CN115947854B

CN115947854B - Anti-human CD40 protein monoclonal antibody, preparation method and application thereof

Info

Publication number: CN115947854B
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: 2023-06-30
Anticipated expiration: 2042-12-30
Also published as: CN115947854A

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 taking commercial human CD40 protein as immunogen and based on monoclonal antibody development technology of single B lymphocyte screening and culture, and has stronger affinity with human CD40 protein to reach 7.2X10 after detection ^‑10 M. The rabbit monoclonal antibodies against human CD40 protein provided herein can be used to develop flow antibodies and related kits for detecting human CD40 protein. The rabbit monoclonal antibody of the anti-human CD40 protein can be used as a human CD40 protein agonist for activating B cells, and the expression level of the 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 field of biotechnology, in particular to an anti-human CD40 protein monoclonal antibody, a preparation method and application thereof.

Background

The human CD40 protein is a type I 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). CD40 protein is widely expressed on the surface of immune cells, including B cells, T cells, monocytes, macrophages and dendritic cells, and is an important functional protein on the surface of cells, involved in the activation of various immune cells. And also expressed on the surface of a portion of non-immune cells such as epithelial cells. In addition, CD40 is also expressed on leukemia B cells and is also expressed on the surface of certain cancer cells, such as epithelial cancer cells.

The ligand CD40L (also known as CD 154) of the human CD40 protein, CD40L, is predominantly expressed on the surface of activated cd4+ T lymphocytes, providing the co-stimulatory signals necessary for B lymphocyte activation. After the recipient cells are activated, CD40L expressed on the cell surface is then excised and flowed into the blood stream to produce a biologically active, soluble, trimeric fragment, also known as soluble CD40L.

The CD40-CD40L co-stimulatory pathway is important for thymus-dependent humoral immunity, and B cell activation, differentiation and immunological memory are all independent of the co-stimulatory pathway. And is also very important for activating Antigen Presenting Cells (APC), which is critical for T cell activation.

Other immune cells expressing CD40 protein can secrete various cytokines after being activated, and play different immune functions. For example, macrophages secrete TNF- α, dendritic cells and macrophages secrete interleukin 12, which are important for T cell and NK cell mediated antitumor immunity.

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

CD40 agonist antibodies in the prior art are mostly derived from murine monoclonal antibodies, with relatively weak affinity. Thus, there is a need in the art to develop a new high affinity, high activation activity monoclonal antibody that provides a new choice for therapeutic antibodies based on CD40 targets.

Disclosure of Invention

The present application provides a high affinity rabbit monoclonal antibody against human CD40 protein to solve one of the above technical problems to some extent. The rabbit monoclonal antibody can recognize human CD40 protein, and the affinity constant of the binding of the antibody and the recombinant expressed human CD40 protein is 7.2 multiplied by 10 ^-10 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 commercial human CD40 protein; the preparation method is 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 preparing a kit for detecting human CD40 protein so as to achieve clinical application value of human CD40 protein detection or related disease diagnosis, and the application provides the following technical scheme for achieving the purpose:

in a first aspect, the present application provides an antibody that specifically binds human CD40 protein, said antibody comprising a light chain variable region (VL) defined according to the Kabat numbering system, said light chain variable region (VL) having three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 5 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with it; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence;

and/or

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

In a second aspect, the present application provides an antibody that specifically binds human CD40 protein, the 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 the amino acid sequence shown in SEQ ID NO. 4.

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

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

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

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

In a seventh aspect, the application discloses the use of an antibody according to any one of the first to third aspects or a kit according to 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, prepares the anti-human CD40 protein rabbit monoclonal antibody based on monoclonal antibody development technology of single B lymphocyte screening and culturing, has stronger affinity with human CD40 protein by detection, reaches 7.2X10 ^-10 M。

The rabbit monoclonal antibodies against human CD40 protein provided herein can be used to develop flow antibodies and related kits for detecting human CD40 protein.

The rabbit monoclonal antibody of the anti-human CD40 protein can be used as a human CD40 protein agonist for activating B cells, and the expression level of the 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 human CD40 protein rabbit monoclonal antibodies provided in the examples herein.

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

FIG. 3 is a graph showing the flow-through binding peaks of human CD40 protein rabbit monoclonal antibody and daudi cells (human B lymphoblastic cell line) provided in the examples of this application.

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

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Interpretation of the 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-type antibodies, so-called full length antibodies, antigen binding portions of Y-type antibodies, and genetic or chemical modifications thereof. Wherein an "antigen binding portion" refers to one or more portions or fragments of a Y-type antibody that retains the ability of the antibody to specifically bind to human CD40 protein.

In this application, the term "monoclonal antibody" (mAb) includes a population of highly homogeneous antibodies having substantially identical antigenic determinants. That is, the individual antibodies are essentially identical in the population, except for the small number of mutations that may occur naturally. Monoclonal antibodies may 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 a polyclonal antibody which typically comprises antibodies directed against different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring preparation by any particular method. The antibodies can be made by monoclonal antibody development techniques based on single B lymphocyte selection and culture.

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

In this application, the term "antibody" refers to an immunoglobulin molecule consisting of four heterologous polypeptide chains, of which 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 isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The heavy and light chains vary widely in about 110 amino acid sequences near the N-terminus, with the other portions of the amino acid sequences being relatively constant. Thus, the regions of the light and heavy chains that vary greatly near the N-terminal amino acid sequence are referred to as variable regions (V) and account for 1/4 and 1/2 of the heavy and light chains, respectively; the region of relatively stable amino acid sequence near the C-terminus is called constant region (C) and occupies 3/4 and 1/2 of the heavy and light chains, respectively.

The V chains of the heavy and light chains are referred to as VH and VL, respectively. Each of VH and VL contains a region of highly variable 3 amino acid composition and arrangement sequence, termed hypervariable region (hypervariable region, HVR) or complementarity determining region (complementarity determining region, CDR), including HVRl (CDRl), HVR2 (CDR 2) and HVR3 (CDR 3), wherein HVR3 (CDR 3) varies to a greater extent. The 3 CDRs of VH and VL together form the antigen-binding site of the antibody, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. In the V region, the amino acid composition and arrangement order of the regions outside the CDRs are relatively conserved, called Framework Regions (FR). VH or VL has four framework regions, denoted FR1, FR2, FR3 and FR4, respectively.

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

In the present application, the term "framework region" or "framework region" residues 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 an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In this 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 tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of 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 such that 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 Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (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 also 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 sequences being compared is occupied by the same base or amino acid monomer subunit, then the molecules are identical at that position. For example, if 8 out 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 a protein/polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., 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 effects of the immune system, which activation is mainly reflected in the early stages of the immune response. The human CD40 protein plays a key role in antigen presentation and plays a first role in immune response. The monoclonal antibody provided by the application is used as an 'agonist' of human CD40 protein, specifically binds to the human CD40 protein, activates the expression of costimulatory molecules such as MHC-II, CD86 and the like in B cells, and induces Ig class conversion in the B cells. Activated B cells migrate to lymphoid organs where they present antigens to T cells, and CD40 activated DCs and B cells support immune responses by releasing immunostimulatory cytokines and chemokines such as IL-6, IL-12p70, ifnγ, CXCL10, TNF- α, and the like.

Antibodies to

An antibody disclosed in the examples of the present application, which specifically binds to human CD40 protein, comprising a light chain variable region (VL) defined according to the Kabat numbering system, said light chain variable region (VL) having three Complementarity Determining Regions (CDRs), respectively: VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 5 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with it; VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO. 6 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence; and VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 7 or a sequence having substitution, deletion or addition of 1 to 3 amino acids as compared with the amino acid sequence;

and/or

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 the 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 in SEQ ID NO. 2.

In certain embodiments, the anti-human CD40 protein antibody may have a Y-type molecular structure. In one embodiment, an antibody against human CD40 protein may comprise a pair of heavy chains and a pair of light chains. The heavy chain may include one heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies generally comprise five types of heavy chains: antibodies of corresponding composition are termed IgG, igD, igA, igM and IgE five antibodies. The light chain may be a smaller polypeptide subunit relative to the heavy chain. The light chain may include a light chain variable region and a light chain constant region. VL is typically the N-terminal part of the light chain, exhibiting higher variability in amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, the heavy chain variable region VH and the light chain variable region VL may both be used to recognize and bind 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 monoclonal antibodies

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

The immunogen used for preparing the human CD40 rabbit monoclonal antibody is derived from outsourced commercial human CD40 protein (Yiqiao Shenzhou Cat: 10774-H02H); the preparation method is monoclonal antibody development technology based on single B lymphocyte screening and culture. In some embodiments, the method of making comprises: human CD40 protein is used as immunogen to immunize New Zealand white rabbits; b lymphocytes are sorted from spleen cells of the large white blood cell line and cultured; extracting RNA in B lymphocytes, and reversely transcribing the RNA into cDNA; the cDNA is amplified by PCR to obtain a natural paired rabbit monoclonal antibody; the heavy chain variable region (VH) gene and the light chain variable region (VL) gene of the rabbit monoclonal antibody which are paired are respectively loaded on an expression vector, the vector is transfected into a host cell, the host cell is cultured, and the monoclonal antibody is obtained by separating and purifying the culture solution of the host cell.

Specifically, the preparation implementation process of the rabbit monoclonal antibody comprises the following steps:

1. immunization of animals

Commercial human CD40 protein is used as immunogen to immunize New Zealand white rabbits, each white rabbit is immunized by 200 mug, the immunogen is mixed with the equivalent amount of complete Freund's adjuvant to prepare an emulsifier before the first immunization, and the emulsifier is injected subcutaneously at the abdomen and back of the rabbits. 150 μg of immunogen is mixed with an equal amount of incomplete Freund's adjuvant every 3 weeks after the primary immunization to prepare an emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of a rabbit for two times of boosting. Serum samples of rabbits were collected after three immunizations, titers against human CD40 protein were determined by ELISA, and serum was purified to antibodies after the last immunization, endogenous samples were tested by WB, IF and IHC, rabbits with high serum titers and best endogenous detection were taken, boosted once by subcutaneous multipoint injection of 200. Mu.g of immunogen, and spleens were taken three days later.

2. Spleen cells were isolated.

3. B lymphocyte sorting

See patent "201910125091.4 for a method of efficiently isolating individual antigen-specific B lymphocytes from spleen cells.

4. Cloning of genes encoding Rabbit monoclonal antibodies

The cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cells of positive clones were collected, lysed, and RNA was 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 cDNA of the corresponding positive clone using PCR and sequenced.

5. Production and purification of monoclonal antibodies

In order to obtain a plurality of rabbit monoclonal antibodies for recognizing human CD40 protein, the invention respectively loads heavy chain and light chain genes of the rabbit monoclonal antibodies on expression vectors, and transfects 293F cells with plasmids; the antibody recognizing human CD40 protein is obtained after 72-96 hours of transfection. Purifying recombinant rabbit monoclonal antibody recognizing human CD40 protein from transfected culture medium supernatant by using protein A affinity gel resin, sub-packaging after the antibody is verified to be qualified, and preserving at low temperature of-20 ℃ for standby.

6. Results

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

Affinity detection of rabbit monoclonal antibodies to human CD40 protein

The affinity of the anti-human CD40 protein rabbit monoclonal antibody is accurately measured by using a Biacore 3000 biological molecular interaction analyzer of GE company; wherein the selected antibody is used at a concentration of 37nM and immobilized on a CM5 chip; then binding was performed with three concentrations of 37.5nM, 75nM and 150nM of recombinant human CD40 protein to obtain an affinity curve; finally, by curve fitting and calculation, the affinity curves are shown in fig. 1, and the fitting results are shown in table 1. The obtained rabbit monoclonal antibody against human CD40 protein has an affinity of 7.2X10 ^-10 M。

TABLE 1

Note that: k (K) _off (dissociation rate constant) represents dissociation rate constants between molecules, representing how fast the molecules dissociate; k (K) _on (association rate constant) is the intermolecular binding rate constant, representing the speed of intermolecular binding

Application of rabbit monoclonal antibody

1. Rabbit monoclonal antibodies provided herein for ELISA binding Activity detection

The detection method comprises the following steps:

the anti-human CD40 protein rabbit monoclonal antibodies and CD40L protein were coated with carbonate buffer (ph 9.4, 0.05M), incubated overnight at 4 degrees; washing the plate with a washing liquid; blocking with phosphate buffer (pH 7.2, 0.05M) containing 5% bovine serum albumin, 0.05% Tween-20; the biotinylated recombinant human CD40 protein is added into an ELISA plate after being diluted in a phosphate buffer solution with 0.05 percent of Tween-20 in a gradient way, incubated for 1 hour under normal temperature conditions, and then the plate is washed by a washing solution; adding avidin-labeled horseradish peroxidase (HRP) diluted with phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20, incubating for 1 hour at normal temperature, and washing the plate with a washing solution; adding TMB color development liquid, developing at normal temperature for 10 minutes, adding oxalic acid to stop color development, and measuring light absorption values at 450nm and 630nm respectively, wherein OD450 is subtracted from OD630 to obtain a corrected light absorption value; the values of the human CD40 protein concentration (Log 10) are plotted on the abscissa and the corrected absorbance values (OD 450) are plotted on the ordinate.

Detection result: as shown in FIG. 2, the binding activity of the anti-CD40 rabbit monoclonal antibody and 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 performed by adopting a flow type binding activity detection method of an antibody, and the specific detection method is as follows:

taking 1.5X10 ⁶ The number of the daudi cells was determined,aliquoted into 3 portions, washed once with PBS+0.1% BSA buffer; 100 μl PBS+0.1% BSA,100 μl Isotype mAb (10 μg/ml), anti-CD40 mAb (10 μg/ml) were added respectively and incubated at 4deg.C for 30 min; 400g, centrifuged for 3 min, and the supernatant discarded, 100. Mu.l PBS+0.1% BSA, 100. Mu. l G, respectively&R_IgG_FITC (1:500 dilution), 100 μ l G&R_igg_fitc (1:500 dilution) resuspended cells and incubated at 4 ℃ for 30 min; 400g, centrifuged for 3 min, the supernatant discarded, and washed twice with 100. Mu.l PBS+0.1% BSA buffer; detecting fluorescent signals on the cell surface using an Agilent NovoCyte Advanteon analytical flow cytometer; the plots were plotted using Novoexpressss software.

The results are shown in FIG. 3, where the flow binding signal of the rabbit monoclonal antibody against human CD40 protein and positive cells has 2 log transitions.

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

In the embodiment of the application, the rabbit monoclonal antibody provided by the application is used for detecting the B cell activation activity by adopting a flow type binding activity detection method of the antibody, and the specific detection method is as follows:

human Peripheral Blood Mononuclear Cells (PBMCs) were thawed and cultured for 4 hours; purifying B cells with a Stemcell Pan-B cell kit; purified B cells were then grown at 2.5X10 ⁶ The individual cells/wells were packed in 96-well flat bottom plates (50. Mu.g/ml polymyxin B was added to 0.2ml B cell medium); crosslinking a rabbit anti-human CD40 monoclonal antibody or a control group of rabbit anti-mAb with 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 and live/dead cells were identified with Zombie NIR (1:1500); staining and fixing Pan-B cells with anti-human CD19, CD86 and MHC-II antibodies; detecting fluorescent signals on the cell surface using an Agilent NovoCyte Advanteon analytical flow cytometer; the plots were plotted using Novoexpressss software.

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

TABLE 2

Experimental grouping	CD 86+/MHC-II+ ratio
		Blank	0.44％
Irrelevant control antibody (5. Mu.g/ml)	0.58％
		Anti-human CD40 monoclonal antibody (5 μg/ml)	5.18％
Anti-human CD40 monoclonal antibody (1 μg/ml)	5.34％
		CD40L(5μg/ml)	1.37％
CD40L(1μg/ml)	0.62％

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims

1. An antibody that specifically binds 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), respectively: VL CDR1 consisting 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;

and

comprising a heavy chain variable region (VH) defined according to the Kabat numbering system, said heavy chain variable region (VH) having three Complementarity Determining Regions (CDRs), respectively: a VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 8; VH CDR2 consisting of the amino acid sequence shown in SEQ ID No. 9; and VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 10.

2. An antibody that specifically binds human CD40 protein, the 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 the amino acid sequence shown in SEQ ID NO. 4.

3. An antibody comprising a light chain and a heavy chain, said light chain consisting of the amino acid sequence set forth in SEQ ID No. 1; 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 1-3, and a detectable label attached to the antibody.

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

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

7. Use of the antibody of any one of claims 1-3 or the kit of claim 5 for detecting human CD40 protein for diagnosis and treatment of non-disease.