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A kind of PD-1 antibody and its preparation method and application

A PD-1 and antibody technology, applied in the field of biomedicine, can solve the problems of low rejection and high antibody titer

Active Publication Date: 2020-11-06
SHANGHAI YUNYI HEALTHCARE MANAGEMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no better PD-1 antibody that properly plays the role of positive and negative regulation to control the balance of the immune system and is applied to tumor immunotherapy, and achieves the effect of low rejection and high antibody titer, and does not meet the above conditions. The better PD-1 antibody can be applied to the preparation of related drugs such as tumor immunotherapy, autoimmune disease, infectious disease and transplant rejection, so it is urgent to solve the above problems

Method used

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  • A kind of PD-1 antibody and its preparation method and application
  • A kind of PD-1 antibody and its preparation method and application
  • A kind of PD-1 antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The preparation of embodiment 1PD-1 antibody

[0083] (1), preparation of immunogen A

[0084] The nucleotide sequence containing the amino acid sequence Leu25-Glu167 (as shown in the sequence table SEQ ID No.31) encoding human PD-1 protein extracellular region was cloned into the pCpC vector (purchased from Invitrogen) with human IgG Fc fragment (hFc) , V044-50) and prepare plasmids according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview , New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (PEI, Polysciences) and used FreeStyle TM 293 (Invitrogen) was expanded at 37°C. After 4 days, the cell culture medium was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular domain of PD-1...

Embodiment 2

[0105] Example 2 Production and Purification of Lead Antibody

[0106] The antibody concentration produced by hybridoma cells is low, only about 1-10 μg / ml, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0107] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridomaserum free medium, purchased from Invitrogen). When the growth state is good, inoculate the cell culture spinner bottle. Add 500 ml of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0╳10 5 / ml. Close the bottle cap tightly, and place the spinner bottle on a spinner ...

Embodiment 3

[0112] The assay of embodiment 3 leading antibody

[0113] A. Enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibody to PD-1 protein

[0114] The purified PD-1 antibody obtained in Example 2 was cross-reacted with human PD1-hFc protein, monkey PD1-hFc and other immune checkpoint proteins of the PD-1 protein family.

[0115] The purified immunogen A (PD1-hFc) obtained in Example 1 and monkey PD1-hFc (for the preparation method, please refer to the preparation of immunogen A in step (1) of Example 1), wherein the extracellular region of the monkey PD-1 protein ( The database accession number of the amino acid sequence of Leu25-Gln167) is B0LAJ3) or other immune checkpoint proteins (CD28, B7.1, ICOS, CTLA4 and NC-Fc) (all purchased from R&D Systems) were diluted with PBS to a final concentration of 1.0 μg / mL, and then added to a 96-well ELISA plate at 100 μl per well. Seal with plastic film and incubate overnight at 4°C, wash the plate twice with plate was...

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Abstract

The invention discloses a PD‑1 antibody, its preparation method and application. The PD‑1 antibody includes one or more of the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the PD‑1 antibody, and the light chain variable region light chain CDR1 of the PD‑1 antibody , one or more of light chain CDR2 and light chain CDR3. The PD-1 antibody is fully human and has high affinity, which significantly increases the expression of IFN-γ and IL-2 in human mixed lymphocytes or T lymphocytes, so it is used to treat tumors, autoimmune diseases, etc. In the preparation of drugs.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a PD-1 antibody and its preparation method and application. Background technique [0002] Programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids, which is one of the main known immune checkpoints (Immune Checkpoint) (Blank et al, 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed in activated T lymphocytes, and it binds to ligands PD-L1 (programmed cell death-ligand 1, programmed cell death-Ligand 1) and PD-L2 (programmed death receptor-ligand 2. Programmed celldeath-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in vivo. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B and T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12P21/00A61K39/395A61P35/00A61P37/00A61P31/00A61P37/06
CPCC07K16/2818C07K2317/21C07K2317/24C07K2317/33C07K2317/732C07K2317/734C07K2317/76C07K2317/92C07K2317/94A61K39/39591A61P35/00A61K39/39558C07K16/2803
Inventor 杨欣秀罗海山刘虎帅正蓉王健钟勤段清顾红专刘礼乐
Owner SHANGHAI YUNYI HEALTHCARE MANAGEMENT
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