WO2017114204A1 - Monoclonal antibody of anti-podocalyxin-like protein precursor subtype 2 and preparation method and use thereof - Google Patents

Abstract

Provided are a monoclonal antibody of a podocalyxin-like protein precursor subtype 2 (PODXL-v2) functionally expressing an anti-gastric cancer cell surface and a preparation method and use thereof, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 2 or the conservative variation sequence thereof, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4 or the conservative variation sequence thereof.

Description

[Invention name established by ISA according to Rule 37.2] Monoclonal antibody against foot-like protein precursor subtype 2, preparation method and use thereof Technical field

The present invention relates to the field of biopharmaceuticals, and in particular to a monoclonal antibody (designated as MS17-38) of an anthraquinone-like protein precursor subtype 2 (PODXL-v2) which is functionally expressed on the surface of gastric cancer cells and a preparation method thereof use.

Background technique

As described in the inventor Lu Meisheng et al., the previously applied patent "monoclonal antibody MS17-57 against cell surface ectopic expression and its preparation method and use" (patent application number: 201310090565.9, published and approved), Gastric cancer (GC) is the most common digestive system malignancy in humans and one of the leading causes of tumor-related death. Countries such as China and Japan are the countries with the highest incidence of gastric cancer in the world, with nearly 600,000 to 700,000 new cases each year and accounting for the majority of the world's cases. Many patients with gastric cancer have been clinically advanced for the first time. Early screening and diagnosis of gastric cancer currently rely mainly on gastroscopy and histopathological biopsy, but the specificity, sensitivity, accuracy and safety of this early screening and diagnosis method are somewhat restrictive and difficult to popularize. The existence of many interfering factors and clinical application effects are not satisfactory. Therefore, most patients with gastric cancer have been transferred to other organs at the time of initial diagnosis and must rely on surgery, radiotherapy and systemic chemotherapy. These traditional treatments or remedies are not only expensive, but also greatly increase the financial burden on individuals and society, and there are problems such as the specificity of treatment and unsatisfactory efficacy. According to incomplete statistics, the five-year survival rate of patients with advanced gastric cancer usually does not exceed 10%, so new specific and effective clinical treatment methods for gastric cancer are imminent. In recent years, although some new chemotherapeutic drugs and combination therapy (individualized medical treatment) have improved the response rate of gastric cancer patients, they have not reached the median survival in randomized clinical research surveys. The treatment goal of more than one year.

Because chemotherapy and radiotherapy are generally insensitive to gastric cancer and lack effective alternative treatment options, the development of new gastric cancer treatment methods and drugs has become a hot topic in clinical and clinical research in recent years. In 2002, cancer experts at the National Cancer Institute (NCI) recommended the definition of molecular analysis of tumors, which would effectively achieve individualized treatment, recurrence detection, and prognosis assessment for cancer patients. Under the guidance of these new concepts, the rapid development of molecular targeted therapy and molecular biological function therapy for gastric cancer has also become a new trend in cancer therapy.

The inventor Lu Meisheng et al., the earlier application of the patent "anti-cell surface ectopic expression of monoclonal antibodies and preparation methods and uses thereof" (patent application number: 201310090565.9, published and approved) also describes tumor marker targeting and Biological functional therapy is the targeting of certain biomarker molecules that are overexpressed or functionally expressed on the surface of tumor cells. Since the 1990s, a variety of molecular targeted therapeutics (including antibody drugs) have been successively entered into clinical trials for cancer treatment and have shown good efficacy. They include two major classes of macromolecular monoclonal antibodies (acting on Extracellular therapeutic antibody drugs) and small molecule tyrosine kinase inhibitors (chemical drugs acting on cells). Because antibody drugs can be high Degree-specific recognition, binding to the corresponding tumor extracellular receptors and then stimulate the biological function of the cells, if not able to stimulate the function, there are several other antibody treatment methods 1. Antibody Drug Conjugation (ADC) 2. Specific radioimmunotherapy with antibodies combined with radioisotopes; 3. T cell immunotherapy with Chimeric Antigen Receptor (CAR). We classify these as Antibody Targeting Therapy (ATT) and Antibody Biofunctional Therapy (ABT), and the latter antibody biologic therapy (ABT) can be divided into antibodies and biological functions. Synergistic agonists (Agonist) and antagonists of antibodies and biological functions (Antagonist). The biological function of the antibody mainly targets the related molecules of tumor cell development to achieve specific killing of the tumor, preventing metastasis or interference with its growth microenvironment. Therefore, compared with traditional chemotherapy, radiotherapy and traditional Chinese medicine, the antibody drug effect is more obvious, the utility period is long and the side effects are low. Antibody biologic therapy of tumors will become the main direction of cancer treatment development.

The inventor Lu Meisheng et al., the earlier application of the patent "monoclonal antibody against ectopic expression on the cell surface and its preparation method and use" (patent application number: 201310090565.9, published and approved) also describes the background of gastric cancer antibody therapy. Antibody drugs have rapidly developed in recent years in the treatment of gastric cancer. At present, some antibody drugs approved by the US FDA have been approved. Their preclinical research toxicology tests and clinical reports of various stages show that the specificity is not high, the targeting is not strong, and the actual clinical treatment effect is generally not ideal. Drugs include the anti-epidermal growth factor receptor (EGFR) Cetuximab monoclonal antibody Erbitux (Erbitux), anti-EGF receptor-2 (EGFR2) or HER2 as the target of trastuzum ( Trastuzumab) Herceptin, a monoclonal antibody, and Avastin, a target of anti-vascular endothelial growth factor (VEGF) targeting Bevacizumab. Therefore, we must prepare high-efficiency monoclonal antibody drugs that target the biological functions of gastric cancer, in order to meet the urgent needs of current cancer clinical medicine. In the world, scholar Brichory first introduced proteomic peptide synthesis or construction methods into the field of identification and screening of tumor-associated antigens and anti-tumor autoantibodies. Through this idea and method, many new intracellular and extracellular tumors have been revealed. Marker protein molecules such as Glucose Regulated Protein (GRP78), Heat Shock Protein-27, -60, -70, etc. (Heat Shock Protein, HSP-27, -60, -70etc.) and fibrin peptides A (Fibrin Peptide-A) and the like. However, this approach also brings a lot of limitations, because tumor biological functional targets not only have cell surface specific antigens, but also must function with protein spatial structure or natural conformation, which greatly limits the further thinking of Brichory. Research and application. Undoubtedly, the research invention is to identify the surface specific antigen of gastric cancer or other tumor cells by live cell immunization and live cell high-throughput flow cytometry (FACS) screening in the natural state of human organisms, so as to capture and prepare specific and efficient. Therapeutic antibody drugs provide a powerful tool for the identification of gastric cancer molecular markers and the clinical application of gastric cancer targeted therapy monoclonal antibodies.

Podocalyxin (PC) is mainly expressed on the surface of glomerular basement membrane podocytes (Podocyte). Glycoprotein, protein isoelectric point (pI) is less than 7, is acid and negatively charged. It plays a role in maintaining the filtration gap on the glomerular podocytes. Foot glycoprotein has played a role in the pathogenesis of platelet coagulation and early diabetes. However, Podocalyxin-Like protein (PODXL) is a member protein of the salivary mucin family unique to humans and encoded by the PODXL gene. The ankle-like protein has a complex with a sodium pump/hydrogen pump exchange factor of intracellular structural elements and plays a role in the differentiation of hematopoietic cells. It can express on vascular endothelial cells and bind to L-selectin. PODXL has a number of different amino acid sequences, subtypes of different lengths of polypeptide fragments and is expressed on different tissues, cells and tumors.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an anthraquinone-like protein precursor subtype 2 (Simplified as: PODXL-v2; The monoclonal antibody of Precursor, PODXL-v2) and its preparation method and use are used to solve the problems in the prior art.

To achieve the above and other related objects, a first aspect of the present invention provides a monoclonal antibody (MS17-38) of an anthraquinone-like protein precursor subtype 2 (PODXL-v2) which is functionally expressed on the surface of a gastric cancer cell. The amino acid sequence of the variable region of the antibody light chain is SEQ ID NO: 2 or a conservative variant thereof, and the amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 4 or a conservative variant thereof.

Preferably, the coding sequence of the light chain variable region is SEQ ID NO: 1 or a conservative variant thereof, and the coding sequence of the heavy chain variable region is SEQ ID NO: 3 or a conservative variant thereof.

Preferably, the MS17-38 monoclonal antibody is murine.

More preferably, the MS17-38 monoclonal antibody is an immunoglobulin of the IgGl heavy chain and the kappa light chain subtype.

The MS17-38 monoclonal antibody provided by the present invention is a PODXL-v2 antigen (or receptor, or epitope), or a partial anti-binding (binding or acting) PODXL which is resistant to (binding or acting on) the surface functional expression of a tumor cell. A monoclonal antibody that is antigen, or partially resistant (binding or acting) to the PODXL-v1 antigen.

The present invention further provides a derivative of the MS17-38 monoclonal antibody, which may be the MS17-38 monoclonal antibody fragment, or the MS17-38 monoclonal antibody or the MS17-38 monoclonal antibody fragment. The fusion protein, the fragment of the monoclonal antibody is Fab, Fab', F(ab') ₂ , Fv or scFv, and the like.

A second aspect of the invention provides an isolated DNA molecule encoding a variable or full length amino acid of the heavy and/or light chain of the MS17-38 monoclonal antibody.

A third aspect of the invention provides a construct comprising the isolated DNA molecule.

Preferably, the DNA vector expression construct is inserted into the expression vector by the isolated antibody DNA molecule The dragon site is constructed.

The expression vector may specifically be a conventional expression vector well known to those skilled in the art, and specific expression vectors include, but not limited to, a pET series expression vector, a pGEX series expression vector, a pcDNA series expression vector, and the like.

A fourth aspect of the invention provides a monoclonal antibody expression system constructed by transfecting the construct into a host cell.

Any cell suitable for expression vectors (constructs) for expression of the antibodies described in this patent can be used as a host cell. For example, cells of yeast, insects, plants, and the like. Preferably, the host cell is a eukaryotic cell, and a mammalian host cell line which does not produce an antibody may be used, and specific cell lines include, but not limited to, Chinese hamster ovary cells (CHO), kidney cells of baby hamsters. (BHK, ATCC CCL 10), Sertoli cells of young rats, kidney cells of monkeys (COS cells), kidney CVI cells of monkeys transformed by SV40 (COS-7, ATCC CRL 165 1), Human embryonic kidney cells (HEK-293), monkey kidney cells (CVI, ATCC CCL-70), kidney cells of African green monkeys (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC) CCL-2) and so on.

The invention generates an antibody by high-throughput screening of tumor living cells by mixing a human gastric cancer cell line with a living cell to immunize a mouse and selecting a high immunoreactive mouse spleen and a mouse SP2/0 cell line to produce an antibody. Hybridoma cells are obtained by screening high-throughput live cells of hybridoma antibody anti-cancer cell lines and screening for normal human peripheral blood mononuclear leukocytes (PBMC) to obtain hybridoma cells with high gastric cancer cells. A monoclonal antibody MS17-38 that specifically reacts. In the present invention, the gene coding sequence of the antibody of interest is screened from the monoclonal cultured cell line to construct an expression vector, and the activity of the antibody can be reconstructed by expression system to obtain the MS17-38 monoclonal antibody.

The MS17-38 monoclonal antibody was obtained by screening according to the following steps: four mixed gastric cancer cell lines SGC7901, BGC823, MKN28, MKN45 live cells were used to immunize mice, and the immunized mouse spleen cells and mouse myeloma cells were used. Fusion, screening for a hybridoma cell strain capable of binding to the above four gastric cancer living cell surface antigens without reacting with human normal peripheral blood mononuclear cells, subcloning the hybridoma cell line to obtain the cultured hybridization The monoclonal antibody MS17-38 was obtained by affinity purification of the supernatant of the tumor cells. In an embodiment of the present invention, the four mixed gastric cancer cell lines SGC7901, BGC823, MKN28, and MKN45 are preferably mixed in equal proportion, and the mouse myeloma cells are SP2/0 mouse myeloma cells, and the fusion is performed. The specific method is to chemically fuse by PEG, and the hybridoma cell culture supernatant is purified by immunoaffinity chromatography.

A fifth aspect of the present invention provides a method for producing the MS17-38 monoclonal antibody, comprising the steps of: culturing an expression system of the monoclonal antibody under conditions suitable for expressing the antibody, thereby expressing the single The antibody was cloned and the monoclonal antibody was isolated by purification.

After obtaining the nucleic acid sequence encoding the antibody of the present invention, the antibody of interest can be produced by the following method. For example, a vector containing a nucleic acid encoding an antibody of interest is directly introduced into a host cell, and the cells are cultured under appropriate conditions to induce expression of the encoded antibody. The expression vector and the host cell used in the present invention are both prior art and can be directly obtained by a commercial method. The DMEM medium of 10% FBS used in the culture is also various conventional mammalian cell culture media, and those skilled in the art can The appropriate DMEM medium is selected empirically and cultured under conditions suitable for host cell growth. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time. The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. Once the monoclonal antibodies of the present invention are obtained, the monoclonal antibodies can be isolated 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 such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

A sixth aspect of the present invention provides the use of the MS17-38 monoclonal antibody in the preparation or screening of a therapeutic drug for tumors, or the use of a medicament for preparing a tumor diagnostic.

The tumor therapeutic drug specifically refers to a drug which treats cancer and prevents primary cancer metastasis by using PODXL-v2 functionally expressed on the surface of a tumor cell as an antigen, binding or acting on the antigen PODXL-v2. The binding or acting on the antigen PODXL-v2 specifically includes, but is not limited to, immunosuppressing the PODXL-v2 antigen, or recognizing the tumor antigen by a specific monoclonal antibody, and concentrating the tumor therapeutic drug to the tumor site, selecting Sexual killing of tumor cells and the like.

Therefore, the tumor diagnostic drug specific target for tumor cells target PODXL-v2, with PODXL-v2 as a biomarker, differential diagnosis, pathological diagnosis, early diagnosis, imaging diagnosis and prognosis diagnosis and other diagnostic reagents.

Preferably, the tumor is gastric cancer.

More preferably, the gastric cancer is gastric squamous cell carcinoma, gastric adenocarcinoma, gastric small cell carcinoma, gastric adenosquamous carcinoma, gastric carcinoid or gastric and duodenal cancer.

A seventh aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the MS17-38 monoclonal antibody or an immunoconjugate thereof.

The immunoconjugates include, but are not limited to, conjugates of the monoclonal antibodies or fragments thereof in combination with drugs, toxins, cytokines, radionuclides, enzymes, or other diagnostic agents.

The pharmaceutical composition binds or acts on the antigen with PODXL-v2 functionally expressed on the surface of a tumor cell, thereby treating cancer and/or preventing primary cancer metastasis.

The MS17-38 monoclonal antibody of the present invention can be used by formulating a pharmaceutical composition by any means known in the art. Such compositions have the monoclonal antibody as the active ingredient plus one or more pharmaceutically acceptable carriers or excipients which are compatible with the monoclonal antibody.

The pharmaceutical composition is for treating gastric cancer, preferably for treating one or more of gastric squamous cell carcinoma, gastric adenocarcinoma, gastric small cell carcinoma, gastric adenosquamous carcinoma, gastric carcinoid or gastric and duodenal cancer The combination. When the pharmaceutical composition is used for preventing or treating a tumor in a subject, an effective amount of the pharmaceutical composition can be administered to a subject.

An eighth aspect of the invention provides a diagnostic kit comprising a diagnostically effective amount of the MS17-38 monoclonal antibody or an immunoconjugate thereof.

Therefore, the diagnostic kit targets the target cell of PODXL-v2, and uses PODXL-v2 as a biomarker for differential diagnosis, pathological diagnosis, early diagnosis, imaging diagnosis and prognosis diagnosis.

Preferably, the diagnostic kit further comprises a marker for the MS17-38 monoclonal antibody.

The label of the MS17-38 monoclonal antibody binds to the MS17-38 monoclonal antibody, and the types of labels that can be used include, but are not limited to, fluorescent labels, radioactive labels, enzyme labeling agents, chemiluminescent labels. One or more combinations.

Depending on the detection principle of the kit, the kit may also contain one or more reagents required for detection. In addition, the kit may also include, as needed, a container, a control (negative or positive), a buffer, an adjuvant, and the like.

The invention obtains an anti-cancer monoclonal antibody, and further screens and identifies the antigen on the surface of the gastric cancer cell, and obtains a new biomarker antigen. Specifically, the present invention has prepared a specific MS17-38 monoclonal antibody against this marker antigen (the natural conformation of the gastric cancer cell surface PODXL-v2 antigen) while discovering and identifying a tumor biomarker, and according to the specific antibody Screening identified its corresponding specific gastric cancer molecular marker: cell membrane expressed protein PODXL-v2.

The present invention aims to realize the screening and identification of new monoclonal antibodies specific for surface molecular markers against gastric cancer cells by using a high-throughput screening (HTS) antibody to reversely identify a tumor cell surface-expressing antigen-binding strategy. Molecular markers of gastric cancer. Monoclonal antibodies are a good proteomics research tool. The traditional monoclonal antibody preparation pathway is an antibody hybridoma method, usually immunized with non-molecular markers, and less HTS method using a large number of plates after fusion, thus obtaining anti-cells. The probability of antigenic natural epitope antibodies is low (traditional antibody screening methods are difficult to obtain antibodies that recognize native conformational antigens on the surface of tumor living cells). The invention adopts a "shot-Gun" method for living cell immunization, combines antibody hybridoma technology with high-throughput detection by flow cytometry, and immunizes mice through tumor living cells, using a unique antibody hybridization. Tumor high fusion rate method for fusion, large-scale plating (50 to 80 plates each time) combined with live cell flow cytometry (FACS)-HTS detection screening method, direct screening of anti-cell surface at the living cell level Monoclonal antibody with a conformational epitope (Conformational Epitope) molecular marker, and by review and identification of antibody hybridoma cells Cloning, finally selected specific high affinity monoclonal antibody: MS17-38 specific antibody against the natural conformation PODXL-v2 antigen on the surface of gastric cancer cells. After the identification of MS17-38 antibody and the correlation analysis between MS17-38 antibody and clinicopathological parameters of gastric cancer, the specific binding target of MS17-38 antibody was prepared by Western blotting, immunoprecipitation and protein profiling. The rapid identification of antigenic proteins led to the screening and discovery of this PODXL-v2 new surface marker for gastric cancer cells.

The MS17-38 monoclonal antibody of the present invention can specifically target the foot-like protein precursor subtype 2 (PODXL-v2) molecule which has a molecular weight of 135 kDa and is naturally expressed on the surface of tumor cells and has a spatial isoform. The reaction does not react with the molecular linear body protein of the PODXL-v2 monomer having a molecular weight of about 51 kDa. In the specific monoclonal antibody hybridoma screening design of the present invention, the expressed monoclonal antibody is secreted by a monoclonal antibody hybridoma cell line named MS17-38, and the subtype of the mouse monoclonal antibody belongs to the IgG1 heavy weight. Chain and kappa light chain.

The present invention relates to the correlation between the monoclonal antibody specific to the prepared anti-gastric cancer mixed cell line and the clinicopathological parameters of gastric cancer and other digestive tract tumors, and then acts on the MSD-v2 molecule on the surface of the tumor cell by the MS17-38 monoclonal antibody phase. The function was analyzed and it was found that MS17-38 monoclonal antibody is produced against the surface PODXL antigen of a group of human gastric cancer cells, and can induce specific and functional biological reactions, and can specifically recognize gastric cancer and other digestive tract tumors and target. To the treatment, it can be applied to the diagnosis and imaging of digestive tract tumors. The invention establishes a novel method for preparing high-throughput anti-stomach and natural conformation antigen-specific antibodies on the surface of solid tumor cells of digestive tract, and obtains MS17-38 monoclonal antibody as a proteomics research tool to identify and identify gastric cancer by reverse screening. Isotope tract solid tumor cell surface molecular targeting markers. The monoclonal antibody can be further developed into a biologic agent for the treatment of human gastric cancer after human/mouse chimerization or humanization.

DRAWINGS

Figure 1 is a graph showing the titer reaction of mouse serum after immunization with four mixed gastric cancer cell lines with normal human PBMC and gastric cancer SGC7901 and BGC823 cell lines.

Fig. 2 shows the different degree of binding reaction of MS17-38 monoclonal antibody to four kinds of immune gastric cancer cell lines and other control cell lines by FACS. Among them, the binding reaction of A, MS17-38 mAb and control antibody with SGC-7901 and BGC-823 gastric cancer cell lines. B, MS17-38 mAb and control antibody were combined with MKN-45, BGC-823, normal human peripheral blood PBMC cells, respectively.

Figure 3 and Figure 4 show the different degrees of binding reaction of MS17-38 monoclonal antibody to two gastric cancer cell lines and two control cell lines by FACS. Among them, Figure 3 shows the response of MS17-38 mAb and control antibody to MKN-28, AGS-N cell lines. Figure 4 shows the binding reaction of MS17-38 mAb and control antibody to normal human peripheral blood PBMC cells and fetal gastric mucosal epithelial transformed cell line GES-1, respectively.

Figure 5 is an ELISA assay for the binding of MS17-38 monoclonal antibody to gastric cancer BGC823 cell membrane extracting protein.

Figure 6 is an ELISA assay for the binding of MS17-38 monoclonal antibody to gastric cancer MKN45 cell membrane extracting protein.

Figure 7 shows the immunohistochemical reaction of MS17-38 monoclonal antibody and homologous monoclonal antibody in A. gastric mucosal transformed forming cells GES-1, B. gastric cancer cells MKN-45, C. gastric cancer cells BGC-823.

Figure 8 is a purified band of SDS-PAGE gel of MS17-38 monoclonal antibody against gastric cancer BGC823 and MKN45 cell membrane extracting target proteins.

Figure 9 shows the results of three mass spectrometry analyses of gastric cancer BGC823 and MKN45 cell membrane extracting target proteins by MS17-38 monoclonal antibody.

Figure 10 is an MS17-38 mAb analysis of 6-mer and 8-mer amino acid stacked micromatrix chips.

Figure 11 is a comparison of the amino acid sequences of PODXL, PODXL-v1 and PODXL-v2 and the difference between them. The specific binding of MS17-38 mAb to the PODXL-v2 spatial conformation site is two sites.

Figure 12 is a Western Blot assay showing that PODXL-v2 siRNA interferes with the expression of PODXL-v2 on MKN45 cell membranes.

detailed description

The embodiments of the present invention are described below by way of specific examples, and those skilled in the art can readily understand other advantages and effects of the present invention from the disclosure of the present disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes can be made without departing from the spirit and scope of the invention.

Before the present invention is further described, it is to be understood that the scope of the present invention is not limited to the specific embodiments described below; The singular forms "a", "the", and "the"

When the numerical values are given by the examples, it is to be understood that the two endpoints of each numerical range and any one of the two. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning In addition to the specific methods, devices, and materials used in the embodiments, the methods, devices, and materials described in the embodiments of the present invention may also be used according to the prior art and the description of the present invention by those skilled in the art. Any method, apparatus, and material of the prior art, similar or equivalent, is used to practice the invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and the conventional techniques in the art. Conventional techniques in the related art. These techniques are well described in the prior literature, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin ( PMWassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.

Example 1

Four gastric cancer cells (BGC-823, MKN-28, MKN-45 and SGC-7901, all from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were mixed in four equal proportions in DMEM medium containing 10% FBS. After neutralization in 5% CO ₂ , 37 ° C environment, the collected live cells were mixed in PBS buffer as an immunogen in A/J-JAX mice (purchased at the Experimental Animal Model Center of Nanjing University, mouse source) Immunized in the back subcutaneous and tail veins at The Jackson Laboratory, USA, 3-5 million cells per mouse per point (in 0.1 ml). Immunization once every other week; after 1 week of the third immunization, the mouse serum was taken, and the flow cytometry high-throughput system (FACS-HTS) was used to detect the reaction between the serum and the four mixed gastric cancer cells (see Example 3 for details). Healthy volunteer PBMC as a control cell [Peripheral Blood Mononuclear Cells (PBMC) were isolated from the peripheral blood of healthy volunteers by Ficoll solution and used as a normal high-throughput screening (FACS-HTS) in fluorescence flow cytometry. Human cell antigen control screening]. Pre-fusion booster immunization was performed in mice with a higher serum titer and a lower degree of cross-reactivity with control cell PBMC (the difference between the mean fluorescence intensity values of serum and gastric cancer cells and control cells at the same dilution ratio > 500).

The spleen of the boosted mouse was taken, and the spleen cells of the immunoreactive mouse No. 1 were prepared into a single cell suspension in serum-free DMEM medium; under 50% PEG (pH 7.4), Splenocytes were fused with SP2/0 mouse myeloma cells, fused and plated in large batches (50 plates) and cultured for 10 days in HAT selective medium to the formation of antibody hybridoma cell clones. The above four gastric cancer cell lines were collected and mixed for large-scale culture, and normal human peripheral blood PBMC cells were isolated as control control cells, and two groups of cells (the first group was mixed with four gastric cancer cells in equal proportions, and the second group was Normal human peripheral blood PBMC cells were resuspended in 1.5% BSA/PBS blocking solution pre-cooled on ice cubes and evenly distributed (about 100,000 to 200,000 cells per well). 51 96-well U-types were added. In the plate [total 102 plates, two 51 plates were positive (immunized mouse serum and gradient dilution) and negative (normal mouse serum and gradient dilution and HAT selective medium) control plate].

The supernatant of 50 antibody hybridoma cell fusion plates (96-well plates) (corresponding to the first antibody, ie, the original monoclonal antibody) was transferred to the corresponding screening plate and control plate at 70 μL/well each time and shaken and mixed. After the reaction in an ice bath and washing with a blocking solution, a cell fluorescence fluorescent staining reaction was carried out by adding 100 μl/well of FITC fluorescently labeled goat anti-mouse, thereby high-throughput screening by fluorescent flow cytometry ( FACS-HTS), the FACS parameters were adjusted with the blank control wells and the cells of the isotype control wells as the background, and the cell samples of each well of the 96-well U-shaped plates were subjected to FACS detection one by one. At the same time, the following two conditions are determined as positive cell pores: (1) binding reaction with 4 gastric cancer cell surface antigens (ie, the sample signal peak shift amplitude is greater than one logarithmic value compared with the isotype control signal peak); 2) No binding reaction with PBMC cells (ie, the sample signal peak shift amplitude is less than 5-10% compared to the isotype control signal peak). Selection and selection of unique hybridoma high-throughput screening cells, transition of selective medium and common 10% FBS DMEM medium, subcloning of hybridoma cells and multiple culture supernatants of antibody hybridoma cells For the detection, the hybridoma cell supernatant corresponding to MS-17-38 mAb was affinity-purified and filtered through a 0.2 micron membrane and stored at 4 ° C aseptically, or 50% glycerol was stored at -20 ° C for a long time.

Example 2

Total RNA was extracted from MS17-38 monoclonal antibody hybridoma cell lines using Qiagen (Valencia, CA) RNeasy kit, and mRNA was inverted using Invitrogen (Grand Island, NY) SuperScript III First-Strand kit. A cDNA library of MS17-38 mAb was recorded. 21 specific primer-paired PCR reactions (reactions without λ light chain) were performed using 23 primers and experimental methods contained in the "Mouse IgG Library Primer Set" (F2010) kit from Progen Biotechnik, Germany. Specific light and heavy chain products are subjected to DNA sequencing, translation of amino acid polypeptide sequences, and identification of CDRs (antigenic determinant regions) and FW (skeletal regions). The specific results are as follows:

The coding sequence of the variable region of the MS17-38 mAb light chain is set forth in SEQ ID NO: 1:

The amino acid sequence of the variable region of the MS17-38 mAb light chain is set forth in SEQ ID NO: 2:

The coding sequence of the variable region of the MS17-38 mAb heavy chain is set forth in SEQ ID NO: 3:

The amino acid sequence of the variable region of the MS17-38 mAb heavy chain is set forth in SEQ ID NO: 4:

The coding sequences SEQ ID NO: 1, SEQ ID NO: 3 correspond to the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The underline in the amino acid sequence indicates the position of the CDR region, which is arranged in the order of CDR1, CDR2 and CDR3 and the region sequence between them is the cytoskeletal sequence (FW).

Example 3

Construction of full-length eukaryotic expression vector of MS17-38 mAb and establishment of its expression cell line:

The DNA sequence encoding the variable region of the light chain and the variable region of the heavy chain was carried out using a PCR reaction.

Amplification, introduction of appropriate restriction sites at both ends of the gene of the antibody heavy chain variable region and the light chain variable region. After PCR amplification, the PCR products of the light chain variable region gene and the heavy chain variable region gene were recovered and purified by agarose gel electrophoresis. The amplification products of the light chain and the heavy chain are respectively added to the corresponding restriction enzymes, and the digestion products are purified by the DNA recovery and purification kit, and the heavy chain (VH) and light chain (VL) products obtained by PCR are combined with human IgG1CH1. And the intermediate carrier of CL (pGEM-T) The ligation reaction was carried out to obtain the intermediate vectors pGEM-T-H and pGEM-T-L, respectively, and the obtained VL+CL and VH+CH1 genes were recombined into the vector pcDNA3.1. The ligation product was transformed into DH5α E. coli and plated on 2YT agar medium containing 50 μg/ml carbenicillin. The obtained positive clones were cultured in 2YT liquid medium containing 50 μg/ml carbenicillin, and after positive sequencing by Invitrogen, the positive clone plasmid was extracted using the plasmid large-draw kit.

Linearized plasmid DNA was transfected into CHO cells using Invitrogen's Neon system. The transfected CHO cells were subjected to dilution and cloning culture, and screened in 94113 medium (product of Irvine Scientific) containing 50 μmol (μmol) of methionine sulfoximine (MSX) to obtain a monoclonal antibody. Antibody expressing cell line.

The obtained monoclonal cell line was subjected to shake flask culture in 94113 medium containing 50 μmol of methionine iminosulfone, and the cell culture supernatant was harvested when the viable cell density was less than 30%. The antibody of interest was isolated and purified from the cell culture supernatant using a Protein A affinity chromatography column.

RNA was extracted from the monoclonal cells, and the target gene was detected, and the number of copies of the target gene of the obtained monoclonal cells was confirmed, and the MS17-38 monoclonal antibody was verified. N-terminal sequencing of the antibody protein was carried out, and the results were identical to those of the antibody hybridoma cell line.

Example 4

On a 96-well "U" plate, an average of about 200,000 different gastric cancer cells per cell (SGC7901 and BGC823 cells of gastric cancer, normal human PBMC as a control) / 100 μl were added with 1% BSA/PBS. In the plate, the serum of the gastric cancer live cell immunization (the serum of the immunized mouse obtained in Example 1) was serially diluted in a 5-fold titer, and then 100 μl (μL) was added to each well, and the mixture was iced. Or reaction at 4 ° C for 20 minutes, after 2 washes, add 1:333 diluted goat anti-mouse IgGFc-FITC 100 μL / well, 4 ° C reaction and wash, BD LSR-II fluorescence flow cytometry - HTS The average fluorescence intensity value (MFI) of each well was read on board.

The results showed that the titer response of the serum of the mouse 1 with high immunoreactivity was significantly higher than that of the normal human PBMC. The mouse spleen cells were used for the hybridization of MS17-38 monoclonal antibody. Fusion experiment. (As shown in Fig. 1FACS, the titer response of mouse serum after immunization with four mixed gastric cancer cell lines to normal human PBMC, gastric cancer SGC7901 and BGC823 cell lines is shown).

Example 5

The affinity-purified MS17-38 mAb was subjected to the combined staining reaction of the four immunological gastric cancer cell lines by the U-plate staining method described in Example 4, and was read on the LSR-II FACS instrument. MFI. The other experimental steps were the same as in Example 4.

The results showed that FACS had different degrees of binding reaction to MS17-38 monoclonal antibody and three kinds of immune gastric cancer cell lines, among which MS17-38 monoclonal antibody had the highest response to MKN-45 gastric cancer cell line, but to SGC-7901 cells and BGC. The -823 cell reaction was relatively low, and there was no binding reaction with the normal PBMC reactivity (Fig. 2FACS for MS17-38 monoclonal antibody reacted with different immunosuppressive gastric cancer cell lines and normal PBMC cells, respectively.) A, MS17 -38 monoclonal antibody and control antibody (unrelated homozygous mouse monoclonal antibody, IgG1 heavy chain, Kappa light chain, the same control antibody in the latter statement are the same control monoclonal antibody) combined with SGC-7901 and BGC-823 gastric cancer cell lines Reaction B. MS17-38 mAb and control antibody are shown in the binding reaction with MKN-45, BGC-823, normal human peripheral blood PBMC cells, respectively).

Example 6

The affinity-purified MS17-38 monoclonal antibody was subjected to a binding staining reaction to normal human PBMC, gastric cancer cell lines MKN-28, GES-1, AGS-N by the U-plate staining method described in Example 4. The MFI value of the FITC fluorescence was read on an LSR-II FACS instrument.

The results indicated that MS17-38 monoclonal antibody had high binding reactivity to GES-1 and AGS cell lines, but no binding reaction with normal human PBMC, and the isotype control unrelated monoclonal antibody was a negative control without binding reaction. Figure 3 and Figure 4 show the different degrees of binding reaction of MS17-38 monoclonal antibody to two gastric cancer cell lines and two control cell lines by FACS. Among them, Figure 3 shows the response of MS17-38 mAb and control antibody to MKN-28, AGS-N cell lines. Figure 4 shows the binding reaction of MS17-38 monoclonal antibody and control antibody to normal human peripheral blood PBMC cells and GES-1 (fetal gastric epithelial transformed cells) cell lines, respectively.

Example 7

The affinity-purified MS17-38 monoclonal antibody was extracted from the gastric cancer cell line BGC823 and the gastric cancer cell line MKN-45, respectively (previously coated in the Immunon-II 96-well ELISA plate of Fisher Scientific, USA) A series of enzyme-linked binding reactions such as ELISA were performed, and OD values and mapping were read at 450-nm optical density on an ELISA plate reader.

The results of ELISA showed that MS17-38 monoclonal antibody did not bind to gastric cancer BGC823 and gastric cancer MKN-45 membrane extracting protein, indicating that MS17-38 monoclonal antibody could not react with the bound target protein after degradation on the attached ELISA plate, suggesting MS17- The specific reaction of 38 mAb with the conformational epitope antigen, which was prepared for the specific working conditions of the subsequent antibody co-immunoprecipitation (MS17-38 monoclonal antibody and gastric cancer BGC823 (Fig.-5) and gastric cancer MKN-45 ( As shown in the ELISA assay for cell membrane extracting protein binding as shown in Figure -6).

Example 8

Gastric mucosal transformed cells GES-1 and gastric cancer cells BGC823 and MKN45 were centrifuged (Cytospin) In combination with MS17-38 mAb, the catalase staining reaction showed that the target proteins were distributed on the cell membrane surface (magnification 40x, respectively) (immunohistochemistry). The gastric mucosal transformed cells GES-1 were centrifuged (Cytospin) and then combined with the MS17-38 monoclonal antibody obtained by affinity purification of the culture supernatant. The catalase staining reaction showed that the monoclonal antibody could interact with the surface of the cell membrane. Target protein binding.

The results of immunohistochemistry showed that MS17-38 monoclonal antibody could bind to the surface of cell membrane of gastric mucosal transformed cells GES-1 and gastric cancer cells BGC823 and MKN45, which is also a proof of the targeting of MS17-38 mAb target protein (such as Figure 7 - MS17-38 mAb and isotype-independent monoclonal antibody in A. Gastric mucosal transformation forming cells GES-1, B. Gastric cancer cells MKN-45, C. Gastric cancer cells BGC-823 immunohistochemical reaction detection).

Example 9

Gastric cell line BGC823 and MKN45 cell membrane extracts were purified and co-immunoprecipitated (IP) with different molecular weights by MS17-38 monoclonal antibody affinity column (specifically coupled with MS17-38 monoclonal antibody affinity purification beads). The molecular marker samples were separately loaded and electrophoresed (SDS-PAGE). The results of SDS-PAGE bromophenol blue staining showed that the antigenic purity of the membrane extract proteins of gastric cancer MKN45 and BGC823 purified by MS17-38 monoclonal antibody was very high, which was consistent with further antigen mass spectrometry after excision of antigen strips. Claim. (Figure -8 MS17-38 monoclonal antibody showed a purified band of SDS-PAGE gel for gastric cancer BGC823 and MKN45 cell membrane extraction target protein).

Example 10

As described in Example 9, the indirect immunoprecipitation method is to purify the bound antigen of gastric cancer cells (MKN45 and BGC823) by affinity column of MS17-38 monoclonal antibody, and the Fc end of the antibody and Protein-A magnetic The beads specifically bind to and wash away the non-specifically adsorbed protein, and then the SDS-PAGE is loaded, and the buffer is heated to dissociate. Direct immunoprecipitation was performed by direct coupling of MS17-38 mAb to Dynabeads magnetic beads activated by Invitrogen, Inc. (Grand Island, NY, USA). The subsequent steps were the same as indirect immunoprecipitation. It has been shown that the immunoprecipitation method for MS17-38 mAb must use a direct method to obtain a specific and clear target band (as shown in Figure -8). Subsequent multiple high-sensitivity mass spectrometry confirmed that the corresponding target of MS17-38 monoclonal antibody was PODXL-v2 protein (Figure -9MS17-38 monoclonal antibody for three-dimensional mass spectrometry analysis of gastric cancer BGC823 and MKN45 cell membrane extracting target protein).

Example 11

According to the NCBI large database data, the 8-amino acid and 6-amino acid amino acid sequences of the longest one of the PODXL subtypes were repeatedly and overlapped on a microarray chip, and then fluorescently labeled with MS17-38 mAb. Indirect hybridization reaction of goat antibodies revealed that the two amino acid sequences of 221-224 and 473-477 have strong fluorescence. Light binding strength, indicating that MS17-38 mAb is bound to the isomer PODXL-v2 (Figure 10-MS17-38 mAb against 6-mer and 8-mer amino acid stack micromatrix chip analysis), while MS17-38 The monoclonal antibody specifically binds to the isoform PODXL-v2, because only PODXL-v2 lacks a sequence between the two spatial conformation binding sites, so that the formation of the two conformation binding sites needs to be spatially folded. Neither PODXL nor PODXL-v1 could form a conformational site for the binding of MS17-38 mAb to PODXL-v2 (Figure -11 is the alignment of PODXL, PODXL-v1 and PODXL-v2 amino acid sequences and the difference between them MS17-38 mAb binds specifically to the PODXL-v2 spatial conformation site by two sites).

Example 12

In the MKN-45 cell culture, each 25nM (nanomolar) final concentration of PODXL-v2 (product number s10769,), FAM120B, Sec16A, SMARTC1 and purchased by Ambion Lifetech (now ThermoFisher Scientific) was added. The blank control siRNA was transfected with siRNA to finally inhibit expression of each siRNA target protein. The treated cellular proteins were extracted and the bands of each protein were separated in SDS-PAGE, respectively. Specific reaction with MS17-38 mAb and β-Actin antibody (Western Blot control) revealed that only PODXL-v2 siRNA-treated MKN-45 cell-extracted protein can bind to MS17-38 monoclonal antibody. The v2 band disappeared, so it was demonstrated from another aspect that the MS17-38 mAb specifically reacted with PODXL-v2 (Fig. -12 Western Blot test showed that PODXL-v2 siRNA interfered with the expression of PODXL-v2 on the membrane of MKN45 cells).

In summary, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above-described embodiments are merely illustrative of the principles of the invention and its effects, and are not intended to limit the invention. Modifications or variations of the above-described embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and scope of the invention will be covered by the appended claims.

Claims (13)

1. A monoclonal antibody against the surface protein functional expression of gastric cancer cell surface subtype 2, wherein the amino acid sequence of the antibody light chain variable region is SEQ ID NO: 2 or a conservative variant thereof, and the antibody is heavy The amino acid sequence of the variable region of the chain is SEQ ID NO: 4 or a conservative variant thereof.
2. The monoclonal antibody according to claim 1, wherein the coding sequence of the light chain variable region is SEQ ID NO: 1 or a conservative variant thereof, and the coding sequence of the heavy chain variable region Is SEQ ID NO: 3 or its conservative variant sequence.
3. A monoclonal antibody according to claim 1 wherein said monoclonal antibody is murine.
4. A monoclonal antibody according to claim 1, wherein said monoclonal antibody is an immunoglobulin of an IgG1 heavy chain and a kappa light chain subtype.
5. An isolated DNA molecule encoding the variable or full length amino acid sequence of the heavy and/or light chain of the monoclonal antibody of any of claims 1-4.
6. A construct comprising the isolated DNA molecule of claim 5.
7. An expression system for a monoclonal antibody constructed by transfecting the construct of claim 6 into a host cell.
8. The method for producing a monoclonal antibody according to any one of claims 1 to 4, comprising the step of culturing an expression system of said monoclonal antibody under conditions suitable for expressing said antibody, thereby expressing said The monoclonal antibody is purified and the monoclonal antibody is isolated.
9. The use of the monoclonal antibody according to any one of claims 1 to 4 for the preparation or screening of a medicament for treating a tumor, or the use of a medicament for the diagnosis of a tumor.
10. The use according to claim 9, wherein the tumor is gastric cancer, more preferably gastric squamous cell carcinoma, gastric adenocarcinoma, gastric small cell carcinoma, gastric adenosquamous carcinoma, gastric carcinoid or stomach or stomach and twelve Refers to bowel cancer.
11. A pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody to an anthraquinone-like protein precursor subtype 2 that is functionally expressed on the surface of said gastric cancer cell or an immunoconjugate thereof.
12. The pharmaceutical composition of claim 11 further comprising one or more pharmaceutically acceptable carriers or excipients.
13. A diagnostic kit comprising the monoclonal antibody of the foot-like protein precursor subtype 2 of the surface functional expression of the gastric cancer cell or an immunoconjugate thereof.

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