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Anti-PD-1 monoclonal antibody and application thereof

A monoclonal antibody, PD-1 technology, applied in the fields of application, antibody, anti-tumor drugs, etc., can solve the problems of ineffective activation of the immune system and immune escape of tumor cells, and achieve the recovery of lymphocyte activity, high binding rate, and specificity good sex effect

Active Publication Date: 2018-07-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the tumor microenvironment of the body, tumor cells highly express PD-L1. After combining with PD-1 on T cells, it induces the exhaustion of T cells, inhibits the function of T cells, and cannot effectively activate the immune system. immune escape

Method used

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  • Anti-PD-1 monoclonal antibody and application thereof
  • Anti-PD-1 monoclonal antibody and application thereof
  • Anti-PD-1 monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Animal immunization

[0028] The recombinant human PD-1 protein (dissolved in PBS buffer, which has been preliminarily identified, not the focus of this patent and will not be expanded) prepared in the laboratory in the early stage is mixed with Quick Antibody in an equal volume, and injected intramuscularly to 6-8 weeks old In BAL b / c mice, 50μg / 200μl / mice. The immunization process was carried out according to Quick Antibody instructions. A total of 3-5 immunizations were carried out. After each immunization for a period of time, the orbital blood (50 μl) of the immunized mice was collected by capillary blood sampling. The indirect ELISA was used to determine the serum antibody efficacy of the mice after serial dilution of the serum with the blocking solution. price. The mouse with the highest specific antibody titer in serum was selected for cell fusion. The final test results showed that the serum titer of the immunized mice could reach more than 1:100,0...

Embodiment 2

[0029] Example 2 Preparation of hybridoma cell lines

[0030] 1. Cell fusion

[0031] Cell fusion was performed using the polyethylene glycol method. The specific operations are as follows:

[0032] 1) One week before fusion, the sp2 / 0-Ag14 myeloma cells were recovered, and 8-azaguanine was used to adjust the cell state. Two days before cell fusion, sp2 / 0-Ag14 was expanded and cultured, and the state was observed to make it close to the logarithmic growth phase one day before fusion, and the medium was changed the day before fusion;

[0033] 2) Generally, when the serum titer of immunized mice reaches 100,000, the fusion can be achieved. Select the mouse with the highest PD-1-specific antibody titer in the serum, pulse immunization 3-4 days before fusion, and inject PD-1 protein into the abdominal cavity or tail vein;

[0034] 3) Preparation of feeder layer cells (mouse peritoneal macrophages): feeder layer cells were laid on the day before fusion, and older ICR mice (e.g....

Embodiment 3

[0041] Example 3 Preparation and purification of anti-PD-1 monoclonal antibody

[0042] 1. Ascites Collection

[0043] Take the intraperitoneal injection of 500 μl of paraffin oil (SIGMA) into 8-10 week old female Bal b / c mice. One week later, intraperitoneal injection of 1 × 10 6 well-growing hybridoma cells. About 7-10 days after the hybridoma cells were inoculated, the abdomen of the mice was obviously bulging. At this time, the ascites was collected and centrifuged at 4°C at 12,000 rpm for 5 min to remove cell debris, and the supernatant was collected for subsequent purification.

[0044] 2. Antibody Purification

[0045] Antibody purification was performed using ProteinA / G column packing (Abmart). Specific operation: Ascites supernatant should be diluted with BindingBuffer to a certain volume and filtered and sterilized before loading. After loading, wash with Binding Buffer to remove non-specifically bound impurities, and finally elute, add 5-10ml Elution Buffer to ...

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Abstract

The invention relates to a novel anti-PD-1 monoclonal antibody and a variable region sequence of the antibody and belongs to the technical field of biology. The novel anti-PD-1 monoclonal antibody takes recombinant human PD-1 protein (rhEPD-1) as an immunogen for immunizing a BAL b / c mouse; spleen cells of the mouse are taken and are fused with myeloma cells sp2 / 0-Ag14 to obtain a hybridoma cell line capable of expressing the anti-PD-1 antibody; the cell line can stably secrete the single monoclonal antibody. According to the novel anti-PD-1 monoclonal antibody, variable region nucleotide andamino acid sequences of an H chain and an L chain of the anti-PD-1 monoclonal antibody are cloned at the same time. The anti-PD-1 monoclonal antibody provided by the invention can be specifically combined with PD-1 and the combination of the PD1 and PD-L1 is blocked; functions of T cells are recovered. The novel anti-PD-1 monoclonal antibody can be used as an immune checkpoint inhibitor and is used for treating cancer and infectious diseases.

Description

Technical field: [0001] The invention relates to a monoclonal antibody that can specifically bind to PD-1, and belongs to the field of biotechnology. Specifically, it relates to the preparation method of the monoclonal antibody, its variable region sequence and its application. Background technique: [0002] Hybridoma technique (hybridoma technique), also known as monoclonal antibody technique, in 1975 Koehler and Milstein proved that the fusion of myeloma cells with immunized animal spleen cells can form highly specific monoclonal antibodies against a certain antigen. Hybridoma technology utilizes the characteristics that myeloma cells can be serially passaged in vitro and that lymphocytes can secrete specific antibodies or factors. When two kinds of cells are fused, the successfully fused cells can have the main characteristics of these two kinds of cells. [0003] PD-1 / PD-L1 immunotherapy improves the immune response by blocking the interaction between the two, and has t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P31/00G01N33/68
CPCA61K2039/505C07K16/2818C07K2317/21C07K2317/24C07K2317/56C07K2317/565
Inventor 邱郑王旻邢黎军徐祎凤王红
Owner CHINA PHARM UNIV
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