Preparation method and use of chimeric human anti-murine monoclonal antibody for inhibiting angiostatin acceptor

A monoclonal antibody, anti-vascular technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, application, antibody, etc., can solve problems such as mAb limitation and trough, and achieve the effect of inhibiting ATP synthesis

Inactive Publication Date: 2012-05-23
SUZHOU INDAL PARK HUMAN ANTIBODOMICS DEV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At the end of the 20th century, due to the limitations of the current technology, most of the prepared mouse hybridomas were mostly of mouse origin, and the human anti-mouse antibody (HAMA) reaction was easily generated in the human body, so that mAbs were used for therapeutic purposes. Research is constrained and enters a trough

Method used

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  • Preparation method and use of chimeric human anti-murine monoclonal antibody for inhibiting angiostatin acceptor
  • Preparation method and use of chimeric human anti-murine monoclonal antibody for inhibiting angiostatin acceptor
  • Preparation method and use of chimeric human anti-murine monoclonal antibody for inhibiting angiostatin acceptor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 chimeric antibody

[0038] A hybridoma cell line was obtained by conventional fusion of mouse splenocytes immunized with human ATP synthase antigen and mouse myeloma cells, and a positive clone hybridoma cell line 7E10 was screened out.

[0039] Take positive clone hybridoma 7E10 cell line: 5000-10000 cells, use Trizol (product of Invitrogen Company) to separate total RNA, and use reverse transcriptase (product of Invitrogen Company) to obtain cDNA. The above operations were carried out in accordance with the manufacturer's instructions. PCR was carried out with the following primers and conditions, and the amplicon used was KOD plus (TOYOBO) to ensure that possible mutations were reduced during the amplification process.

[0040] There are 4 sets of amplification primers for the antibody Fab region, respectively mouse IgG-5' / mouse IgG1-3', mouseIgG-5' / mouse IgG2a-3', mouse IgG-5' / mouse IgG3-3', (LCi +LC2+LC3+LC4+LC5+LC6+LC7) / mouse kapp...

Embodiment 2

[0179] Example 2 Anti-tumor effect experiment of anti-angiostatin receptor mouse antibody

[0180] 1. The purpose of the experiment:

[0181] Pharmacodynamic experiment of antitumor activity of 7E10 injection.

[0182] 2. Experimental animals:

[0183] Mice (ICR, 16-21g, male), purchased and pre-raised for 5 days a week before the experiment, were raised in the Experimental Animal Center of Nan Medical University

[0184] Construction of animal model: expand the culture of lung cancer cells A549, inoculate 3 million nude mice subcutaneously, set up high (50mg / kg / time), medium (25mg / kg / time), low (12.5mg / kg / time) Three doses of experimental group and PBS control group and A549 no treatment group.

[0185] 3. Experimental drugs:

[0186] After the 7E10 is obtained through expression and conventional separation and purification, the 7E10 injection is prepared by conventional methods.

[0187] 7E10 injection low dose group: 7E10 concentration is 0.05mg / ml

[0188] 7E10 injec...

Embodiment 3

[0213] Example 3 Effects of Angiostatin Receptor Mouse Antibody on Tumor Cells

[0214] Experimental tumor cells: human lung adenocarcinoma cell line (A549), human highly metastatic lung cancer cell line (95-D)

[0215] 7E10 antibody: the concentration is 100ug / ml, 200ug / ml

[0216] 1. Mouse 7E10 antibody acts on tumor cells to inhibit proliferation

[0217] Experimental steps:

[0218] (1) Collect the logarithmic phase cells, adjust the concentration of the cell suspension, add 100ul to each well, and plate to adjust the density of the cells to be tested to 1000-10000 wells (the edge wells are filled with sterile PBS).

[0219] (2) Incubate at 37°C with 5% CO2 until the cell monolayer covers the bottom of the well (96-well flat bottom plate). / ml, 300ug / ml of 7E10 antibody. Five replicate wells were set up for each concentration.

[0220] (3) 5% CO2, incubate at 37°C for 16-48 hours, observe under an inverted microscope.

[0221] (4) Add 20 ul of MTT solution (5 mg / ml, ...

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Abstract

The invention discloses a chimeric human anti-murine monoclonal antibody for inhibiting an angiostatin acceptor, and its preparation method and use. The chimeric human anti-murine monoclonal antibody for inhibiting the angiostatin acceptor comprises an antibody light-chain unit and an antibody heavy-chain unit, wherein a heavy chain is connected to a light chain through a disulfide bond. The chimeric human anti-murine monoclonal antibody for inhibiting the angiostatin acceptor is a chimeric antibody, can maintain human adenosine triphosphate (ATP) synthase-resistant activity and is suitable for be used in vivo. The chimeric human anti-murine monoclonal antibody for inhibiting the angiostatin acceptor has obvious effects of inhibiting proliferation of a breast cancer cell MDA-MB-231, has certain effects of inhibiting proliferation of a lung cancer cell A549, a liver cancer cell HepG2, and a cell PC-3 and a cell HUVEC of the prostate, causes apoptosis of associated cells, specifically binds with tumor tissue, and specifically identifies a tumor cell membrane and an endothelial cell membrane. In addition, the chimeric human anti-murine monoclonal antibody for inhibiting the angiostatin acceptor can inhibit activity of ATP synthase on surfaces of tumor cell membranes and endothelial cell membranes, formation of vessels in vitro, and moving of the cell HUVEC and the breast cancer cell MDA-MB-231 in vitro, and is meaningful to antitumor therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the preparation and application of an anti-angiostatin receptor human-mouse chimeric monoclonal antibody. Background technique [0002] In recent years, it has been found that ATP synthase is not only present in the inner membrane of mitochondria, but also expressed on the plasma membrane surface of endothelial cells and tumor cells. In 1995, Mozer and Pizzo identified the binding site for angiostatin (ring cake domains 1-3) on the surface of endothelial cells. Angiostatin-binding protein with a size of about 55kD was successfully isolated by ligand hybridization of human umbilical vein endothelial cell (HUVEC) membrane fraction. The amino-terminal sequencing of the protein, peptide fingerprinting and immunological analysis showed that the receptor of the anti-tumor angiogenesis drug angiostatin on the cell surface is the α / β subunit of human F1F0-ATP synthase (the gene names are AT...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08A61K39/395A61P35/00
CPCC07K16/28A61K2039/505C07K2317/24A61P35/00
Inventor 倪健朱向玲
Owner SUZHOU INDAL PARK HUMAN ANTIBODOMICS DEV
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