Blocking CD40 antibody and use thereof

Abstract

Provided are an anti-CD40 antibody and the use thereof. The antibody comprises heavy chain variable region CDR1, CDR2 and CDR3 sequences respectively shown as SEQ ID NO: 1, 2 and 3 or amino acid sequences having at least 80% identity with SEQ ID NO: 1, 2 and 3; and light chain variable region CDR1, CDR2 and CDR3 sequences respectively shown as SEQ ID NO: 4, 5 and 6 or amino acid sequences having at least 80% identity with SEQ ID NO: 4, 5 and 6.

Description

Blocking CD40 antibodies and their applications Technical Field

[0001] The present invention relates to the field of antibodies, and in particular, to an antibody capable of binding to CD40 or an antigen-binding fragment thereof and uses thereof. Background Art

[0002] CD40 is a type I membrane protein, primarily expressed on the surface of antigen-presenting cells (APCs), including B cells, monocytes, macrophages, and dendritic cells. Studies have shown that CD40 is also expressed on the surface of platelets and some non-hematopoietic cells such as fibroblasts, endothelial cells, and smooth muscle cells. Some types of tumor cells also express CD40. Therapies targeting CD40 are used in immune-related diseases such as autoimmune diseases and cancer. CD40L (CD40 Ligand), the ligand for CD40, is a type II membrane protein expressed on the surface of a variety of cells, including activated CD4 T cells, CD8 T cells, NK cells, granulocytes, endothelial cells, smooth muscle cells, macrophages, and platelets.

[0003] The interaction between CD40 and CD40L induces both humoral and cell-mediated immune responses. After binding to CD40, CD40L transmits activation signals to APCs, promoting APC activation, upregulating the expression of co-stimulatory ligands such as CD80 and CD86, and promoting APC proliferation, thereby promoting immune activation. In autoimmune diseases, blocking CD40 antibodies can inhibit CD40 activation signals by binding to CD40, potentially alleviating the progression of autoimmune diseases such as multiple sclerosis.

[0004] Therefore, there is an urgent need for disease drugs that can interfere with the effects of CD40-CD40L binding and block CD40 signaling.

[0005] Summary of the Invention

[0006] The present invention aims to address, at least to some extent, at least one of the technical problems existing in the prior art. Currently, blocking CD40 antibodies such as Iscalimab (Novartis) and Ravagalimab (AbbVie) are in clinical trials. The inventors' research has found that Iscalimab and Ravagalimab trigger APC activation and upregulate the expression of CD80, CD86, and other proteins, contradicting the intended effects of these two antibodies.

[0007] Based on this, the inventors screened and obtained a murine anti-CD40 monoclonal antibody with high binding activity to the human CD40 protein. Furthermore, the inventors humanized the constant region of the monoclonal antibody, retaining the CDRs of the murine anti-CD40 monoclonal antibody, to obtain a chimeric antibody. Furthermore, the framework regions of the light chain variable region or the heavy chain variable region of the chimeric antibody were humanized to obtain a fully humanized anti-CD40 antibody. This CD40 antibody is able to specifically target and bind to the human CD40 protein, effectively blocking the binding of CD40 to CD40L, inhibiting CD40 downstream signaling, and suppressing B cell proliferation without activating APCs (such as B cells). It is expected to play a role in immune-related diseases such as autoimmune diseases, transplant rejection, and cancer.

[0008] Therefore, in the first aspect of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 1, 2, and 3, or amino acid sequences having at least 80% identity with SEQ ID NOs: 1, 2, and 3, respectively; and light chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6, or amino acid sequences having at least 80% identity with SEQ ID NOs: 4, 5, and 6, respectively. The antibody or antigen-binding fragment thereof according to the embodiments of the present invention can bind to human CD40 protein, block the binding of CD40 to CD40L, inhibit the downstream signaling of CD40, inhibit B cell proliferation, and does not cause activation of APC cells (such as B cells), thereby effectively treating or preventing CD40-mediated related diseases and conducting related scientific research.

[0009] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment thereof may further include at least one of the following additional technical features:

[0010] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 as shown in SEQ ID NO: 2, a heavy chain variable region CDR3 as shown in SEQ ID NO: 3, a light chain variable region CDR1 as shown in SEQ ID NO: 4, a light chain variable region CDR2 as shown in SEQ ID NO: 5, and a light chain variable region CDR3 as shown in SEQ ID NO: 6.

[0011] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region as shown in SEQ ID NO: 7 or SEQ ID NO: 11; and / or a light chain variable region as shown in SEQ ID NO: 8 or SEQ ID NO: 12.

[0012] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: 1) a heavy chain variable region as shown in SEQ ID NO: 7, and a light chain variable region as shown in SEQ ID NO: 8; or 2) a heavy chain variable region as shown in SEQ ID NO: 11, and a light chain variable region as shown in SEQ ID NO: 12.

[0013] According to an embodiment of the present invention, the antibody or its antigen-binding fragment contains a heavy chain framework region sequence and a light chain framework region sequence, and at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0014] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody and a murine antibody or a mutant thereof.

[0015] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.

[0016] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from murine IgG1 antibody, IgG2a antibody, IgG4 antibody or mutants thereof or human IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody or mutants thereof.

[0017] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 9 and 13 and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 10 and 14.

[0018] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain with an amino acid sequence as shown in SEQ ID NO: 10.

[0019] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain with an amino acid sequence as shown in SEQ ID NO: 13 and a light chain with an amino acid sequence as shown in SEQ ID NO: 14.

[0020] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof includes at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody and a CDR-grafted antibody.

[0021] As used herein, the term "monoclonal antibody" refers to an antibody that can recognize only one specific antigenic epitope. Common monoclonal antibodies include two light chains with a relatively low molecular weight and two heavy chains with a relatively high molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. The amino acid sequence at the amino terminus (N-terminus) of the heavy or light chain varies greatly and is called the variable region (V region). The carboxyl terminus (C-terminus) of the heavy or light chain is relatively stable and varies very little, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively. In addition, the monoclonal antibody also includes, but is not limited to, single-chain antibodies, Fab antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and at least one of the minimum recognition units.

[0022] As used herein, the term "CDR-grafted antibody" refers to the transplantation of the CDRs of a monoclonal antibody from one species into the variable region of an antibody from another species. For example, the CDRs of a mouse monoclonal antibody can be transplanted into the variable region of a human antibody to replace the human antibody CDRs, allowing the human antibody to acquire the antigen-binding specificity of the mouse monoclonal antibody while reducing its heterologous nature. It should be noted that both polyclonal antibodies and monoclonal antibodies in this application can be CDR-grafted antibodies.

[0023] According to an embodiment of the present invention, the monoclonal antibody includes at least one of a full-length antibody, an Fv antibody, a single-chain antibody, a Fab antibody, a single-domain antibody, and a minimum recognition unit.

[0024] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO: 20.

[0025] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in the first aspect. According to some embodiments of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule is capable of binding to human CD40 protein, effectively treating or preventing CD40-mediated diseases.

[0026] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0027] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0028] In a third aspect, the present invention provides an expression vector carrying the nucleic acid molecule described in the second aspect. The expression vector may include optional control sequences that are operably linked to the nucleic acid molecule. The control sequences are one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vectors provided in the embodiments of the present invention can efficiently and massively express the antibody or antigen-binding fragment thereof in suitable host cells.

[0029] Herein, "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected function of regulating the transcription and translation of the exogenous gene. When the above-mentioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Those skilled in the art will understand that the nucleic acid molecules used to encode antibodies or antigen-binding fragments thereof can be independently inserted into different vectors, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned antibodies or antigen-binding fragments thereof can be effectively achieved under the mediation of the regulatory system, thereby achieving a large amount of antibodies or antigen-binding fragments thereof in vitro.

[0030] In a fourth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect, comprising: introducing the expression vector described in the third aspect into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment thereof. The methods provided in some specific embodiments of the present invention can effectively obtain large quantities of the antibody or antigen-binding fragment thereof in vitro.

[0031] According to some specific embodiments of the present invention, the above method for preparing the aforementioned antibody or antigen-binding fragment thereof may further include at least one of the following additional technical features:

[0032] According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.

[0033] According to some embodiments of the present invention, the cell is a eukaryotic cell.

[0034] According to some embodiments of the present invention, the eukaryotic cell is a mammalian cell. According to some embodiments of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.

[0035] In a fifth aspect, the present invention provides a recombinant cell carrying the nucleic acid described in the second aspect, or the expression vector described in the third aspect, or capable of expressing the antibody or antigen-binding fragment thereof described in the first aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some embodiments of the present invention, the recombinant cell can efficiently and abundantly express the aforementioned antibody or antigen-binding fragment thereof under appropriate conditions.

[0036] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0037] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the antibodies or antigen-binding fragments thereof described herein. It will be readily understood by those skilled in the art that conditions suitable for the expression of antibodies or antigen-binding fragments thereof include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies or antigen-binding fragments thereof according to the specific environment of the laboratory.

[0038] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0039] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0040] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0041] In its sixth aspect, the present invention provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof described in the first aspect, and a therapeutic agent. As previously described, the antibodies or antigen-binding fragments of the embodiments of the present invention are capable of effectively binding to the CD40 protein, effectively treating or preventing CD40-related diseases. Therefore, immunoconjugates comprising such antibodies or antigen-binding fragments are also capable of binding to human CD40 protein, and such immunoconjugates are effective in preventing and / or treating CD40-mediated diseases.

[0042] According to an embodiment of the present invention, the immunoconjugate may further include at least one of the following additional technical features:

[0043] According to an embodiment of the present invention, the therapeutic agent can prevent and / or treat CD40-mediated diseases or other diseases.

[0044] According to an embodiment of the present invention, the CD40-mediated disease is selected from transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0045] In a seventh aspect, the present invention provides a composition comprising the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the immunoconjugate described in the sixth aspect. As previously mentioned, the antibody or antigen-binding fragment thereof in some specific embodiments of the present invention can effectively bind to the human CD40 protein and effectively inhibit CD40-mediated B cell activation. Therefore, a composition comprising the above substances can also effectively bind to the human CD40 protein and have a good effect in preventing and / or treating CD40-mediated diseases. The type of the composition is not particularly limited and can be a food composition or a pharmaceutical composition.

[0046] The compositions of the present invention can also be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the compositions can also contain a chemotherapeutic agent. The antibodies, antigen-binding fragments thereof, or immunoconjugates of the present invention can also be combined with a second therapeutic agent, exemplary of which include, but are not limited to, other agents that inhibit CD40 activity (including other antibodies, antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with CD40 upstream or downstream signaling.

[0047] It should be noted that the composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0048] In an eighth aspect, the present invention provides a medicament comprising the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect, or the composition of the seventh aspect. As previously mentioned, the antibody or antigen-binding fragment thereof of certain embodiments of the present invention can effectively bind to human CD40 protein. Therefore, a medicament comprising an effective amount of the active ingredient of the antibody or antigen-binding fragment thereof, or a series of substances thereof, can also effectively bind to human CD40 protein and have a good effect in preventing and / or treating CD40-mediated diseases.

[0049] According to an embodiment of the present invention, the above-mentioned medicine may further include at least one of the following additional technical features:

[0050] According to an embodiment of the present invention, the drug may further include a pharmaceutically acceptable carrier.

[0051] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0052] The effective amount of the antibody or antigen-binding fragment thereof described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0053] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0054] The drug of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be compatible with the mode of administration, wherein the mode of administration can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, or intraperitoneal administration. The dosage form of the drug of the present invention is an injection, an oral preparation (tablet, capsule, oral solution), a transdermal agent, or a sustained-release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The drug is preferably manufactured under sterile conditions. The antibody or its antigen-binding fragment can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0055] In a ninth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, the immunoconjugate described in the sixth aspect, or the composition described in the seventh aspect in the preparation of a medicament for preventing and / or treating CD40-mediated diseases. As previously mentioned, the antibody or antigen-binding fragment thereof in certain embodiments of the present invention can effectively bind to the human CD40 protein. Therefore, a medicament comprising an effective amount of the antibody or antigen-binding fragment thereof, or a series of substances thereof, can also effectively bind to the human CD40 protein and have a good effect in preventing and / or treating CD40-mediated diseases.

[0056] In the tenth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect, the composition of the seventh aspect, or the medicament of the eighth aspect for preventing and / or treating CD40-mediated diseases. As previously mentioned, the antibody or antigen-binding fragment thereof in some embodiments of the present invention can effectively bind to the human CD40 protein. Therefore, an effective amount of the antibody or antigen-binding fragment thereof, or a series of substances thereof, can also effectively bind to the human CD40 protein, thereby effectively preventing and / or treating CD40-mediated diseases.

[0057] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0058] According to an embodiment of the present invention, the CD40-mediated related diseases include transplant rejection, autoimmune diseases, infectious diseases and cancer.

[0059] According to an embodiment of the present invention, the autoimmune disease includes but is not limited to at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0060] According to an embodiment of the present invention, the cancer includes but is not limited to at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0061] In its eleventh aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, and the recombinant cell described in the fifth aspect. As previously mentioned, the antibody or antigen-binding fragment thereof according to some embodiments of the present invention can effectively bind to human CD40 protein. Therefore, a kit comprising the antibody or antigen-binding fragment thereof can effectively detect human CD40 protein qualitatively or quantitatively. The kit provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of human CD40 and antibodies. These kits may include any one or more of the following: antagonists, anti-CD40 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-CD40 antibodies can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, the test can be performed on serum or blood of a subject to detect related diseases. The kit can also be used for scientific research to detect human CD40 protein in a test sample. Such related diseases may include CD40-related diseases, such as cancer. Of course, the antibodies or antigen-binding fragments thereof provided herein can also be used for radioimmunoassay and radioimmunotherapy of the above-mentioned diseases. For the above-mentioned application scenarios, the binding molecules are also applicable and will not be repeated here.

[0062] The kit may also include conventional materials for detecting CD40, such as coating solution.

[0063] In a twelfth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect in preparing a kit for detecting CD40. As previously mentioned, the antibodies or antigen-binding fragments thereof according to certain embodiments of the present invention are capable of effectively binding to human CD40 protein and blocking the binding of the CD40 protein to CD40L. Therefore, the antibodies or antigen-binding fragments thereof can be used to prepare a kit for detecting CD40 protein, which is capable of effectively performing qualitative or quantitative detection of human CD40 protein.

[0064] In a thirteenth aspect, the present invention provides a method for treating and / or preventing CD40-mediated diseases. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of at least one of the following: 1) the antibody or antigen-binding fragment thereof described in the first aspect; 2) the nucleic acid molecule described in the second aspect; 3) the expression vector described in the third aspect; 4) the recombinant cell described in the fifth aspect; 5) the immunoconjugate described in the sixth aspect; 6) the composition described in the seventh aspect; and 7) the drug described in the eighth aspect. As described above, the antibody or antigen-binding fragment thereof is capable of binding to human CD40 protein and is capable of effectively treating or preventing CD40-mediated diseases, preferably transplant rejection, autoimmune diseases, infectious diseases, and cancer. Therefore, the method according to an embodiment of the present invention is capable of effectively treating or preventing CD40-mediated diseases.

[0065] According to an embodiment of the present invention, the above method for treating or preventing a disease may further include at least one of the following additional technical features:

[0066] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0067] According to an embodiment of the present invention, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0068] The effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0069] In a fourteenth aspect, the present invention provides a method for detecting CD40. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment thereof described in the first aspect, or the kit described in the eleventh aspect, to detect a sample to be tested. As previously mentioned, the antibody or antigen-binding fragment can effectively bind to CD40. Thus, the method of the present invention can effectively detect CD40, such as qualitatively or quantitatively detecting CD40 in a biological sample. It can also be used to determine the status of an individual, such as determining whether the individual's CD40 level is excessively high or low after obtaining the individual's CD40 level. The biological sample can be cells, tissues, blood, etc.

[0070] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof forms an immune complex with CD40 in the sample to be detected; the presence of the immune complex is detected, and based on the presence of the immune complex, it is determined whether the sample to be detected contains CD40.

[0071] According to an embodiment of the present invention, the presence of the immune complex indicates the presence of CD40 in the sample to be tested.

[0072] In the fifteenth aspect of the present invention, a method for inhibiting the growth of cells expressing human CD40 antigen is provided, comprising administering the antibody or antigen-binding fragment thereof according to the first aspect to the cells, wherein the antibody or antigen-binding fragment thereof specifically binds to the CD40 antigen on the surface of human cells, wherein the binding of the antibody or antigen-binding fragment thereof to the CD40 antigen inhibits the growth or differentiation of the cells.

[0073] In the sixteenth aspect of the present invention, the present invention provides a method for inducing clearance of peripheral B cells, comprising administering the antibody or antigen-binding fragment thereof according to the first aspect to the cells, wherein the antibody or antigen-binding fragment thereof specifically binds to the CD40 antigen on the surface of human cells, wherein the binding of the antibody or antigen-binding fragment thereof to the CD40 antigen induces clearance of the cells.

[0074] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0076] FIG1 is a graph showing the ELISA results of the binding of 21G6 antibody, competing antibody Iscalimab, and Ravagalimab to human CD40 protein according to a specific embodiment of the present invention;

[0077] FIG2 is a graph showing the ELISA results of the binding of 21G6 antibody, competing Iscalimab antibody, and Ravagalimab antibody to monkey CD40 protein according to a specific embodiment of the present invention;

[0078] FIG3 is a flow cytometry result diagram of the binding of 21G6 antibody, competing antibody Iscalimab, and Ravagalimab to HCT-116 human colorectal cancer cells according to a specific embodiment of the present invention;

[0079] FIG4 is a flow cytometry result diagram of the binding of 21G6 antibody and humanized h21G6 antibody to HCT-116 human colorectal cancer cells according to a specific embodiment of the present invention;

[0080] FIG5 is a flow cytometry result diagram of the binding of 21G6 antibody, Iscalimab antibody and Ravagalimab antibody to human peripheral blood B cells according to a specific embodiment of the present invention;

[0081] FIG6 is a flow cytometry result diagram showing the blocking effect of 21G6 antibody, Iscalimab antibody and Ravagalimab antibody on the binding of CD40L-mFc to HCT-116 human colorectal cancer cells according to a specific embodiment of the present invention;

[0082] 7 is a flow cytometry result diagram showing that 21G6 antibody and humanized h21G6 antibody blocked the binding of CD40L-mFc to HCT-116 human colorectal cancer cells according to a specific embodiment of the present invention;

[0083] FIG8 is a graph showing the results of the inhibition of CD40L-mFc-mediated CD40 reporter cell activation by 21G6 antibody, competitor Iscalimab antibody, and Ravagalimab antibody according to specific embodiments of the present invention;

[0084] FIG9 is a graph showing the results of 21G6 antibody, competing Iscalimab antibody, and Ravagalimab antibody promoting up-regulation of CD86 molecule expression in human peripheral blood B cells according to a specific embodiment of the present invention;

[0085] FIG10 is a graph showing the results of the inhibition of CD40L-mFc-mediated upregulation of CD80 expression by B cells by 21G6 antibody, competitor Iscalimab antibody, and Ravagalimab antibody according to a specific embodiment of the present invention;

[0086] FIG11 is a graph showing the results of the inhibition of CD40L-mFc-mediated upregulation of CD86 expression by B cells by 21G6 antibody, competitor Iscalimab antibody, and Ravagalimab antibody according to specific embodiments of the present invention;

[0087] FIG12 is a graph showing the results of the inhibition of CD40L-mFc-mediated B cell proliferation by the 21G6 antibody, the competitor Iscalimab antibody, and the Ravagalimab antibody according to specific embodiments of the present invention. DETAILED DESCRIPTION

[0088] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0089] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0090] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0091] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0092] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0093] The antibodies or antigen-binding fragments thereof described herein are typically prepared by biosynthetic methods. Based on the nucleotide sequences described herein, those skilled in the art can readily prepare the encoding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook, "Molecular Cloning Laboratory Manual." As one embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing a nucleotide sequence in segments and then performing overlap extension PCR. The antibodies or antigen fragments are numbered and defined using the Kabat numbering system.

[0094] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0095] The term "murine antibody," as used herein, refers to a monoclonal antibody against human CD40 prepared according to the knowledge and skill in the art. This antibody is prepared by injecting a test subject with an antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional properties. In a preferred embodiment of the present invention, the murine CD40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or variants thereof.

[0096] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. The variable region gene is cloned from mouse hybridoma cells, and then the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are connected to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic expression system or a prokaryotic expression system. In a preferred embodiment of the present invention, the antibody light chain of the CD40 chimeric antibody further comprises a light chain Fc region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the CD40 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0097] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database. In order to avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0098] As used herein, the term "monoclonal antibody" refers to an antibody that has a single antigen-binding site.

[0099] Herein, the term "polyantibody" refers to an antibody that has two or more different antigen-binding sites.

[0100] As used herein, the term "mutant" or "variant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0101] The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence of an antibody that is responsible for antigen binding, for example, generally including: amino acid residues 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and amino acid residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or from the "hypervariable loops" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and around 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0102] As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0103] Under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art can replace, add and / or delete one or more (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0104] As previously described, the monoclonal antibodies of the present invention may be full-length antibodies or may comprise only functional fragments thereof (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-CD40 antibodies with modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites may be useful, or antibodies lacking fucose moieties on oligosaccharide chains may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0105] As used herein, a "full-length antibody" is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). The same class of immunoglobulins can also be divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are classified as either kappa or lambda chains based on their constant regions.

[0106] As used herein, the term "functional fragment" refers specifically to antibody fragments such as CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, or any fragment whose half-life can be increased by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" means polyethylene glycol), or by incorporation into liposomes, wherein the fragment has CD40 binding activity. Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, and more preferably at least 1 / 10, of the affinity of the antibody from which it is derived. Such functional fragments will comprise a minimum of 3 amino acids, and preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

[0107] In the present invention, unless otherwise specified, the term "antigen-binding fragment" generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, generally an antigen-binding region or a variable region, illustratively including CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.

[0108] As used herein, the term "CDR-grafted antibody" refers to the transplantation of CDRs from a monoclonal antibody (mAb) from one species into the variable region of an antibody from another species. For example, CDRs from a murine monoclonal antibody can be transplanted into the variable region of a human antibody to replace the human antibody CDRs, allowing the human antibody to acquire the antigen-binding specificity of the murine monoclonal antibody while reducing its heterologous nature.

[0109] As used herein, the term "Fab antibody" or "Fab" generally refers to an antibody containing only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0110] As used herein, the term "nanoantibody" (single domain antibody or VHH antibody), which was originally described as an antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e., "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprises only a heavy chain variable region (VH) and conventional CH2 and CH3 regions, and specifically binds to an antigen through the heavy chain variable region.

[0111] In this article, the term "Fv antibody" generally refers to an antibody composed only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0112] As used herein, the term "single-chain antibody" or "scFv" refers to a fragment consisting of the variable regions of the heavy and light chains of an antibody connected by a short peptide.

[0113] The amino acid or nucleic acid sequences involved in the present invention are shown in Table 1.

[0114] Table 1

[0115] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0116] Example 1: Preparation of antibodies

[0117] (1) Mouse monoclonal antibodies against human CD40 were generated. Purified recombinant CD40 extracellular region His-tagged fusion protein (CD40-His) (purchased from Acro) was used as an antigen to immunize Balb / c mice (9 weeks old, purchased from Shanghai Lesk, weighing approximately 20 g).

[0118] Balb / c mice were immunized three times with purified antigen and complete and incomplete Freund's adjuvants. Immune responses were assessed by tail vein bleeding. Serum was screened by ELISA and flow cytometry to identify mice expressing anti-human CD40 immunoglobulins. Splenocytes were removed from mice with the highest anti-CD40 immunoglobulin expression and fused with murine myeloma SP2 / 0 cells (ATCC No. CRL-1581). The resulting hybridomas were screened for antibodies to generate mouse monoclonal antibodies.

[0119] Culture the candidate hybridoma cells to a total number of 10 6 The cells were collected by centrifugation at 800 rpm for 10 minutes, and total RNA was extracted using a Trizol kit (purchased from Invitrogen). Using the total RNA as a template, a cDNA library was synthesized by reverse transcription. The cDNA was then used as a template for PCR amplification of the variable region nucleic acid sequences corresponding to the hybridoma cells. The primer sequences used in the PCR amplification reaction were complementary to the first framework region or signal peptide region and constant region of the antibody variable region (for specific sequence selection, see Larrick, J.W., et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, J.J., et al., (1991) BioTechniques, 11, 152-156). PCR amplification was performed in a 50 μl reaction system, with 2 μl of cDNA, 5 μl of 10× PCR buffer, 2 μl of upstream and downstream primers (5 μM), 2 μl of dNTPs, 1 μl of Taq enzyme (Takara, Ex Taq), and 38 μl of H₂O. The reaction was pre-denatured at 95°C for 5 min, followed by temperature cycling for PCR amplification. The reaction conditions were: denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 50 s, for a total of 32 cycles, followed by extension at 72°C for 7 min. The amplified products were sequenced to obtain the heavy and light chain variable region sequences of the mouse monoclonal antibody 21G6 (heavy chain variable region sequence, SEQ ID NO: 7, and light chain variable region sequence, SEQ ID NO: 8).

[0120] (2) Mouse antibody humanization experiment: Referring to the light chain variable region sequence and heavy chain variable region sequence of CD40 antibody 21G6, a humanized template with the best match to its non-CDR region was selected. The CDR region of the mouse antibody was transplanted onto the selected humanized template, replacing the CDR region of the human template, to obtain a humanized antibody. Then, based on the three-dimensional structure of the mouse antibody, the buried residues, residues that directly interact with the CDR region, and residues that have an important influence on the conformation of VL and VH were back-mutated to obtain the humanized antibody h21G6 antibody. The sequence of the heavy chain variable region of the humanized antibody h21G6 is shown in SEQ ID NO: 11, and the sequence of the light chain variable region is shown in SEQ ID NO: 12.

[0121] Example 2: Production of antibodies

[0122] The specific experimental procedures for antibody production are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution. (2) The pcDNA3.4 vector containing the antibody heavy chain and antibody light chain (commissioned to Nanjing GenScript for synthesis) was added to 2mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain solution A. (3) 160μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) was added to 2mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution B. (4) Solution A and solution B were then mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50mL ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C and 5% CO2 for 1 day, 8mL Feed and 300μL Enhancer (purchased from Thermo Fisher) were added, and the culture was transferred to 32°C and 5% CO2. 8mL Feed was added on the 5th day, and the culture supernatant was harvested after 9 days of culture. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from NanoMicro).

[0123] Example 3: 21G6 antibody ELISA binding experiment

[0124] ELISA assays are used to test the binding properties of CD40 antibodies. CD40 extracellular domain His-tag fusion proteins are coated onto 96-well plates. The strength of the signal after antibody addition is used to determine the binding properties of the antibody to the CD40 protein.

[0125] Human CD40-His protein or monkey CD40-His protein (purchased from Acro) was diluted to 2 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μl / well. The plate was incubated at 4°C overnight. The PBS buffer was aspirated from the 96-well plate, and the plate was washed six times with PBST (PBS pH 7.2 containing 0.1% Tween 20). Then, 200 μl / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μl / well of the CD40 antibody 21G6 (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), the competitor Iscalimab antibody (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), the Ravagalimab antibody (heavy chain sequence SEQ ID NO: 17, and light chain sequence SEQ ID NO: 18), and hIgG1LALA (purchased from Bio-Bio) were added in a serial dilution of PBST / 0.05% BSA and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μl / well of HRP (horseradish peroxidase)-labeled anti-human IgG secondary antibody (purchased from Jacksonlab) was added in a serial dilution of PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, add 80 μl / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 min. Then, add 80 μl / well of 4 M sulfuric acid to terminate the reaction. The absorbance is read at 450 nm using a microplate reader.

[0126] The experimental results are shown in Figures 1 and 2, indicating that the CD40 antibody of the present invention can bind not only to human CD40 protein, but also to monkey CD40 protein, and the binding activity of the two is similar to that of competing antibodies Iscalimab and Ravagalimab.

[0127] Example 4: 21G6 antibody flow cytometry binding experiment

[0128] Flow cytometry experiments were used to detect the binding properties of CD40 antibodies. CD40 antibodies were added to the cells, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the mouse antibody and CD40.

[0129] (1) HCT-116 human colorectal cancer cells were diluted to 2×10 6 / ml, added to a volume of 100μl / tube in a 1.5ml EP tube, and 10μl / tube of goat serum was added. The tube was blocked at 4°C for 30 minutes. Different concentrations of 21G6 antibody (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), competitor Iscalimab antibody (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), Ravagalimab antibody (heavy chain sequence SEQ ID NO: 17, and light chain sequence SEQ ID NO: 18), humanized h21G6 antibody (heavy chain sequence SEQ ID NO: 13, and light chain sequence SEQ ID NO: 14), and hIgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30 minutes. 1ml of PBS was added to the EP tube, and the tube was centrifuged at 3500rpm for 5 minutes at 4°C. The supernatant was discarded and the tube was washed once with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μl / tube of PBS. Add 1 μl / tube of APC-conjugated goat anti-human IgG secondary antibody (purchased from Jackson Lab) and incubate at 4°C in the dark for 30 min. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0130] The experimental results are shown in Figure 3, demonstrating that the 21G6 antibody of the present invention can bind to HCT-116 human colorectal cancer cells with stronger binding activity than competing antibodies: Iscalimab and Ravagalimab. The binding activity of the humanized h21G6 antibody to HCT-116 human colorectal cancer cells is shown in Figure 4, further demonstrating that the humanized h21G6 antibody of the present invention can bind to HCT-116 human colorectal cancer cells with similar binding activity to that of the 21G6 antibody.

[0131] (2) Dilute human peripheral blood mononuclear cells (PBMC) to 2×10 6 / ml (purchased from Heyousheng Bio), added to a volume of 100μl / tube in a 1.5ml EP tube, to which 10μl / tube goat serum was added, and blocked at 4°C for 30min. Different concentration gradients of 21G6 antibody (heavy chain sequence SEQ ID NO:9, and light chain sequence SEQ ID NO:10), competitor Iscalimab antibody (heavy chain sequence SEQ ID NO:15, and light chain sequence SEQ ID NO:16), Ravagalimab antibody (heavy chain sequence SEQ ID NO:17, and light chain sequence SEQ ID NO:18), and hIgG1LALA (purchased from Baiying Bio) were added and incubated at 4°C for 30min. 1ml PBS was added to the EP tube, centrifuged at 4°C, 3500rpm×5min, the supernatant was discarded, and the tube was washed again with PBS. After centrifugation, discard the supernatant and resuspend the cells with 100 μl / tube PBS. Add 1 μl / tube APC-labeled goat anti-human IgG secondary antibody (purchased from Jackson lab) and FITC-labeled CD19 antibody (purchased from Biolegend) and incubate at 4°C in the dark for 30 minutes. Wash twice with PBS and discard the supernatant after centrifugation. Resuspend the cells with 200 μl / tube PBS and detect using flow cytometry.

[0132] The experimental results are shown in FIG5 , which further demonstrate that the 21G6 antibody of the present invention can bind to human peripheral blood B cells, and its binding activity is stronger than that of its competitors, the Iscalimab antibody and the Ravagalimab antibody.

[0133] Example 5: Detection of CD40 Antibody Blocking Ability

[0134] Flow cytometry was used to examine the blocking properties of CD40 antibodies. CD40 antibodies and CD40L-mFc proteins were added to HCT-116 cells. The signal intensity after the addition of APC-labeled anti-mouse IgG was used to determine the effect of the antibodies on CD40L-mFc binding to HCT-116 cells.

[0135] HCT-116 human colorectal cancer cells were diluted to 2 × 10 6 / ml, added to a volume of 100 μl / tube in a 1.5 ml EP tube, and 10 μl / tube of goat serum was added. The tube was blocked at 4°C for 30 min. Different concentrations of 21G6 antibody (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), competitor Iscalimab antibody (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), Ravagalimab antibody (heavy chain sequence SEQ ID NO: 17, and light chain sequence SEQ ID NO: 18), hIgG1LALA (purchased from Bio-Bio), and 0.5 μg / ml CD40L-mFc protein (amino acid sequence shown in SEQ ID NO: 19) were added and incubated at 4°C for 30 min. 1 ml of PBS was added to the EP tube, and the tube was centrifuged at 3500 rpm for 5 min at 4°C. The supernatant was discarded and the tube was washed once with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μl / tube of PBS. Add 1 μl / tube of APC-conjugated goat anti-mouse IgG secondary antibody (Biolegend) and incubate at 4°C in the dark for 30 min. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0136] The experimental results are shown in Figures 6 and 7, indicating that the 21G6 antibody of the present invention can block the binding of CD40L to HCT-116 cells, and the blocking activity is similar to that of competing products Iscalimab antibody and Ravagalimab antibody; and the humanized h21G6 antibody can also block the binding of CD40L to HCT-116 cells, and the blocking activity is similar to that of the 21G6 antibody.

[0137] Example 6: 21G6 antibody inhibits CD40 reporter cell activation experiment

[0138] The reporter cell experiment was used to examine the characteristics of CD40 antibodies in affecting CD40 downstream activation signals. CD40L-mFc and CD40 antibodies were added to CD40 reporter cells. The strength of the luciferase signal after the addition of the antibody was used to determine the effect of the CD40 antibody on CD40 downstream activation signals.

[0139] Dilute the CD40 reporter cells to 2 × 10 cells / mL using complete DMEM medium. 5 / ml (purchased from Kebai Bio), added to a 96-well plate at a volume of 100μl / tube, 0.5μg / ml CD40L-mFc and gradient dilutions of the CD40 antibody to be tested, 21G6 antibody (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), competitor Iscalimab (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), Ravagalimab antibody (heavy chain sequence SEQ ID NO: 17, and light chain sequence SEQ ID NO: 18), and hIgG1LALA (purchased from Baiying Bio) were added and incubated at 37°C for 6h. Luciferase substrate was added and detected using a microplate reader.

[0140] The experimental results are shown in Figure 8, which show that the CD40 antibody 21G6 of the present invention can inhibit the activation of CD40 reporter cells, while the competing products Iscalimab and Ravagalimab antibodies cannot achieve the same effect. The competing monoclonal antibodies only have inhibitory function at certain concentrations, and the inhibitory function is weaker than that of 21G6.

[0141] Example 7: Experiment on the Effect of CD40 Antibody on B Cell Activation

[0142] Flow cytometry experiments were used to examine the effect of CD40 antibodies on the expression of CD86 by B cells. CD40 antibodies were added to human peripheral blood PBMCs, and the expression of CD86 after the addition of the antibody was used to determine whether the CD40 antibody induced B cell activation.

[0143] Human peripheral blood PBMCs were diluted to 2 × 10 6 / ml (purchased from Heyousheng Bio), added to a 96-well plate at a volume of 100μl / tube, to which the CD40 antibody 21G6 antibody (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), the competitor Iscalimab antibody (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), Ravagalimab antibody (heavy chain sequence SEQ ID NO: 17, and light chain sequence SEQ ID NO: 18), and hIgG1LALA (purchased from Baiying Bio) were added and incubated at 37°C for 24h. CD19 and CD86 fluorescent antibodies were labeled (purchased from Biolegend), and the cells were then resuspended in 200μl / tube PBS and detected by flow cytometry.

[0144] The experimental results are shown in Figure 9, which show that the CD40 antibody 21G6 of the present invention does not promote B cells to express CD86, while the competing antibodies Iscalimab and Ravagalimab promote B cells to upregulate the expression of CD86. The research and development objectives of 21G6 and competing antibodies are both CD40 antagonists, and competing antibodies cause B cell activation, which is very detrimental to antibody function.

[0145] Example 8: CD40 Antibody Inhibits CD40L-Mediated B Cell Activation

[0146] Flow cytometry was used to examine the effect of CD40 antibodies on CD40L-mediated B cell upregulation of CD80 and CD86. CD40L-mFc and CD40 antibodies were added to human peripheral blood PBMCs. The expression of CD80 and CD86 after antibody addition was used to determine whether CD40 antibodies inhibited CD40L-mediated B cell activation.

[0147] Human peripheral blood PBMCs were diluted to 2 × 10 6 / ml (purchased from Heyousheng Bio), added to a volume of 100μl / tube in a 96-well plate, to which the CD40 antibody 21G6 antibody (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), the competitor Iscalimab antibody (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), the Ravagalimab antibody (heavy chain sequence SEQ ID NO: 17, and light chain sequence SEQ ID NO: 18), hIgG1LALA (purchased from Baiying Bio) and 0.5μg / ml CD40L-mFc were added and incubated at 37°C for 24h. CD19, CD80 and CD86 fluorescent antibodies (purchased from Biolegend) were labeled, and the cells were then resuspended in 200μl / tube PBS and detected by flow cytometry.

[0148] The experimental results are shown in Figures 10 and 11. Figure 10 shows that the CD40 antibody 21G6 of the present invention inhibits the expression of CD80 by B cells, and the inhibitory effect is stronger than that of the competing antibodies Iscalimab and Ravagalimab, and the inhibitory effect of the competing antibodies is further weakened at high concentrations; Figure 11 shows that the CD40 antibody 21G6 of the present invention inhibits the expression of CD86 by B cells, and the inhibitory effect is stronger than that of the competing antibodies Iscalimab and Ravagalimab, and the inhibitory effect of the competing antibodies is further weakened at high concentrations.

[0149] Example 9: CD40 antibody inhibition of B cell proliferation experiment

[0150] Flow cytometry experiments were used to detect the inhibitory effect of CD40 antibodies on B cell proliferation. Human peripheral blood PBMCs were labeled with CFSE, and CD40 antibodies and CD40L-mFc were added and cultured for 120 hours. The strength of the CFSE signal was used to determine the effect of CD40 antibodies on B cell proliferation.

[0151] PBMC cells were labeled with 5 μM CFSE. After labeling, human peripheral blood PBMC were diluted to 2×10 5 / ml (purchased from Saili Bio), added to a volume of 100μl / tube in a 96-well plate, to which 0.5μg / ml CD40L-mFc, as well as 21G6 antibody (heavy chain sequence SEQ ID NO: 9, and light chain sequence SEQ ID NO: 10), competitor Iscalimab antibody (heavy chain sequence SEQ ID NO: 15, and light chain sequence SEQ ID NO: 16), and hIgG1LALA (purchased from Baiying Bio) were added and incubated at 37°C for 120h. Then, the results were detected by flow cytometry.

[0152] The results are shown in FIG12 , which show that the CD40 antibody 21G6 of the present invention can effectively inhibit CD40L-mediated B cell proliferation, while the competitor Iscalimab antibody has a significantly weaker inhibitory effect on B cell proliferation than the 21G6 antibody.

[0153] The above experimental results demonstrate that the antibodies obtained by the present invention can bind to human and monkey CD40, block the binding of CD40 and CD40L, and inhibit CD40 downstream activation signals, thereby suppressing B cell proliferation. Therefore, the antibodies can interfere with signaling between immune cells, modulating immune activity and inflammatory responses. These functions give them great potential for treating or preventing CD40-mediated diseases, providing new options and strategies for disease treatment and immune regulation.

[0154] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

[0155] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that: include: The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences that are at least 80% identical to SEQ ID NOs: 1, 2 and 3; and The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences that are at least 80% identical to SEQ ID NOs: 4, 5 and 6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: include: The heavy chain variable region CDR1 sequence as shown in SEQ ID NO:1, the heavy chain variable region CDR2 as shown in SEQ ID NO:2, the heavy chain variable region CDR3 as shown in SEQ ID NO:3, the light chain variable region CDR1 as shown in SEQ ID NO:4, the light chain variable region CDR2 as shown in SEQ ID NO:5, and the light chain variable region CDR3 as shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: include: The heavy chain variable region shown in SEQ ID NO:7 or SEQ ID NO:11; and / or The light chain variable region is shown in SEQ ID NO:8 or SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: include: 1) a heavy chain variable region as shown in SEQ ID NO: 7, and a light chain variable region as shown in SEQ ID NO: 8; or 2) the heavy chain variable region shown in SEQ ID NO:11, and the light chain variable region shown in SEQ ID NO:

12.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof contains a heavy chain framework region sequence and a light chain framework region sequence, and at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The light chain constant region and the heavy chain constant region are both derived from mouse IgG antibody or its mutant or human IgG antibody or its mutant.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The light chain constant region and the heavy chain constant region are both derived from mouse IgG1 antibody, IgG2a antibody, IgG4 antibody or mutants thereof or human IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody or mutants thereof.

9. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 9 and 13 and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 10 and 14.

10. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain with an amino acid sequence as shown in SEQ ID NO:9 and a light chain with an amino acid sequence as shown in SEQ ID NO:

10.

11. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain with an amino acid sequence as shown in SEQ ID NO:13 and a light chain with an amino acid sequence as shown in SEQ ID NO:

14.

12. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof includes at least one of a monoclonal antibody, a polyclonal antibody, a multimeric antibody and a CDR-grafted antibody.

13. The antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The monoclonal antibody includes at least one of a full-length antibody, a Fv antibody, a single-chain antibody, a Fab antibody, a single-domain antibody and a minimum recognition unit.

14. The antibody or antigen-binding fragment thereof according to claim 13, characterized in that ,, the antibody or its antigen-binding fragment can bind to the amino acid sequence shown in SEQ ID NO:

20.

15. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. An expression vector, characterized in that: Carrying the nucleic acid molecule of claim 15.

17. A recombinant cell, characterized in that Carrying the nucleic acid molecule of claim 15, the expression vector of claim 16, or being capable of expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

18. The recombinant cell according to claim 17, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 16 into a host cell.

19. The recombinant cell according to claim 18, characterized in that The recombinant cell is a eukaryotic cell; Alternatively, the recombinant cell is a mammalian cell.

20. An immunoconjugate, characterized in that include: The antibody or antigen-binding fragment thereof and the therapeutic agent according to any one of claims 1 to 14; the antibody or antigen-binding fragment thereof is coupled to the therapeutic agent; The therapeutic agent is capable of preventing and / or treating CD40-mediated diseases; The CD40-mediated disease is selected from transplant rejection, autoimmune disease, infectious disease and cancer.

21. A composition, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 19, or the immunoconjugate according to claim 20.

22. A drug, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 19, the immunoconjugate according to claim 20, or the composition according to claim 21; Optionally, the medicament further comprises a pharmaceutically acceptable excipient.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 19, the immunoconjugate according to claim 20, the composition according to claim 21 or the medicament according to claim 22, comprising: For preventing and / or treating CD40-mediated diseases; For preparing a drug for preventing and / or treating a CD40-mediated disease; Optionally, the CD40-mediated disease is selected from transplant rejection, autoimmune disease, infectious disease and cancer.

24. A kit, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14; The nucleic acid molecule according to claim 15; The expression vector of claim 16; or The recombinant cell according to any one of claims 17 to 19.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, or the recombinant cell according to any one of claims 17 to 19, comprising: Used to detect CD40; Used for preparing a kit for detecting CD40.

26. A method for preventing and / or treating a CD40-mediated disease, characterized in that: include: Administering a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 19, the immunoconjugate according to claim 20, the composition according to claim 21, or the drug according to claim 22 to a subject.

27. A method for detecting CD40, characterized in that: include: Using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, or the kit according to claim 24 to detect the sample to be detected; Optionally, the antibody or antigen-binding fragment thereof forms an immune complex with CD40 in the sample to be detected; The presence of the immune complex is detected, and based on the presence of the immune complex, it is determined whether the sample to be detected contains CD40.

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