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PD (Programmed Cell Death)-1 antibody as well as preparation method and application thereof

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

Active Publication Date: 2017-05-24
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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  • PD (Programmed Cell Death)-1 antibody as well as preparation method and application thereof
  • PD (Programmed Cell Death)-1 antibody as well as preparation method and application thereof
  • PD (Programmed Cell Death)-1 antibody as well as preparation method and application thereof

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 is cloned into the pCpC vector with human IgG Fc fragment (hFc) (purchased from Invitrogen, V044-50) and prepare the plasmid according to the established standard molecular biology method. For specific methods, refer to 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...

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 (Hybridoma serum 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 lead 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 washin...

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Abstract

The invention discloses a PD (Programmed Cell Death)-1 antibody as well as a preparation method and application thereof. The PD-1 antibody comprises one or more of a heavy chain CDR (Complementarity-Determining Region)1, a heavy chain CDR 2 and a heavy chain CDR 3 in a heavy chain variable region of the PD-1 antibody as well as one or more of a light chain CDR 1, a light chain CDR 2 and a light chain CDR 3 in a light chain variable region of the PD-1 antibody; the PD-1 antibody is completely humanized and high in affinity; according to the application of the PD-1 antibody in a human mixed lymphocyte or a T lymphocyte, the expressive quantities of IFN (Interferon)-gamma and IL(Interleukin)-2 are remarkably increased, therefore, the PD-1 antibody is applied to preparation of drugs for treating tumors, autoimmune diseases and the like.

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 cell death-Ligand 2) Binding 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 that t...

Claims

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Application Information

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Patent Type & Authority Applications(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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