A monoclonal antibody that recognizes recombinant EGF-CRM197 vaccine and its preparation method

Abstract

The present invention provides a monoclonal antibody that recognizes a recombinant EGF-CRM197 vaccine and a preparation method thereof. Based on the research and development and preparation of a recombinant EGF-CRM197 vaccine, the present invention further immunizes mice with the recombinant EGF-CRM197 vaccine, fuses the mouse spleen cells with mouse myeloma cells, screens and produces a hybridoma cell line of specific monoclonal antibodies that recognize the recombinant EGF-CRM197 vaccine, and obtains the antibody sequence after sequencing. After the hybridoma cells are cultured in mice, monoclonal antibodies that recognize the recombinant EGF-CRM197 vaccine can be prepared in vitro by methods such as chromatography purification. The present invention can be used 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.

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, thereby 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] The recombinant EGF-CRM197 therapeutic tumor vaccine (hereinafter referred to as the "recombinant EGF-CRM197 vaccine") is a novel conjugate vaccine composed of EGF cross-linked with the carrier protein CRM197. Developed by Shanghai Huidun Biotechnology Co., Ltd., it has completed Phase I clinical trials and is currently undergoing Phase II clinical trials. The vaccine's primary mechanism is to induce the production of high levels of anti-EGF antibodies in patients, neutralizing EGF in the body and thereby inhibiting the EGF / EGFR signaling pathway, thereby suppressing the growth of tumor cells that rely on this signaling pathway, ultimately achieving the goal of treating tumors.

[0004] Vaccine potency is the most important release quality standard for recombinant EGF-CRM197 vaccines. 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 potency 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. This 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 of 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, and then evaluate the effectiveness of the recombinant EGF-CRM197 vaccine, providing a basis for strengthening patients' immunity.

[0006] In summary, the development of a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine can be used to measure the relative potency of the recombinant EGF-CRM197 vaccine in vitro, providing a powerful tool for quality control of recombinant EGF-CRM197 before clinical use. Furthermore, a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine can be used in the preparation of a quantitative ELISA kit for human EGF. Therefore, the development of a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine is of great significance for the development and clinical application of the recombinant EGF-CRM197 vaccine. Summary of the Invention

[0007] In response to the deficiencies of the existing technology and actual needs, the present invention provides a monoclonal antibody for recognizing the 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 the recombinant EGF-CRM197 vaccine.

[0010] The heavy chain variable region of the antibody has the following complementarity determining regions 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 regions 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: Six-week-old BALB / c mice were used. The antigen was the recombinant EGF-CRM197 vaccine. Immunization was by subcutaneous injection, administered once weekly for a total of four doses.

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

[0020] Step 3: Screening of hybridoma cells: Amplify the hybridoma cells from step (2), collect the supernatant and 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 stable antibody production, perform 3-4 cloning cycles, and select clones that are 100% positive in all 3-4 screenings.

[0021] Step 4: Cloning of hybridoma cells: The positive wells screened in step (3) were cloned using a conventional limiting dilution method to screen for hybridoma cell lines that produced anti-EGF specific monoclonal antibodies, which were numbered 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, 200 μl was injected intraperitoneally. 7-10 days later, ascites was collected from the mice. The ascites was purified via a Protein A chromatography column. The column was first equilibrated with Solution A (PB, pH 7.0). After loading the ascites sample onto the column, the column was linearly eluted with Solution B (0.1 M glycine-HCl, pH 2.7) until the antibody peak appeared. Finally, the purified antibody was adjusted to pH 7.2 with 1 M Tris-HCl, pH 8.0, and sterilized by filtration to obtain the S-172-4 monoclonal antibody.

[0023] Compared with existing technologies, the present invention offers advantages in that the monoclonal antibodies provided herein can specifically and highly affinity recognize EGF, a component of the recombinant EGF-CRM197 vaccine. These antibodies can be used to measure the relative efficacy of the recombinant EGF-CRM197 vaccine in vitro with high accuracy and reproducibility, providing an effective means for quality control of recombinant EGF-CRM197 prior to clinical use. Furthermore, in clinical use, neutralizing antibodies induced by immunization with the recombinant EGF-CRM197 vaccine neutralize EGF in patient serum, thereby exerting an anti-tumor effect. Therefore, a reduction in serum EGF concentration is also an important indicator for evaluating vaccine efficacy. The present invention provides a monoclonal antibody that specifically recognizes recombinant EGF-CRM197 and can be used as a capture or detection antibody for an EGF quantitative ELISA detection kit to prepare an EGF quantitative ELISA detection kit. The kit has high sensitivity and good repeatability, and can accurately detect changes in EGF concentration 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 strengthening immunity for patients, and playing an important role in evaluating the immune efficacy of the vaccine.

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

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

[0026] After extensive and in-depth research and a large amount of screening, the inventors have 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 use of the purified and prepared monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine in the release potency detection of the recombinant EGF-CRM197 vaccine and in the evaluation of the immune efficacy after immunization with the recombinant EGF-CRM197 vaccine.

[0027] the term

[0028] In order to make the present invention easier to understand, certain 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 generally 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 change. 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 to refer to the antibody of the present invention.

[0032] The "recombinant EGF-CRM197 vaccine" described herein is a recombinant human epidermal growth factor (hEGF)-diphtheria toxin mutant CRM197 conjugate, obtained by coupling recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant CRM197 via the chemical crosslinker glutaraldehyde. The conjugate has the structure hEGF-L-CRM197, where L represents a linker linking recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant CRM197, formed by covalent bonds via glutaraldehyde crosslinking. The preparation method and composition of the "recombinant EGF-CRM197 vaccine" are based on our company's patent application: Recombinant hEGF-CRM197 Tumor Therapeutic Vaccine Formulation, Patent Grant Number: CN113855792A.

[0033] Antibody

[0034] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 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 varies between heavy chains of different immunoglobulin isotypes. 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 the first constant region of the heavy chain, and the variable region of the light chain is opposite 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" refers to certain parts of the variable region in an antibody that are different in sequence, which form 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 segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0036] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to 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 are 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 structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

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

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

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

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

[0045] In another preferred embodiment, 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] Encoding polynucleotide

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

[0053] More preferably, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO: 17; and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in 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 will appreciate that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog. The polynucleotide homologs of the present invention can be prepared by substituting, deleting, or adding one or more bases in the gene encoding the protein sequence while maintaining 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 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, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0060] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This 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 sequences of the present invention through 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 and HEK-293 cells.

[0063] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are 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, among other conventional separation and purification methods well known to those skilled in the art.

[0064] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using 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, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0065] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the antibodies can be separated and purified by various separation methods utilizing 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 a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other 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 present invention provides a monoclonal antibody that recognizes the recombinant EGF-CRM197 vaccine, which can bind to the antigen component EGF in the recombinant EGF-CRM197 vaccine with high affinity, and has a significantly higher affinity for the EGF protein than commercially available anti-EGF monoclonal antibodies. This antibody can be used for a more sensitive enzyme-linked immunosorbent assay to detect the EGF content of the recombinant EGF-CRM197 vaccine, as well as changes in EGF content in patient serum after immunization with the recombinant EGF-CRM197 vaccine. It 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 intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally 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 as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

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

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

[0074] (1) Animal immunization: Six-week-old BALB / c mice were used, and the antigen was the recombinant EGF-CRM197 vaccine (the preparation method is described in our patent application: Recombinant hEGF-CRM197 Tumor Therapeutic Vaccine Formulation, Patent Authorization Announcement No.: CN113855792A). 10 μg of recombinant EGF-CRM197 vaccine was mixed with equal volumes of Montanide ISA51VG adjuvant (Seppic Chemical Reagent Company, France, Product No.: T02621) and emulsified. The mixture was then subcutaneously injected into the back of the mice. The vaccine was administered once a week for a total of four times. One week after the last immunization, the spleen was harvested 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 were taken, and blood was collected from their eyeballs. The serum was separated and used as the positive control serum for antibody detection. At the same time, the mice were killed by cervical dislocation, and their spleens were removed to prepare the spleen cell suspension.

[0077] Preparation of myeloma cell suspension: thaw 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 mixed in a ratio of 5:1 in serum-free DMEM medium, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and the centrifuge tube was vigorously shaken to disperse the cells. Within 1 minute, 1 ml of 50% PEG (pH 8.0, 40°C) was added while shaking. After addition, it was allowed to stand for 90 seconds. Serum-free DMEM medium was added to terminate the fusion. The cells were centrifuged at 1500 rpm for 5 minutes. The precipitate was suspended in HAT medium and distributed into 96-well cell plates containing feeder cells. The cells were 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. On the 10th day, the medium was replaced with HT medium. When the confluent cells covered 10%-50% of the well bottom, positive wells were screened using a conventional indirect ELISA method. Plates were coated with EGF and the hybridoma culture supernatant was tested using an enzyme-labeled goat anti-mouse antibody as the secondary antibody and mouse antiserum as the positive control. Hybridomas expressing the highest antibody levels were screened.

[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 different in nature. In order to obtain completely homogeneous monoclonal antibodies, the hybridoma cells must be cloned.

[0085] Prepare a suspension of hybridoma cells to be cloned and dilute to 8 cells / ml in HT medium supplemented with 20% serum. Add mouse peritoneal cells to the hybridoma suspension at a density of 5E4 cells / ml. Seed 0.1 ml of the cell suspension per well, seeding 0.8 hybridoma cells per well. Culture in a cell culture incubator at 37°C, 5% CO₂ for 7-10 days. Antibody testing is available upon the emergence of visible colonies. Observe under an inverted microscope, mark wells with only single colonies, and remove the supernatant for antibody testing.

[0086] The cells from the antibody-positive wells were cultured and frozen. At the same time, the cells from the positive wells were cloned and the cloned monoclonal antibodies were identified by ELISA. The monoclonal cell line with the highest titer of anti-EGF antibodies 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, 200 μl was injected intraperitoneally. 7-10 days later, ascites was collected from the mice. The ascites was purified via a Protein A chromatography column. The column was first equilibrated with Solution A (PB, pH 7.0). After loading the ascites sample onto the column, the column was linearly eluted with Solution B (0.1 M glycine-HCl, pH 2.7) until the antibody peak appeared. Finally, the purified antibody was adjusted to pH 7.2 with 1 M Tris-HCl, pH 8.0, and sterilized by filtration to obtain the purified S-172-4 monoclonal antibody.

[0089] Example 2: Monoclonal Antibody S-172-4 / Commercial Monoclonal Antibody Used to Detect 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 using Na2CO3-NaHCO3 buffer, respectively. Coat the ELISA plate with eight gradients of the recombinant EGF-CRM197 vaccine test sample and reference sample, with two 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 and add the 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, 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 the plate, add 100 μl / well of TMB, incubate in the dark for 15 min, add 50 μl / well of stop solution, and read at 450 nm.

[0093] (4) Result calculation

[0094] The OD values ​​of the recombinant EGF-CRM197 vaccine test product and the physical and chemical reference product 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 ranged from 7.8 to 1000 pg / ml, showing a good linear relationship; whereas the commercially available anti-human monoclonal antibody only showed a certain linear relationship between 125 and 1000 pg / ml, and its sensitivity was much lower than that of the S-172-4 antibody of the present invention.

[0101] Recombinant EGF-CRM197 samples were used as test samples. Three individuals were tested three times per batch, for a total of nine tests per batch. The analysis showed that the S-172-4 monoclonal antibody of the present invention, when used to detect the in vitro relative potency of the recombinant EGF-CRM197 vaccine, produced stable and reproducible results, with a coefficient of variation (CV) within 10%. In contrast, commercially available anti-human EGF antibodies exhibited poor reproducibility, with a coefficient of variation (CV) exceeding 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 individuals ranges from approximately 0 to 300 pg / ml (median approximately 150 pg / ml), while the EGF concentration in the peripheral blood serum of lung cancer patients ranges from approximately 0 to 500 pg / ml (median approximately 250 pg / ml). The mechanism of action of the recombinant EGF-CRM197 vaccine is to induce the production of neutralizing antibodies against EGF, thereby reducing serum EGF concentrations and achieving anti-tumor effects. Therefore, monitoring serum EGF levels is an important clinical indicator for evaluating the immune efficacy of the recombinant EGF-CRM197 vaccine. Currently available anti-human EGF antibodies are used to prepare quantitative ELISA kits for human EGF, but they generally only recognize nanogram (ng / ml) levels of EGF, which is insufficient for clinical use. Quantitative ELISA kits from imported R&D companies can be used to measure EGF levels in human serum, but they are priced high and intended for scientific research only. They do not have medical device approval and are therefore unsuitable for post-market clinical testing of the recombinant EGF-CRM197 vaccine. 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 a commercially available rabbit anti-human EGF polyclonal antibody (Cat. No. ab9695, abcam) as the detection antibody. The following procedures were also performed using a commercially available rabbit anti-human EGF monoclonal antibody (Cat. No. AF5148, affinitybioseience) as the capture antibody and a commercially available rabbit anti-human EGF polyclonal antibody (Cat. No. ab9695, abcam) as the detection antibody.

[0107] Dilute the S-172-4 monoclonal antibody to a final concentration of 5 μg / ml using NaCO3-NaHCO3 buffer (pH 9.6), add it to a 96-well microplate at 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. The plate was 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, and 7.8 pg / ml, with the buffer 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, and perform 2 wells in parallel. Cover with the 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 (100 μl / well) was added, and incubated at 37°C for 1 hour.

[0113] The plate was washed three times with PBST, patted dry, and 100 μl / well of TMB was added. After incubation for 15 min in the dark, 50 μl / well of stop solution was added and the plate was 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 average, 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 with 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 with 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 standards measured by human EGF quantitative ELISA kit prepared with S-172-4 monoclonal antibody and commercially available anti-human EGF antibody

[0117]

[0118] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection 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 regions 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 regions CDR: VL-CDR1 of SEQ ID NO: 10, VL-CDR2 of the amino acid sequence LAS, and VL-CDR3 of SEQ ID NO:

12.

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

1.

3. The antibody according to claim 1, wherein The amino acid sequence of the antibody light chain variable region is SEQ ID NO: 9.

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