Monoclonal antibody against podocalyx-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells and its preparation method and use

A monoclonal antibody, functional technology, applied in the field of biopharmaceuticals, can solve problems such as limiting the research and application of Brichory ideas

Active Publication Date: 2018-10-26
陆梅生 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also brings many limitations, because the tumor biological function target not only has cell surface specific antigens, but also must have protein space structure or natural conformation, which greatly limits the further development of Brichory's thinking. research and application

Method used

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  • Monoclonal antibody against podocalyx-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells and its preparation method and use
  • Monoclonal antibody against podocalyx-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells and its preparation method and use
  • Monoclonal antibody against podocalyx-like protein precursor subtype 2 functionally expressed on the surface of gastric cancer cells and its preparation method and use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Four gastric cancer cell lines mixed in equal proportions (BGC-823, MKN-28, MKN-45 and SGC-7901, all from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in DMEM medium containing 10% FBS. Neutralized at 5% CO 2 , After culturing at 37°C, the collected living cells were mixed in PBS buffer as an immunogen, and tested in A / J-JAX mice (purchased from the Experimental Animal Model Center of Nanjing University, and the mice were from The Jackson Laboratory, the United States) Immunization was carried out by subcutaneous injection in the back and tail vein, 3-5 million cells (in 0.1 ml) per mouse per spot. , immunized once every other week; one week after 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 4 strains of mixed gastric cancer cells (see Example 3 for details). ), PBMC of healthy volunteers were used as control cells [...

Embodiment 2

[0063] Total RNA was extracted from the MS17-38 monoclonal antibody hybridoma cell line with the RNeasy kit of Qiagen (Valencia, California, USA), and the mRNA was reversed with the SuperScript III First-Strand kit of Invitrogen (Grand Island, New York, USA). The cDNA library of MS17-38 monoclonal antibody was recorded. Using 23 primers and experimental methods contained in the Progen Biotechnik company "Mouse IgG Library Primer Set" (F2010) kit in Germany to carry out 21 specific primer pairing PCR reactions (reactions that do not contain λ light chain), the resulting Specific light and heavy chain products were subjected to DNA sequencing, translation of amino acid polypeptide sequences, and identification of CDRs (antigen determinant regions) and FW (framework regions). The specific results are as follows: The coding sequence of the variable region of the MS17-38 mAb light chain As shown in SEQ ID NO: 1:

[0064]

[0065] (SEQ ID NO: 1)

[0066] The amino acid sequence...

Embodiment 3

[0075] Construction of full-length eukaryotic expression vector of MS17-38 monoclonal antibody and establishment of expression cell line:

[0076] The DNA sequences encoding the light chain variable region and the heavy chain variable region described above were subjected to PCR reactions.

[0077] Amplification, introducing appropriate enzyme cleavage sites at both ends of the gene of the antibody heavy chain variable region and light chain variable region. After PCR amplification, the PCR products of the light chain variable region gene and the heavy chain variable region gene are recovered and purified by agarose gel electrophoresis. The amplified products of light chain and heavy chain were respectively added with corresponding restriction endonucleases, and the digested products were purified by DNA recovery and purification kit. The heavy chain (VH) and light chain (VL) products obtained by PCR were mixed with human IgG1CH1 The intermediate vectors (pGEM-T) of and CL we...

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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

technical field [0001] The present invention relates to the field of biopharmaceuticals, in particular to a monoclonal antibody (named MS17-38) against the functional expression of procalyx-like protein subtype 2 (PODXL-v2) on the surface of gastric cancer cells and its preparation method and use. Background technique [0002] As the inventor Lu Meisheng et al. previously applied for the patent "anti-cell surface ectopically expressed monoclonal antibody MS17-57 and its preparation method and use" (patent application number: 201310090565.9, published and approved), Gastric cancer (GC) is the most common malignant tumor of the digestive system in humans and one of the leading causes of tumor-related death. Countries such as China and Japan have the highest incidence rate of gastric cancer in the world, with nearly 600,000 to 700,000 new cases each year, accounting for the majority of the world's cases. Many patients with gastric cancer are already in the late clinical stage...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63A61K39/395A61P35/00G01N33/577
CPCA61K2039/505C07K16/28A61K39/395C12N15/63G01N33/577
Inventor 陆梅生杰弗瑞·李张冬青刘炳亚
Owner 陆梅生
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