Monoclonal antibody for recognizing recombinant EGF-CRM197 vaccine and preparation method thereof

Abstract

The invention provides a monoclonal antibody capable of recognizing a recombinant EGF-CRM197 vaccine and a preparation method thereof, on the basis of researching, developing and preparing the recombinant EGF-CRM197 vaccine, after the recombinant EGF-CRM197 vaccine is further immunized and inoculated to a mouse, mouse spleen cells and mouse myeloma cells are fused, and the monoclonal antibody capable of recognizing the recombinant EGF-CRM197 vaccine is obtained. And screening a hybridoma cell strain which generates a specific monoclonal antibody for recognizing the recombinant EGF-CRM197 vaccine, and sequencing to obtain an antibody sequence. After the hybridoma cells are cultured in a mouse body, the monoclonal antibody for recognizing the recombinant EGF-CRM197 vaccine can be prepared in vitro through methods of chromatographic purification and the like. The method can be used for detecting the release titer of the recombinant EGF-CRM197 vaccine and evaluating the immune efficacy after the recombinant EGF-CRM197 vaccine is immunized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody for identifying a recombinant EGF-CRM197 vaccine and a preparation method thereof. Background Art

[0002] Human epidermal growth factor (EGF) is a single-chain low molecular weight polypeptide composed of 53 amino acids, which is mainly distributed in the extracellular fluid of various tissues of the human body. EGF mainly exerts its function through EGFR. After binding to EGFR, EGF can activate multiple signaling pathways including Ras-Raf-MAPK, JAK-STAT, PI(3)K-Akt, etc. In recent years, studies have shown that EGF and EGFR are overexpressed in many solid tumors such as glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, gastric cancer, etc. Many tumors produce a large amount of EGF through autocrine and paracrine means, thereby overactivating the EGFR pathway. Overactivated EGFR enhances transcription, post-transcriptional activity or gene expression through multiple signaling pathways, causing cell phenotype transformation, and then leading to the occurrence and development of tumors. Therefore, reducing the content of EGF in tumor cells plays a vital role in inhibiting the proliferation of tumor cells, signal transduction between tumor cells, and thus inhibiting the development of tumors.

[0003] Recombinant EGF-CRM197 tumor therapeutic vaccine (hereinafter referred to as "recombinant EGF-CRM197 vaccine") is a new type of conjugate vaccine formed by cross-linking EGF and carrier protein CRM197. It was developed by Shanghai Huidun Biotechnology Co., Ltd. and has completed Phase I clinical trials and is currently conducting Phase II clinical trials. The main mechanism of this vaccine is to induce high levels of anti-EGF antibodies in patients, neutralize EGF in the body, thereby inhibiting the EGF / EGFR signaling pathway, and then inhibiting the growth of tumor cells that rely on this signaling pathway, achieving the purpose of treating tumors.

[0004] Vaccine potency is the most important release quality standard for the recombinant EGF-CRM197 vaccine. The commonly used method for detecting vaccine potency is to immunize mice or guinea pigs with the vaccine, conduct in vivo challenge experiments or obtain serum for antibody titer determination. This method is complicated and time-consuming. Referring to the "In vitro Relative Efficacy Test of Inactivated Hepatitis A Vaccine" in the 2020 edition of the Chinese Pharmacopoeia, by comparing the ratio of the amount of antigen (EGF) in different batches of recombinant EGF-CRM197 vaccine to physical and chemical reference substances, we established a method for determining the in vitro relative potency of the recombinant EGF-CRM197 vaccine. The capture antibody used in this method is a monoclonal antibody prepared after immunizing mice with the recombinant EGF-CRM197 vaccine. The antibody can effectively bind to the antigen in the coated vaccine, and its sensitivity is much higher than that of commercially available monoclonal antibodies.

[0005] In addition, in clinical use, the neutralizing antibodies induced by immunization with the recombinant EGF-CRM197 vaccine neutralize EGF in the patient's serum, thereby exerting an anti-tumor effect. Therefore, the reduction in EGF concentration in serum is also an important measurement indicator for evaluating the immune efficacy of the vaccine. A monoclonal antibody that specifically recognizes recombinant EGF-CRM197 has been developed as a capture or detection antibody for the EGF quantitative ELISA detection kit, which can be used to quantitatively detect the concentration of EGF in the serum of patients immunized with the recombinant EGF-CRM197 vaccine, thereby evaluating the effectiveness of the recombinant EGF-CRM197 vaccine and providing a basis for strengthening immunity for patients.

[0006] In summary, the development of a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine can be used to detect the in vitro relative efficacy of the recombinant EGF-CRM197 vaccine, providing a powerful means for quality control of the recombinant EGF-CRM197 before clinical use; in addition, a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine can be used for the preparation of a human EGF quantitative ELISA detection kit. Therefore, with the development and clinical application of the recombinant EGF-CRM197 vaccine, the development of a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine is of great significance. Summary of the invention

[0007] In view of the deficiencies in the prior art and actual needs, the present invention provides a monoclonal antibody for recognizing a recombinant EGF-CRM197 vaccine and its application. The monoclonal antibody can bind to the recombinant EGF-CRM197 vaccine and plays an important role in evaluating the relative potency of the recombinant EGF-CRM197 and evaluating the immune efficacy of the vaccine in clinical applications.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a monoclonal antibody that recognizes a recombinant EGF-CRM197 vaccine.

[0010] The heavy chain variable region of the antibody has the following complementarity determining region CDR:

[0011] VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, and VH-CDR3 of SEQ ID NO:4;

[0012] The light chain variable region of the antibody has the following complementarity determining region CDR:

[0013] VL-CDR1 of SEQ ID NO: 10, VL-CDR2 of SEQ ID NO: 11, and VL-CDR3 of SEQ ID NO: 12;

[0014] Furthermore, the amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 1.

[0015] Furthermore, the amino acid sequence of the antibody light chain variable region is SEQ ID NO:9.

[0016] Furthermore, the monoclonal antibody is used to prepare a kit for evaluating the relative efficacy of the recombinant EGF-CRM197 vaccine in vitro and an ELISA kit for quantitatively detecting the EGF concentration in serum.

[0017] The present invention also provides a method for preparing a monoclonal antibody, comprising the following steps:

[0018] Step 1: Animal immunization: 6-week-old BALB / c mice were used, and the antigen was the recombinant EGF-CRM197 vaccine. The immunization route was subcutaneous injection; the drug was administered once a week for a total of 4 times.

[0019] Step 2: Cell fusion: Prepare spleen cell suspension from the mice selected in step (1). Take spleen cells and start fusing with SP2 / 0 myeloma cells using PEG-mediated fusion method.

[0020] Step 3: Screening of hybridoma cells: Amplify the hybridoma cells in step (2), take the supernatant to test the ELISA titer and antibody subtype, and select cells that are positive for EGF antigen for cloning. To ensure the positive rate of the hybridoma cell line and the stable production of antibodies, perform 3-4 clonings and select clones that are 100% positive in 3-4 screenings.

[0021] Step 4: Cloning of hybridoma cells: The positive wells screened in step (3) were cloned by conventional limiting dilution method to screen for hybridoma cell lines producing anti-EGF specific monoclonal antibodies, which were numbered as S-172-4 cells;

[0022] Step 5: Preparation of monoclonal antibodies: Nude mice were intraperitoneally injected with 0.5 ml of liquid paraffin one week in advance. The S-172-4 cells obtained in step (4) were adjusted to 5×10 6 / ml, 200μl was injected intraperitoneally, and 7-10 days later, the mouse ascites was collected. The ascites was purified by Protein A chromatography column, and the chromatography column was first equilibrated with A liquid PB PH7.0. After the ascites sample was loaded on the column, it was linearly eluted with B liquid 0.1M glycine-HCl PH2.7 until the peak of the antibody appeared. Finally, the pH of the purified antibody was adjusted to 7.2 with 1M PH 8.0 Tris-Hcl, and sterilized by membrane to obtain S-172-4 monoclonal antibody.

[0023] Compared with the prior art, the advantages of the present invention are that the monoclonal antibody provided by the present invention can specifically and highly identify the recombinant EGF-CRM197 vaccine component EGF, and can be used to detect the in vitro relative efficacy of the recombinant EGF-CRM197 vaccine, with high accuracy and good repeatability, and provides an effective means for quality control of recombinant EGF-CRM197 before clinical use. In addition, in clinical use, the neutralizing antibodies induced by the recombinant EGF-CRM197 vaccine after immunization neutralize the EGF in the patient's serum, thereby exerting an anti-tumor effect. Therefore, the reduction of the EGF concentration in the serum is also an important indicator for evaluating the immune efficacy of the vaccine. The present invention provides a monoclonal antibody that specifically recognizes recombinant EGF-CRM197, which can be used as a capture or detection antibody of an EGF quantitative ELISA detection kit and used to prepare an EGF quantitative ELISA detection kit. The kit has high sensitivity and good repeatability, and can accurately detect the concentration change of EGF in the serum of patients immunized with the recombinant EGF-CRM197 vaccine, thereby evaluating the effectiveness of the recombinant EGF-CRM197 vaccine, providing a basis for patients to strengthen immunity, and playing an important role in evaluating the immune efficacy of the vaccine.

[0024] Figure 1 The figure shows the relative efficacy results of the S-172-4 monoclonal antibody of the present invention and the commercially purchased anti-human EGF antibody in detecting the recombinant EGF-CRM197 vaccine in vitro.

[0025] Figure 2 A human EGF quantitative ELISA detection kit prepared for the S-172-4 monoclonal antibody of the present invention, and a standard curve. DETAILED DESCRIPTION

[0026] The present inventors have conducted extensive and in-depth research and a large number of screenings and developed a monoclonal antibody (number: S-172-4) that recognizes the recombinant EGF-CRM197 vaccine, provided a preparation method and antibody sequence of the antibody, and further verified the application effect of using the purified monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine for the release potency detection of the recombinant EGF-CRM197 vaccine and for evaluating the immune efficacy after immunization with the recombinant EGF-CRM197 vaccine.

[0027] the term

[0028] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0031] As described herein, "a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine" or "S-172-4 monoclonal antibody" can be used interchangeably and both refer to the antibody of the present invention.

[0032] As described herein, the "recombinant EGF-CRM197 vaccine" is a recombinant human epidermal growth factor hEGF-diphtheria toxin mutant CRM197 conjugate, and the conjugate is obtained by coupling the recombinant human epidermal growth factor hEGF with the diphtheria toxin CRM197 through the chemical cross-linking agent glutaraldehyde. The conjugate has the structure shown in hEGF-L-CRM197, wherein L is a linker linking the recombinant human epidermal growth factor hEGF and the diphtheria toxin mutant CRM197, and the linker is a covalent bond formed by cross-linking with glutaraldehyde. The preparation method and composition of the "recombinant EGF-CRM197 vaccine" refer to the patent application of our company: Recombinant hEGF-CRM197 tumor therapeutic vaccine formulation, patent authorization announcement number: CN113855792A.

[0033] Antibody

[0034] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0035] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0036] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0037] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0038] In the present invention, "VH-CDR1" and "CDR-H1" are used interchangeably, both referring to the CDR1 of the heavy chain variable region; "VH-CDR2" and "CDR-H2" are used interchangeably, both referring to the CDR2 of the heavy chain variable region; "VH-CDR3" and "CDR-H3" are used interchangeably, both referring to the CDR3 of the heavy chain variable region. "VL-CDR1" and "CDR-L1" are used interchangeably, both referring to the CDR1 of the light chain variable region; "VL-CDR2" and "CDR-L2" are used interchangeably, both referring to the CDR2 of the light chain variable region; "VL-CDR3" and "CDR-L3" are used interchangeably, both referring to the CDR3 of the light chain variable region.

[0039] In the present invention, the antibody can specifically recognize the recombinant EGF-CRM197 vaccine, which includes a heavy chain and a light chain, wherein the heavy chain contains a heavy chain variable region (VH) amino acid sequence, and the light chain contains a light chain variable region (VL) amino acid sequence.

[0040] The heavy chain variable region (VH) has a complementarity determining region CDR selected from the following group:

[0041] VH-CDR1 shown in SEQ ID NO:2, VH-CDR2 shown in SEQ ID NO:3, and VH-CDR3 shown in SEQ ID NO:4;

[0042] The light chain variable region (VL) has a complementarity determining region (CDR) selected from the group consisting of:

[0043] VL-CDR1 shown in SEQ ID NO:10, VL-CDR2 shown in SEQ ID NO:11, and VL-CDR3 shown in SEQ ID NO:12.

[0044] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:1.

[0045] In another preferred example, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:9.

[0046] In a more preferred embodiment, each antibody or recombinant protein of the present invention specifically includes the following VL and VH sequences, as well as CDR and FR sequences.

[0047] Table 1 Summary of antibody sequences of the present invention

[0048]

[0049]

[0050] Polynucleotide encoding

[0051] The present invention also provides a polynucleotide encoding the above-mentioned antibody or its heavy chain variable region or light chain variable region.

[0052] Preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in the sequence listing SEQ ID NO: 17; and / or, the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in the sequence listing SEQ ID NO: 18.

[0053] More preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in the sequence listing SEQ ID NO: 17; and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in the sequence listing SEQ ID NO: 18.

[0054] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0055] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0056] Antibody preparation

[0057] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0058] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0059] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0060] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0061] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0062] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO cells, HEK-293 cells.

[0063] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, and other conventional separation and purification methods well known to those skilled in the art to obtain the antibodies of the present invention.

[0064] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0065] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the antibodies can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0066] application

[0067] The present invention provides a monoclonal antibody for recognizing a recombinant EGF-CRM197 vaccine and its application. The monoclonal antibody can specifically recognize EGF in the recombinant EGF-CRM197 vaccine and plays an important role in evaluating the relative potency of the recombinant EGF-CRM197 and clinically evaluating the immune efficacy of the vaccine.

[0068] The main advantages of the present invention include:

[0069] 1) Specificity. The antibody can specifically bind to the EGF protein and can specifically recognize the EGF component in the recombinant EGF-CRM197 vaccine.

[0070] 2) High affinity. The monoclonal antibody for recognizing the recombinant EGF-CRM197 vaccine provided by the present invention can bind to the antigen component EGF in the recombinant EGF-CRM197 vaccine with high affinity, and its affinity to the EGF protein is significantly higher than that of the commercially available anti-EGF monoclonal antibody. The antibody can be used for more sensitive enzyme-linked immunosorbent assay to detect the content of EGF in the recombinant EGF-CRM197 vaccine, as well as the change in the EGF content in the patient's serum after immunization with the recombinant EGF-CRM197 vaccine, and can be used to better evaluate the effective components and batch consistency of the vaccine, as well as the clinical immune efficacy of the recombinant EGF-CRM197 vaccine.

[0071] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0072] Example 1: Preparation of a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine

[0073] The main steps of this embodiment include: animal immunization, cell fusion, screening of hybridoma cells, cloning of hybridoma cells and antibody preparation.

[0074] (1) Animal immunization: 6-week-old BALB / c mice were used, and the antigen was the recombinant EGF-CRM197 vaccine (the preparation method refers to the patent application of our company: Recombinant hEGF-CRM197 Tumor Therapeutic Vaccine Formulation, Patent Authorization Announcement No.: CN113855792A). 10 μg of recombinant EGF-CRM197 vaccine and Montanide ISA51VG adjuvant (Seppic Chemical Reagent Company, France, Product No.: T02621) were mixed and emulsified in equal volumes, and then injected subcutaneously on the back of the mice; the drug was administered once a week for a total of 4 times. One week after the last immunization, the spleen was taken for fusion.

[0075] (2) Cell fusion: prepared one week after the last immunization;

[0076] Preparation of spleen cell suspension: On the day of cell fusion, BALB / c mice that have been immunized with the recombinant EGF-CRM197 vaccine are taken, the eyeballs are removed for blood collection, and the serum is separated as the positive control serum for antibody detection. At the same time, the mice are killed by cervical dislocation, and the spleens are removed to prepare spleen cell suspension.

[0077] Preparation of myeloma cell suspension: revive the cells two weeks in advance to ensure that the cells are in the logarithmic growth phase when used;

[0078] Preparation of feeder layer cells: Obtain mouse peritoneal macrophages two days in advance and culture them in 96-well plates;

[0079] Cell fusion: Using the PEG-mediated fusion cell method, spleen cells and myeloma cells were taken in a ratio of 5:1, mixed in serum-free DMEM medium, centrifuged at 1500rpm for 5 minutes, the supernatant was removed, the centrifuge tube was vigorously shaken to disperse the cells, and 1 ml of 50% PEG (pH8.0, 40°C) was added within 1 minute, shaking while adding, and allowed to stand for 90 seconds after adding, and serum-free DMEM medium was added to terminate the fusion. The tube was centrifuged at 1500rpm for 5 minutes, and the precipitate was suspended in HAT medium and distributed into 96-well cell plates containing feeder cells, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0080] (3) Screening of hybridoma cells

[0081] After 5 days of cell culture, the medium was replaced with HAT medium once, and on the 10th day, the medium was replaced with HT medium. When the fused cells covered 10%-50% of the bottom of the well, the positive wells were screened by conventional indirect ELISA method. The plate was coated with EGF, and the culture supernatant of hybridoma cells was detected. The secondary antibody was enzyme-labeled goat anti-mouse antibody, and mouse antiserum was used as a positive control to screen the hybridoma cells with the highest antibody expression.

[0082] (4) Hybridoma cell cloning

[0083] The specific strong positive wells were screened and cloned using the conventional limiting dilution method to obtain monoclonal antibody cell lines.

[0084] The positive hybridoma cells obtained from the original well may come from two or more hybridoma cells, and the antibodies they secrete are of different nature. In order to obtain completely homogeneous monoclonal antibodies, the hybridoma cells must be cloned.

[0085] Prepare the hybridoma cell suspension to be cloned, dilute it to 8 cells / ml with HT medium containing 20% ​​serum, and add mouse peritoneal cells to the hybridoma cell suspension at 5E4 cells per ml. Inoculate 0.1ml of cell suspension per well, i.e. 0.8 hybridoma cells per well; culture in a cell culture incubator at 37°C and 5% CO2 for 7-10 days, and detect antibodies when clones visible to the naked eye appear; observe under an inverted microscope, mark the wells where only a single clone grows, and remove the supernatant for antibody detection.

[0086] The cells from the antibody-positive wells were expanded and cultured, and then frozen. Meanwhile, the cells from the positive wells were further cloned, and the cloned monoclonal antibodies were identified by ELISA. The monoclonal cell line with the highest anti-EGF antibody titer was screened out and numbered as S-172-4 cells.

[0087] (5) Preparation of antibodies

[0088] The selected hybridoma cells were expanded and cultured. Nude mice of about 6 weeks old were taken and injected with 0.5 ml of liquid paraffin into the abdominal cavity of the nude mice one week in advance. The S-172-4 cells obtained in step (4) were adjusted to 5×10 6 / ml, 200μl was injected intraperitoneally, and 7-10 days later, the mouse ascites was collected. The ascites was purified by Protein A chromatography column, and the chromatography column was first equilibrated with A liquid PB PH7.0. After the ascites sample was loaded on the column, it was linearly eluted with B liquid 0.1M glycine-HCl PH2.7 until the peak of the antibody appeared. Finally, the pH of the purified antibody was adjusted to 7.2 with 1M PH 8.0 Tris-Hcl, and sterilized by membrane, and finally the purified S-172-4 monoclonal antibody was obtained.

[0089] Example 2: Monoclonal antibody S-172-4 / commercial monoclonal antibody used to detect the relative efficacy of recombinant EGF-CRM197 vaccine in vitro

[0090] (1) Dilute the recombinant EGF-CRM197 vaccine test sample and the recombinant EGF-CRM197 vaccine reference sample to 1000, 500, 250, 125, 62.5, 31.25, 15.6, and 7.8 pg / ml with Na2CO3-NaHCO3 buffer, and coat the ELISA plate with 8 gradients of the diluted recombinant EGF-CRM197 vaccine test sample and reference sample, with 2 replicate wells for each dilution, 100 μl per well, and incubate at 2-8°C overnight. Then wash the plate with PBST and pat dry. Block with 200 μl of blocking solution and incubate at 37°C for 2 h.

[0091] (2) Take the coated ELISA plate, add appropriate concentration of S-172-4 hybridoma monoclonal antibody or commercial rabbit anti-human EGF monoclonal antibody No. 1 (Cat. No. ab184265, abcam), or commercial rabbit anti-human EGF monoclonal antibody No. 2 (Cat. No. AF5148, affinitybioseience), and incubate at 37°C for 1 hour. After washing the plate, add 1 μg / ml goat anti-mouse IgG-HRP or goat anti-rabbit IgG-HRP, 100 μl per well, and incubate at 37°C for 1 hour.

[0092] (3) After washing, add 100 μl / well TMB, incubate in the dark for 15 min, add 50 μl / well stop solution, and read at 450 nm.

[0093] (4) Calculation of results

[0094] The OD values ​​of the recombinant EGF-CRM197 vaccine test sample and the physical and chemical reference substance at 5 concentrations selected from the 8 concentration points with the best linearity are recorded in Table 2 below.

[0095]

[0096] In vitro relative potency = test sample antigen content / reference vaccine antigen relative content

[0097] Test sample antigen content / reference antigen content = antilg (V / W × lg2)

[0098] V=0.2(T1+T2+T3+T4+T5-S1-S2-S3-S4-S5)

[0099] W=0.1(T5-T1+S5-S1)+0.05(T4-T2+S4-S2)

[0100] The S-172-4 monoclonal antibody of the present invention and the commercially available anti-human EGF antibody were used to detect the recombinant EGF-CRM197 vaccine, and the OD response values ​​were as follows: Figure 1As shown: using the S-172-4 monoclonal antibody of the present invention, the recombinant EGF-CRM197 concentration between 7.8 and 1000 pg / ml showed a good linear relationship; while the commercially purchased anti-human monoclonal antibody only had a certain linear relationship between 125 and 1000 pg / ml, and the sensitivity was much lower than the S-172-4 antibody of the present invention.

[0101] Recombinant EGF-CRM197 samples were used as test samples, and 3 people were tested, each person was tested 3 times, and each batch of samples was tested 9 times in total. The analysis results showed that the in vitro relative efficacy of the recombinant EGF-CRM197 vaccine was detected by the S-172-4 monoclonal antibody of the present invention, and the results were stable, with good repeatability and a coefficient of variation CV within 10%. However, the commercial anti-human EGF antibody had poor repeatability and a coefficient of variation CV of more than 25%.

[0102] Table 3. Comparison of the in vitro relative efficacy of the recombinant EGF-CRM197 vaccine detected by S-172-4 monoclonal antibody and commercially available anti-human EGF antibodies

[0103]

[0104] Example 2: Monoclonal antibody S-172-4 is used to prepare a human EGF detection kit

[0105] The EGF concentration in the peripheral blood serum of healthy people is about 0-300pg / ml (median value is about 150pg / ml), and the EGF concentration in the peripheral blood serum of lung cancer patients is about 0-500pg / ml (median value is about 250pg / ml). The mechanism of action of the recombinant EGF-CRM197 vaccine is to induce the body to produce neutralizing antibodies against EGF, thereby reducing the concentration of EGF in the serum, thereby achieving an anti-tumor effect. Therefore, the monitoring of EGF in serum is an important clinical indicator for evaluating the immune efficacy of the recombinant EGF-CRM197 vaccine. Currently, the anti-human EGF antibodies on the market are used to prepare human EGF quantitative ELISA detection kits, which can generally only identify EGF at the nanogram level (ng / ml), which cannot meet clinical needs. The quantitative ELISA detection kits of R&D companies imported on the market can be used to determine the EGF content in human serum, but the price is high and is only used for scientific research. It has not obtained a medical device license and cannot be used for clinical testing after the recombinant EGF-CRM197 vaccine is launched. Therefore, it is of great significance to develop a quantitative detection kit for human EGF suitable for the clinical application of the recombinant EGF-CRM197 vaccine.

[0106] In this example, we used the S-172-4 monoclonal antibody of the present invention as the capture antibody, and selected the commercially available rabbit anti-human EGF polyclonal antibody (Cat. No. ab9695, abcam) as the detection antibody. The commercially available rabbit anti-human EGF monoclonal antibody (Cat. No. AF5148, affinitybioseience) was selected as the capture antibody, and the commercially available rabbit anti-human EGF polyclonal antibody (Cat. No. ab9695, abcam) was selected as the detection antibody and the following operation was performed.

[0107] Use NaCO3-NaHCO3 buffer at pH 9.6 to dilute S-172-4 monoclonal antibody to a final concentration of 5 μg / ml, add to a 96-well microplate, 100 μl / well, and coat overnight at 2-8°C.

[0108] The plate coated with S-172-4 monoclonal antibody was washed with PBST, patted dry, and blocked with 200 μl of blocking solution, and incubated at 37°C for 2 h.

[0109] Preparation of standards / samples: EGF standard was diluted with buffer to a gradient of 500, 250, 125, 62.5, 31.3, 15.6, 7.8 pg / ml, and the buffer was used as the blank "0" point.

[0110] Add the standard and the serum sample to be tested into the blocked 96-well plate, 200 μl / well, do 2 wells in parallel, cover with sealing film, and incubate at 37°C for 1 hour.

[0111] The plate was washed three times with PBST, patted dry, and 10 μg / ml commercially available rabbit anti-human EGF polyclonal antibody was added as detection antibody, 100 μl / well, and incubated at 37°C for 1 hour.

[0112] The plate was washed three times with PBST, patted dry, and 1 μg / ml goat anti-mouse IgG-HRP was added, 100 μl / well, and incubated at 37°C for 1 hour.

[0113] Wash the plate three times with PBST, pat dry, add 100 μl / well TMB, incubate for 15 min in the dark, add 50 μl / well stop solution, and read at 450 nm / 650 nm.

[0114] Result calculation: Take the average OD value of the standard solution and the test sample, deduct the OD value of the blank control, use the standard solution to make a four-parameter logistic curve, substitute the OD value of the test sample, and calculate the EGF concentration of the sample well.

[0115] The OD readings of the standard curve drawn by the human EGF quantitative detection kit prepared by the monoclonal antibody S-172-4 of the present invention are shown in the following table. The OD readings of the standard product of the human EGF quantitative ELISA detection kit prepared by the S-172-4 monoclonal antibody are shown in the following table. Figure 2 The results showed that the human EGF quantitative detection kit prepared with S-172-4 had a good linear relationship between 15.6-500pg / ml, R 2 >0.95, while the commercially available anti-EGF monoclonal antibody only responded at a concentration of 500 pg / ml.

[0116] Table 4. Comparison of OD values ​​of EGF standard products measured by human EGF quantitative ELISA detection kit prepared by S-172-4 monoclonal antibody and commercially available anti-human EGF antibody

[0117]

[0118] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A monoclonal antibody that recognizes a recombinant EGF-CRM197 vaccine, characterized in that: The heavy chain variable region of the antibody has the following complementarity determining region CDR: VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, and VH-CDR3 of SEQ ID NO:4; The light chain variable region of the antibody has the following complementarity determining region CDR: VL-CDR1 of SEQ ID NO:10, VL-CDR2 of SEQ ID NO:11 and VL-CDR3 of SEQ ID NO:

12.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the antibody heavy chain variable region is SEQ ID NO:

1.

3. The antibody according to claim 1, characterized in that The amino acid sequence of the antibody light chain variable region is SEQ ID NO:

9.

4. A method for preparing a monoclonal antibody that recognizes a recombinant EGF-CRM197 vaccine, comprising preparing a monoclonal antibody that recognizes a recombinant EGF-CRM197 vaccine as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: Animal immunization: The antigen is a recombinant EGF-CRM197 vaccine, which is injected subcutaneously into mice to achieve animal immunization; Step 2: Cell fusion: taking spleen cells from immunized mice and starting to fuse with SP2 / 0 myeloma cells using a PEG-mediated fusion method; Step 3: Screening of hybridoma cells: Amplify the hybridoma cells in step 2, take the supernatant to test the ELISA titer and antibody subtype, and select cells that are positive for EGF antigen for cloning. To ensure the positive rate of the hybridoma cell line and the stable production of antibodies, perform 3-4 clonings, and select clones that are 100% positive in 3-4 screenings; Step 4: The positive wells screened in step 3 are cloned by conventional limiting dilution method to obtain a monoclonal antibody hybrid cell line, numbered: S-172-4 cell; Step 5: Nude mice were intraperitoneally injected with 0.5 ml of liquid paraffin one week in advance. The S-172-4 cells obtained in step 4 were adjusted to 5×10 with physiological saline. 6 / ml, 200μl was injected intraperitoneally, and 7-10 days later, the mouse ascites was collected. The ascites was purified by Protein A chromatography column, and the chromatography column was first equilibrated with A liquid PB PH7.

0. After the ascites sample was loaded on the column, it was linearly eluted with B liquid 0.1M glycine-HCl PH2.7 until the peak of the antibody appeared. Finally, the purified antibody pH was adjusted to 7.2 with 1M PH 8.0 Tris-Hcl, and sterilized by membrane to obtain S-72-4 monoclonal antibody.

5. The monoclonal antibody for recognizing the recombinant EGF-CRM197 vaccine according to claim 1, characterized in that: The monoclonal antibody is used to prepare a kit for evaluating the in vitro relative efficacy of a recombinant EGF-CRM197 vaccine and an ELISA kit for quantitatively detecting the EGF concentration in serum.

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