A kind of human-mouse chimeric monoclonal antibody and its preparation method and application

A monoclonal antibody and antibody heavy chain technology, applied in the biological field, can solve the problems of mAb limitation and trough, and achieve the effect of inhibiting ATP synthesis

Active Publication Date: 2018-07-10
优锐生物医药科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At the end of the 20th century, due to the limitations of the current technology, most of the prepared mouse hybridomas were mostly mouse-derived mAbs, and human anti-mouse antibody (HAMA) reactions were easily produced in humans, making mAbs used for therapeutic purposes. Research is constrained and enters a trough

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  • A kind of human-mouse chimeric monoclonal antibody and its preparation method and application
  • A kind of human-mouse chimeric monoclonal antibody and its preparation method and application
  • A kind of human-mouse chimeric monoclonal antibody and its preparation method and application

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Embodiment 1

[0058] Preparation of chimeric antibodies

[0059] 1) Preparation of mouse-specific monoclonal antibodies

[0060] 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 was screened out and named: 7E10 (McAb178).

[0061] 2) Obtaining the 7E10Fab gene of mouse-specific monoclonal antibody clone

[0062] Take the positive clone hybridoma 7E10 cell line: 5000-10000 cells, use Trizol (product of Invitrogen Company) to isolate 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 amplification enzyme used was KOD plus (TOYOBO) to ensure that possible mutations were reduced during the amplification process.

[0063] There are 4 sets of amplification primer...

Embodiment 2

[0120] HAI178 antibody inhibits the activity of ATP synthase in tumor cell and vascular endothelial cell membrane

[0121] An important biological function of ATP synthase is to synthesize ATP. The amount of ATP produced can be used to measure the activity of ATP synthase on the cell membrane surface, so as to determine the effect of anti-HAI-178mAb on the function of ATP synthase. Cells at 5 x 10 4 Plant each well into a 96-well plate, suck off the culture supernatant after overnight, and carefully wash the cells once with PBS. Add 100 μL / well of phosphate buffer solution (pH7.2, also used as the drug diluent of the following groups) to the normal group, add different concentrations of oligomycin to the positive control group, add different concentrations of antibody diluent to the test group, and add corresponding concentrations to the negative control group Mouse anti-human IgG1, each group has 6 multiple wells, three of which are placed in 5% CO 2 (neutral pH), the other...

Embodiment 3

[0123] Analysis of the ability of HAI178 antibody to inhibit the proliferation of leukemia cells

[0124] HAI178 antibody treated leukemia cells, and analyzed its ability to inhibit the synthesis of ATP in cells. The experimental results showed that HAI178 antibody can significantly inhibit the ability of cells to synthesize ATP. When the antibody concentration reaches 200ug / ml, it can inhibit both HL-60 and MV4-11 cells. About 40% of ATP is synthesized. Further analysis of cell proliferation experiments showed that treatment with HAI178 antibody could significantly inhibit the growth of leukemia cells, and the proliferation inhibition rates of HL-60 and MV4-11 cells both reached about 50%, which was significantly dose-dependent. Such as image 3 Shown: The ATP synthesis experiments shown in Figures A and B were used to analyze the biological activity of HAI178. The experiments showed that in HL-60 (A) and MV4-11 (B) cells, HAI178 treatment can significantly inhibit the ATP s...

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Abstract

The invention relates to the field of biotechnology, in particular to an anti-angiostatin receptor human-mouse chimeric monoclonal antibody and its preparation method and application. The present invention provides an anti-angiostatin receptor human-mouse chimeric monoclonal antibody, comprising an antibody light chain and an antibody heavy chain, the amino acid sequence of the antibody light chain is SEQ ID NO: 1 or its conservative variant sequence, and its anti-angiostatin receptor The amino acid sequence of the heavy chain is SEQ ID NO: 2 or its conservative variant sequence, and the heavy chain and the light chain are connected by a disulfide bond. The anti-angiostatin receptor monoclonal antibody provided by the invention is a chimeric antibody, which makes it more suitable for in vivo application while maintaining its anti-angiostatin receptor activity. The monoclonal antibody has the activity of binding to the natural antigen on the surface of the tumor cell membrane, and can obviously inhibit the synthesis of ATP on the surface of the tumor cell membrane, which is of great significance for anti-tumor therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-angiostatin receptor human-mouse chimeric monoclonal antibody and its preparation method and application. Background technique [0002] Angiostatin Receptor F1F0-ATP synthetase plays an important role in ATP synthesis in tumor cells. In normal human tissues, angiostatin receptors are mainly located on the inner membrane of mitochondria, but a small amount is also present on the cell membrane of endothelial cells. In contrast, many tumor cells overexpress angiostatin receptors, and a large number of them are located on the cell membrane. Therefore, the angiostatin receptor on the tumor cell membrane is a potential antitumor drug target. In recent years, it has been found that ATP synthase is not only present in the inner mitochondrial membrane, but also expressed on the plasma membrane surface of endothelial cells and tumor cells. [0003] Moser and Pizzo (1995) identified ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08A61K39/395A61P35/00G01N33/577G01N33/574
Inventor 倪健梁辉潘健陈建鹤廖新梅
Owner 优锐生物医药科技(深圳)有限公司
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