115] The 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 the expression level of CD95 was significantly reduced in a dose-dependent manner.

[0116] Example 7 Antibody R5C7 inhibits B cell proliferation

[0117] There are some important membrane proteins on the surface of B cells, which have important physiological functions. Among them, CD19 is one of the more specific biomarkers on the surface of B cell membranes. It begins to be expressed in precursor B cells and loses expression until it differentiates into plasma cells. The density of CD19 on the B cell membrane is highly regulated during the development and maturation of B cells. Its expression in mature B cells is 3 times higher than that in immature B cells. The 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 clear tumor cells.

[0118] In this example, the expression of CD19 on B cells in vitro was detected by flow cytometry (FCM) to reflect the ability of candidate hCD40 antibodies to antagonize B cell proliferation. The experimental method is briefly described as follows:

[0119] First, PBMCs were collected from peripheral blood and B cells were purified using the Dynabeads Untouched Human B cells kit according to the instructions. B cells were then resuspended in 1640 complete medium (containing 10% FBS) and counted. 5 cells / wells. Plate the cells in a 6-well plate. Add the prepared antibody R5C7 and isotype control antibody. The antibodies were set at three concentration gradients of 1μg / mL, 5μg / mL and 15μg / mL, and the diluent was 1640 complete medium. The 6-well plate was placed in a 37°C, 5% CO2 cell culture incubator for 48h.

[0120] After the culture is completed, collect the cells in the corresponding numbered 1.5mL EP tube, centrifuge at 1000rpm for 5min, and discard the supernatant. Add 50μL CD19-APC (1:50) solution to each tube of cells (except CD19-group), resuspend the cells and mix well. Incubate at 4℃ in the dark for 30min. After the incubation, add 1mL PBS to wash the cells. Centrifuge at 1000rpm for 5min and discard the supernatant. Resuspend the cells with 150μL PBS and detect with a flow cytometer. Use a flow cytometer to detect the samples. Use FlowJo software for data processing.

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

[0122] From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects: the antibody R5C7 against CD40 provided by the present application can competitively bind to CD40 with CD40L, block the binding of CD40 to its ligand CD40L, thereby inhibiting CD40 / CD40L signal transduction, inhibiting B cell proliferation and activation (i.e., inhibiting memory B cell maturation and development, T cell-dependent immunoglobulin type conversion, etc.), and thus being able to inhibit immune excitation and rejection. Therefore, the antibody is used in the preparation of drugs for the treatment of autoimmune diseases, which has the advantages of strong specificity and few side effects.

[0123] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An anti-CD40 monoclonal antibody, characterized in that: It includes three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3, among which, The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1: GYAFTTTG; The amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2: INTHSGVP; The amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3: VRSGNGNYDLAYFAY; The amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4: QSISDY; The amino acid sequence of the LCDR2 is: YAS; The amino acid sequence of the 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 comprises a heavy chain variable region and a light chain variable region, wherein 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 GYFTTTG 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 comprises a heavy chain constant region and / or a light chain constant region, The heavy chain constant region is a constant region of an immunoglobulin of a human, mouse, monkey, rabbit or sheep, preferably any one of the following mouse constant regions: IgG, IgA, IgM, IgE or IgD, more preferably any one of the following mouse IgG constant regions: IgG1, IgG2a, IgG2b or IgG3; The light chain constant region is a murine κ type constant region.

4. The anti-CD40 monoclonal antibody according to any one of claims 1 to 3, characterized in that The anti-CD40 monoclonal antibody is any one of the following: a murinized antibody molecule, a humanized antibody molecule or a chimeric antibody; Preferably, the anti-CD40 monoclonal antibody is a full-length antibody or an antibody fragment. Preferably, the antibody fragment is selected from any one of the following: Fab, F(ab)2, Fv or ScFv.

5. A drug, characterized in that The method comprises the anti-CD40 monoclonal antibody according to any one of claims 1 to 4.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD40 monoclonal antibody according to any one of claims 1 to 4.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 6.

8. A host cell, characterized in that The host cell is transfected with the recombinant expression vector according to claim 7; Preferably, the host cell is selected from prokaryotic cells, yeast cells, insect cells or mammalian cells; Preferably, the mammalian cell is a HEK293 cell, a CHO cell or a NS0 cell.

9. Use of the anti-CD40 monoclonal antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 6, the recombinant expression vector according to claim 7 or the host cell according to claim 8 in the preparation of a drug for treating and / or preventing diseases associated with CD40 activation.

10. The use according to claim 9, characterized in that: The diseases associated with CD40 activation include autoimmune diseases or immune rejection; Preferably, the autoimmune disease includes one or more of connective tissue diseases, neuromuscular diseases, endocrine diseases, digestive system diseases, urinary system diseases and blood system diseases; Preferably, the connective tissue disease includes any one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis or scleroderma; Preferably, the neuromuscular disease includes any one or more of the following: multiple sclerosis, myasthenia gravis or demyelinating disease; Preferably, the endocrine disease includes any one or more of the following: primary adrenal cortical atrophy, chronic thyroiditis or hyperthyroidism; Preferably, the digestive system disease includes any one or more of the following: ulcerative colitis, primary biliary cirrhosis or autoimmune hepatitis; Preferably, the urinary system disease includes autoimmune glomerulonephritis or pulmonary-renal hemorrhagic syndrome; Preferably, the blood system disease includes autoimmune hemolysis, idiopathic thrombocytopenic purpura or idiopathic leukopenia; Preferably, the immune rejection includes immune rejection after organ transplantation, more preferably includes immune rejection after allogeneic organ transplantation or immune rejection after xenogeneic organ transplantation.

Images