CN115947854A

CN115947854A - Anti-human CD40 protein monoclonal antibody, preparation method and application thereof

Info

Publication number: CN115947854A
Application number: CN202211730821.1A
Authority: CN
Inventors: 娄阳; 黄海斌; 程昊; 陈嘉琛; 彭晓旺; 岳涵
Original assignee: Yourui Seth Wuhan Biotechnology Co ltd
Current assignee: Yourui Seth Wuhan Biotechnology Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-04-11
Anticipated expiration: 2042-12-30
Also published as: CN115947854B

Abstract

The application discloses an anti-human CD40 protein monoclonal antibody, a preparation method and application thereof. The monoclonal antibody against human CD40 protein is prepared by using commercial human CD40 protein as immunogen and based on monoclonal antibody development technology of single B lymphocyte screening and culture, and has strong affinity with human CD40 protein through detection, and the affinity reaches 7.2 multiplied by 10 ^‑10 And M. The rabbit monoclonal antibody for resisting human CD40 protein provided by the application can be used for developing a flow type antibody for detecting the human CD40 protein and a related kit. The rabbit monoclonal antibody of anti-human CD40 protein provided by the application can be used as a human CD40 protein agonist to activate B cells, and the expression level of CD86 and MHC-II of the B cells activated by the antibody is 3-9 times that of the B cells activated by CD40L.

Description

Anti-human CD40 protein monoclonal antibody, preparation method and application thereof

Technical Field

The application relates to the technical field of biology, in particular to an anti-human CD40 protein monoclonal antibody, a preparation method and application thereof.

Background

The human CD40 protein is an I-type transmembrane glycoprotein and consists of an N-terminal signal peptide (20 aa), an extracellular region (193 aa), a transmembrane region (22 aa) and a cytoplasmic region (62 aa). The CD40 protein is widely expressed on the surfaces of immune cells, comprises B cells, T cells, monocytes, macrophages and dendritic cells, is an important functional protein on the cell surface and is involved in the activation of various immune cells. And also on the surface of some non-immune cells such as epithelial cells. In addition, CD40 is also expressed on leukemic B cells, and also on the surface of certain cancer cells, such as epithelial cancer cells.

The ligand of human CD40 protein, CD40L (also known as CD 154), is expressed predominantly on the surface of activated CD4+ T lymphocytes, providing a costimulatory signal necessary for B lymphocyte activation. After the recipient cells are activated, the CD40L expressed on the cell surface is then excised and flowed into the blood stream, resulting in a still biologically active, soluble, trimeric fragment, also known as soluble CD40L.

The CD40-CD40L costimulatory pathway is important for thymus-dependent humoral immunity, and activation, differentiation, and immunological memory of B cells are not well separated from this costimulatory pathway. It is also important to activate Antigen Presenting Cells (APC), and is the key to T cell activation.

Other immune cells expressing the CD40 protein are activated to secrete various cytokines to perform different immune functions. For example, macrophages secrete TNF- α, dendritic cells and macrophages secrete interleukin 12, and these cytokines are important for anti-tumor immunity mediated by T cells and NK cells.

It is because of the important role played by the CD40 protein in the immune system that CD40 protein is one of the most potential targets in immune cell co-stimulatory molecules. Pharmaceutical enterprises at home and abroad have development of CD40 agonist antibodies, 7 in second clinical stage and 6 in first clinical stage at present, the main indications are melanoma, non-small cell lung cancer, pancreatic cancer and the like, and the administration mode is independent administration or combined administration with other treatment modes.

Most of the CD40 agonist antibodies in the prior art are derived from murine monoclonal antibodies, and the affinity is relatively weak. Therefore, there is a need in the art to develop a new high affinity, high activating monoclonal antibody that offers a new choice for CD40 target-based therapeutic antibodies.

Disclosure of Invention

The present application provides a high affinity rabbit monoclonal antibody against human CD40 protein to address one of the above technical problems to some extent. The rabbit monoclonal antibody can identify human CD40 protein, and the affinity constant of the antibody combined with the human CD40 protein expressed by recombination is 7.2 multiplied by 10 ^-10 And M. The antibody can be combined with CD40 protein expressed on the surface of human B cells and activate the B cells, and the activation effect is better than that of CD40L under the same experimental conditions. The immunogen of the anti-human CD40 protein rabbit monoclonal antibody is commercialized human CD40 protein; the preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture. The monoclonal antibody can be specifically combined with human CD40 protein, so that the monoclonal antibody can be applied to the preparation of a kit for detecting the human CD40 protein so as to achieve the clinical application value of human CD40 protein detection or related disease diagnosis, and the application provides the following technical scheme for realizing the purpose:

in a first aspect, the present application provides an antibody that specifically binds to a human CD40 protein, the antibody comprising a light chain variable region (VL) defined according to the numbering system of Kabat, the light chain variable region (VL) having three Complementarity Determining Regions (CDRs) of: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO. 5, or has a sequence with 1-3 amino acid substitutions, deletions or additions compared with the same; VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO. 6, or has a sequence with 1-3 amino acid substitutions, deletions or additions compared with the amino acid sequence; and a VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having 1 to 3 amino acid substitutions, deletions or additions thereto;

and/or

Comprising a heavy chain variable region (VH) defined according to the Kabat numbering system, the heavy chain variable region (VH) having three Complementarity Determining Regions (CDRs) of: a VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 8 or a sequence having 1-3 amino acid substitutions, deletions or additions compared with the same; a VH CDR2 consisting of an amino acid sequence shown by SEQ ID NO. 9 or a sequence having 1-3 amino acid substitutions, deletions or additions compared with the VH CDR 2; and a VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10 or a sequence having 1-3 amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

In a second aspect, the present application provides an antibody that specifically binds to human CD40 protein, said antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH); the light chain variable region (VL) consists of an amino acid sequence shown in SEQ ID NO: 3; the heavy chain variable region (VH) consists of an amino acid sequence shown in SEQ ID NO: 4.

In a third aspect, the present application provides an antibody comprising a light chain consisting of the amino acid sequence shown in SEQ ID NO. 1 and a heavy chain; the heavy chain consists of an amino acid sequence shown as SEQ ID NO. 2.

In a fourth aspect, the present application provides a conjugate comprising the antibody of any one of the first to third aspects, and a detectable label linked to the antibody.

In a fifth aspect, the present application provides a kit for detecting human CD40 protein, the kit comprising the antibody of any one of the first to third aspects or the conjugate of the fourth aspect.

In a sixth aspect, the present application discloses a human CD40 protein agonist comprising the antibody of any one of the first to third aspects, which protein agonist activates B cells expressing a costimulatory signaling molecule (CD 86) and an antigen presenting molecule (MHC-ii).

In a seventh aspect, the application discloses the use of the antibody of any one of the first to third aspects or the kit of the fifth aspect for detecting human CD40 protein.

Compared with the prior art, the application has the advantages and positive effects that at least:

the application adopts commercial human CD40 protein as immunogen, and prepares the anti-human CD40 protein rabbit monoclonal antibody based on the monoclonal antibody development technology of single B lymphocyte screening and culture, and the detection shows that the rabbit monoclonal antibody has stronger affinity with the human CD40 protein and reaches 7.2 multiplied by 10 ^-10 M。

The rabbit monoclonal antibody for resisting human CD40 protein provided by the application can be used for developing a flow type antibody for detecting human CD40 protein and a related kit.

The rabbit monoclonal antibody of anti-human CD40 protein provided by the application can be used as a human CD40 protein agonist to activate B cells, and the expression level of CD86 and MHC-II of the B cells activated by the antibody is 3-9 times that of the B cells activated by CD40L.

Drawings

FIG. 1 shows the results of affinity assays for rabbit monoclonal antibodies to human CD40 protein, provided in the examples herein.

FIG. 2 is an ELISA binding curve of human CD40 protein rabbit monoclonal antibody and human CD40 protein provided in the examples of the present application.

FIG. 3 is a graph of flow binding peaks of human CD40 protein rabbit monoclonal antibodies and daudi cells (human B lymphoblastoid cell line) provided in the examples herein.

FIG. 4 is a graph showing the expression of CD86 and MHC-II on the surface of human B cells after activation with rabbit monoclonal antibody to human CD40 protein, as provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually specified in detail in this application are conventional and commercially available; methods which are not specified in detail are all customary experimental methods and are known from the prior art.

Interpretation of terms

In the present application, the term "antibody" is to be interpreted in the broadest sense, having a variety of antibody structures, including, but not limited to, Y-antibodies, so-called full-length antibodies, antigen-binding portions of Y-antibodies, and genetic or chemical modifications thereof. By "antigen binding portion" is meant one or more portions or fragments of a Y-type antibody that retain the ability of the antibody to specifically bind to human CD40 protein.

In the present application, the term "monoclonal antibody" (mAb) includes a highly homogeneous population of antibodies having substantially identical antigenic determinants. That is, within the population, the individual antibodies are essentially identical, except for a small number of mutations that may occur naturally. Monoclonal antibodies can exhibit a single binding specificity and affinity for a particular epitope on an antigen. Each monoclonal antibody may be directed against the same or substantially the same epitope on the antigen, as compared to polyclonal antibodies that typically comprise antibodies directed against different epitopes. The modifier "monoclonal" indicates that the property of the antibody is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. The antibody can be prepared by monoclonal antibody development techniques based on single B lymphocyte screening and culture.

In the present application, the term "rabbit antibody" or "anti-human CD40 protein monoclonal antibody" or the modifier "rabbit" in similar terms means that the Complementarity Determining Regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. In one embodiment, a rabbit monoclonal antibody to human CD40 protein may comprise the CDRs and Framework Regions (FRs) of the antibody from rabbit immunoglobulin sequences of rabbit origin. In one embodiment, a rabbit antibody or rabbit monoclonal antibody against human CD40 protein may comprise CDRs from an antibody from a rabbit immunoglobulin sequence.

In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four heterologous polypeptide chains, wherein the two chains with the larger molecular weight are referred to as heavy chains (H) and the two chains with the smaller molecular weight are referred to as Light chains (L). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, igD, igG, igA, and IgE, respectively. The approximately 110 amino acid sequences of the heavy and light chains near the N-terminus vary widely, with the amino acid sequences of the other portions being relatively constant. Thus, the regions of the light and heavy chains which vary widely in amino acid sequence near the N-terminus are referred to as variable regions (V) and account for 1/4 and 1/2 of the heavy and light chains, respectively; the regions where the amino acid sequence near the C-terminus is relatively stable are called constant regions (C), and account for 3/4 and 1/2 of the heavy and light chains, respectively.

The V regions of the heavy and light chains are referred to as VH and VL, respectively. Each of VH and VL contains a highly variable region of 3 amino acid composition and arrangement, called hypervariable region (HVR) or Complementarity Determining Region (CDR), including HVRl (CDRl), HVR2 (CDR 2), and HVR3 (CDR 3), with the HVR3 (CDR 3) varying to a greater extent. The 3 CDRs of VH and VL together make up the antigen-binding site of the antibody, which determines the specificity of the antibody, and is the site where the antibody recognizes and binds antigen. In the V region, the amino acid composition and arrangement sequence of the region outside the CDR are relatively conserved, and are called Framework Regions (FRs). VH or VL each have four framework regions, designated FR1, FR2, FR3 and FR4, respectively.

The C regions of the heavy and light chains are referred to as CH and CL, respectively. The CL for different classes (kappa or lambda) of Ig is essentially identical in length, but the CH for different classes of Ig is different in length, e.g., igG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CHl, CH2, CH3 and CH4.

In this application, the residues of the term "framework region" or "framework region" refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

In the present application, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin.

In the present application, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit, then the molecules are identical at that position. For example, if 8 of 10 positions of two sequences match, then the two sequences have 80% identity.

In the present application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of the protein/polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar (of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue.

In the present application, the term "agonist" is a class of compounds that bind to human CD40 protein and activate the immune system, the activation of which is primarily manifested in the early stages of the immune response. The human CD40 protein described herein, because of its critical role in the antigen presentation process, plays its first role in the immune response. The monoclonal antibody provided by the application, as an agonist of human CD40 protein, specifically binds to the human CD40 protein, activates the expression of costimulatory molecules such as MHC-II and CD86 in B cells, and induces Ig class switching in the B cells. Activated B cells migrate to lymphoid organs where they present antigens to T cells, and CD40 activated DC and B cells support the immune response by releasing immunostimulatory cytokines and chemokines such as IL-6, IL-12p70, IFN γ, CXCL10, TNF- α, and the like.

Antibodies

An antibody disclosed in the examples of the present application, which specifically binds to a human CD40 protein, the antibody comprising a light chain variable region (VL) defined according to the Kabat numbering system, the light chain variable region (VL) having three Complementarity Determining Regions (CDRs) of: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO. 5, or has a sequence with 1-3 amino acid substitutions, deletions or additions compared with the same; VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO. 6, or has a sequence with 1-3 amino acid substitutions, deletions or additions compared with the amino acid sequence; and a VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having 1 to 3 amino acid substitutions, deletions or additions thereto;

and/or

Comprising a heavy chain variable region (VH) as defined according to the numbering system of Kabat, the heavy chain variable region (VH) having three Complementarity Determining Regions (CDRs) of: VH CDR1, it is composed of amino acid sequence shown in SEQ ID NO. 8, or has sequence of 1-3 amino acid substitution, deletion or addition compared with it; a VH CDR2 consisting of an amino acid sequence shown by SEQ ID NO. 9 or a sequence having 1-3 amino acid substitutions, deletions or additions compared with the VH CDR 2; and a VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10 or a sequence having 1-3 amino acid substitutions, deletions or additions compared thereto; preferably, the substitution is a conservative substitution.

In certain embodiments, the light chain variable region (VL) of the antibody consists of the amino acid sequence set forth in SEQ ID NO. 3; the heavy chain variable region (VH) of the antibody consists of an amino acid sequence shown in SEQ ID NO. 4.

In certain embodiments, the light chain of the antibody consists of the amino acid sequence set forth in SEQ ID NO. 1; the heavy chain consists of an amino acid sequence shown as SEQ ID NO. 2.

In certain embodiments, the anti-human CD40 protein antibody may have a Y-type molecular structure. In one embodiment, the anti-human CD40 protein antibody may include a pair of heavy chains and a pair of light chains. The heavy chain may comprise a heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies typically include five types of heavy chains: gamma, delta, alpha, mu and epsilon, and the corresponding constituent antibodies are called IgG, igD, igA, igM and IgE antibodies. The light chain may be a polypeptide subunit that is smaller relative to the heavy chain. The light chain may comprise a light chain variable region and a light chain constant region. VL is typically the N-terminal portion of the light chain and exhibits greater variability in amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, both the heavy chain variable region VH and the light chain variable region VL can be used to recognize and bind to human CD40 protein. In one embodiment, the light chain constant region of the antibody is a kappa chain and the heavy chain constant region of the antibody is of the IgG1 type.

Preparation of Rabbit-derived monoclonal antibody

The present application discloses methods for preparing the above antibodies, and the monoclonal antibodies of the present application can be prepared by various methods known in the art, such as by genetic engineering recombination techniques; DNA molecules encoding the heavy and light chain genes of the antibody of the present application are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into an expression vector, and then the host cell is transfected, and the transfected host cell is cultured under specific conditions and the antibody of the present application is expressed.

The immunogen used for preparing the human CD40 rabbit monoclonal antibody is from an purchased commercial human CD40 protein (Cat of Cassia angustifolia: 10774-H02H); the preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture. In some embodiments, the method of making comprises: immunizing a New Zealand white rabbit by taking human CD40 protein as immunogen; b lymphocytes are sorted from splenocytes of the rat immunization and cultured; extracting RNA in B lymphocyte, and performing reverse transcription to obtain cDNA; carrying out PCR amplification on the cDNA to obtain a naturally paired rabbit monoclonal antibody; respectively loading the heavy chain variable region (VH) gene and the light chain variable region (VL) gene of the naturally-paired rabbit monoclonal antibody onto an expression vector, transfecting the vector with a host cell, culturing the host cell, separating from the culture solution of the host cell, and purifying to obtain the monoclonal antibody.

Specifically, the implementation process of preparing the rabbit-derived monoclonal antibody comprises the following steps:

1. animal immunization

The commercial human CD40 protein is used as immunogen to immunize New Zealand white rabbits, each white rabbit is immunized with 200 mug, the immunogen is mixed with an equal amount of complete Freund adjuvant to prepare an emulsifier before first immunization, and the emulsifier is injected into the abdomen and back of the rabbit at multiple subcutaneous points. After the first immunization, 150 mu g of immunogen and an equivalent amount of incomplete Freund's adjuvant are taken at intervals of 3 weeks and mixed to prepare an emulsifier, and the emulsifier is injected into the abdomen and the back of the rabbit at multiple subcutaneous points to strengthen the immunization twice. And (3) collecting a rabbit serum sample after three times of immunization, measuring the titer of the rabbit serum sample aiming at the human CD40 protein by using an ELISA method, purifying the serum after the last immunization into an antibody, detecting an endogenous sample by using WB, IF and IHC, taking the rabbit with high serum titer and best endogenous detection, performing subcutaneous multi-point injection for boosting immunization once by using 200 mu g of immunogen, and taking the spleen after three days.

2. Spleen cells were isolated.

3. B lymphocyte sorting

See the patent "201910125091.4 method for efficient isolation of single antigen-specific B lymphocytes from spleen cells".

4. Cloning of genes encoding rabbit monoclonal antibodies

The cultured B cell supernatants were identified as positive clones by antigen-coated ELISA. The positive cloned cells are collected, lysed, and the RNA is extracted and reverse transcribed into cDNA. The naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the corresponding positively cloned cDNA by PCR and sequenced to determine the sequence.

5. Production and purification of monoclonal antibodies

In order to obtain a plurality of rabbit monoclonal antibodies for identifying human CD40 protein, heavy chain genes and light chain genes of the rabbit monoclonal antibodies are respectively loaded on expression vectors, and plasmids are transfected into 293F cells; the recombinant antibody recognizing the human CD40 protein contained in the culture supernatant is obtained after transfection for 72-96 hours. Purifying recombinant rabbit monoclonal antibody for recognizing human CD40 protein from the supernatant of the transfected culture medium by using protein A affinity gel resin, subpackaging after the antibody is qualified, and storing at low temperature of-20 ℃ for later use.

6. Results

In the embodiment, the monoclonal antibody capable of identifying the human CD40 protein is obtained by screening, wherein the light chain of the monoclonal antibody consists of an amino acid sequence shown in SEQ ID NO. 1, and the heavy chain consists of an amino acid sequence shown in SEQ ID NO. 2; the light chain variable region (VL) of the monoclonal antibody consists of an amino acid sequence shown by SEQ ID NO. 3; the heavy chain variable region (VH) consists of an amino acid sequence shown in SEQ ID NO: 4; the light chain variable region (VL) has three Complementarity Determining Regions (CDRs) which are: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO. 5; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7; the heavy chain variable region (VH) has three Complementarity Determining Regions (CDRs) which are: VH CDR1, which consists of an amino acid sequence shown in SEQ ID NO. 8; VH CDR2 consisting of an amino acid sequence shown as SEQ ID NO. 9; and a VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10.

Affinity detection of rabbit monoclonal antibodies to human CD40 protein

Analysis of biomolecular interactions Using Biacore 3000 from GEThe affinity of the anti-human CD40 protein rabbit monoclonal antibody is accurately measured; wherein the selected antibody is used at a concentration of 37nM and immobilized on a CM5 chip; then recombinant human CD40 protein with three concentrations of 37.5nM, 75nM and 150nM is used for binding to obtain an affinity curve; finally, through curve fitting and calculation, the affinity curve is shown in FIG. 1, and the fitting result is shown in Table 1. The affinity of the obtained rabbit monoclonal antibody against human CD40 protein is 7.2X 10 ^-10 M。

TABLE 1

Note: k _off (dissociation rate constant) represents the dissociation rate constant between molecules, and represents the speed of dissociation between molecules; k _on (association rate constant) is an intermolecular association rate constant representing how fast or slow intermolecular association is

Application of rabbit monoclonal antibody

1. Use of rabbit monoclonal antibodies provided herein for ELISA binding Activity detection

The detection method comprises the following steps:

coating rabbit monoclonal antibody of anti-human CD40 protein and CD40L protein with carbonate buffer (pH9.4, 0.05M), and incubating at 4 deg.C overnight; washing the plate with a washing solution; blocking with phosphate buffer (pH7.2, 0.05M) containing 5% bovine serum albumin, 0.05% Tween-20; after the biotinylation recombinant human CD40 protein is diluted by phosphate buffer solution with the concentration of 0.05 percent Tween-20 in a gradient manner, the diluted solution is added into an enzyme label plate, the incubation is carried out for 1 hour under the normal temperature condition, and then the plate is washed by washing liquid; adding avidin-labeled Horse Radish Peroxidase (HRP) diluted with phosphate buffer containing 1% bovine serum albumin, 0.05% Tween-20, incubating at room temperature for 1 hour, and washing the plate with washing solution; adding TMB color development liquid, developing for 10 minutes at normal temperature, adding oxalic acid to stop developing, and then respectively measuring light absorption values at 450nm and 630nm, wherein OD450 minus OD630 is the corrected light absorption value; the Log of the concentration of human CD40 protein (Log 10) is plotted on the abscissa and the corrected value of the absorbance (OD 450) is plotted on the ordinate.

And (3) detection results: as shown in FIG. 2, the binding activity of the anti-CD40 rabbit monoclonal antibody and the CD40 protein was substantially the same as that of CD40L.

2. Rabbit monoclonal antibody and positive cell binding assay

In the embodiment of the application, the detection is carried out by adopting a flow type binding activity detection method of an antibody, and the specific detection method comprises the following steps:

take 1.5X 10 ⁶ Each daudi cell, divided into 3 portions, was washed once with PBS +0.1% bsa buffer; 100. Mu.l of PBS +0.1% BSA, 100. Mu.l of Isotype mAb (10. Mu.g/ml), anti-CD40 mAb (10. Mu.g/ml) were added, respectively, and incubated at 4 ℃ for 30 minutes; 400g, centrifuged for 3 minutes, the supernatant was discarded, 100. Mu.l of PBS +0.1% of BSA, 100. Mu. l G, respectively&R _ IgG _ FITC (1 diluted 500), 100 μ l G&R _ IgG _ FITC (1 diluted 500) resuspended cells, incubated at 4 ℃ for 30 min; 400g, centrifuged for 3 minutes, the supernatant discarded, washed twice with 100. Mu.l PBS +0.1% BSA buffer; detecting a fluorescent signal on the cell surface using an Agilent NovoCyte Advanteon analysis type flow cytometer; the Novoexpress software was used for mapping.

The results are shown in FIG. 3, which shows that there are 2 log transitions in the flow-binding signals of rabbit monoclonal antibodies against human CD40 protein and positive cells.

3. Application of rabbit monoclonal antibody as human CD40 protein agonist

In the embodiment of the present application, the B cell activation activity of the rabbit monoclonal antibody provided by the present application is detected by using a flow-type binding activity detection method of the antibody, and the specific detection method is as follows:

thawing human Peripheral Blood Mononuclear Cells (PBMCs) and culturing for 4 hours; purifying the B cells by using a Stemcell Pan-B cell kit; purified B cells were cultured at 2.5X 10 ⁶ One cell/well was packed into a 96-well flat bottom plate (50. Mu.g/ml polymyxin B in 0.2ml B cell culture medium); crosslinking rabbit anti-human CD40 monoclonal antibody or control rabbit anti-mAb and goat anti-rabbit Fc at RT for 30 min; adding cross-linked rabbit anti-human CD40 monoclonal antibody or control antibody, or CD40L (Kactus, 5. Mu.g/ml or 1. Mu.g/ml) to human Pan-B cells, and culturing for 18 hours; pan-B cells were collected using Zombie NIR (1; pan-B cells were stained with anti-human CD19, CD86 and MHC-II antibodies and fixed; detecting a fluorescent signal on the cell surface using an Agilent NovoCyte Advanteon analysis type flow cytometer; the Novoexpress software was used for mapping.

Fig. 4 is a graph of fluorescence signals detected on the cell surface by a flow cytometer, and specific detection results are shown in table 2, in which the B cell activation effect of the anti-human CD40 protein rabbit monoclonal antibody is 3 to 9 times that of CD40L.

TABLE 2

Experiment grouping	CD86 +/MHC-II + ratio
		Blank	0.44％
Irrelevant control antibody (5. Mu.g/ml)	0.58％
		Anti-human CD40 monoclonal antibody (5. Mu.g/ml)	5.18％
Anti-human CD40 monoclonal antibody (1. Mu.g/ml)	5.34％
		CD40L(5μg/ml)	1.37％
CD40L(1μg/ml)	0.62％

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Claims

1. An antibody that specifically binds a human CD40 protein, the antibody comprising a light chain variable region (VL) as defined according to the Kabat numbering system, the light chain variable region (VL) having three Complementarity Determining Regions (CDRs) of: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO. 5, or has a sequence with 1-3 amino acid substitutions, deletions or additions compared with the same; VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO. 6, or has a sequence with 1-3 amino acid substitutions, deletions or additions compared with the amino acid sequence; and a VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having 1 to 3 amino acid substitutions, deletions or additions thereto;

and/or

Comprising a heavy chain variable region (VH) defined according to the Kabat numbering system, the heavy chain variable region (VH) having three Complementarity Determining Regions (CDRs) of: a VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 8 or a sequence having 1-3 amino acid substitutions, deletions or additions compared with the same; VH CDR2, it is made up of amino acid sequence shown in SEQ ID NO. 9, or have 1-3 amino acid substitution, deletion or sequence added to compare with it; and a VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10 or a sequence having 1-3 amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

2. An antibody that specifically binds to human CD40 protein, said antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH); the light chain variable region (VL) consists of an amino acid sequence shown in SEQ ID NO: 3; the heavy chain variable region (VH) consists of an amino acid sequence shown in SEQ ID NO. 4.

3. An antibody comprising a light chain consisting of the amino acid sequence shown in SEQ ID NO. 1 and a heavy chain; the heavy chain consists of an amino acid sequence shown in SEQ ID NO. 2.

4. A conjugate comprising the antibody of any one of claims 1 to 3, and a detectable label linked to the antibody.

5. A kit for detecting human CD40 protein, the kit comprising the antibody of any one of claims 1 to 3 or the conjugate of claim 4.

6. A human CD40 protein agonist comprising the antibody of any one of claims 1 to 3, which protein agonist activates B cells to express CD86 and MHC-ii.

7. Use of the antibody of any one of claims 1 to 3 or the kit of claim 5 for the detection of human CD40 protein.