Monoclonal antibody against PD-1, and preparation method and application thereof

A monoclonal antibody, PD-1 technology, applied in the field of biomedicine, can solve the problems of low affinity and weak selectivity, and achieve the effect of good biological activity

Inactive Publication Date: 2018-07-31
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a new anti-human PD-1 monoclonal antibody and provide The preparation method and application of this monoclonal antibody have been understood, thereby completing the present invention

Method used

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  • Monoclonal antibody against PD-1, and preparation method and application thereof
  • Monoclonal antibody against PD-1, and preparation method and application thereof
  • Monoclonal antibody against PD-1, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of PD-1 antigen and PD-1 ligand PD-L1

[0060] The cDNA of human PD-1 was purchased from Origene (catalogue number: SC117011). Design primers, use the cDNA of PD-1 as a template to amplify the coding region of the extracellular segment, and use the cDNA of human IgG1 as a template to amplify the Fc coding region; recover the amplified PCR fragments, and recombine the above fragments by Overlap PCR Together, the signal peptide (MGVKVLFALICIAVAEA) coding region was introduced at the 5-end when designing the primers, and the corresponding restriction sites were introduced at both ends; the pCHO1.0 vector and the aforementioned recombinant PCR fragment were digested with Avr II and Pac I; After the product was purified, it was ligated and transformed into TOP10 competent cells, spread on LB (Amp) plate medium and cultured overnight; the colony was picked, and the plasmid was extracted, and Avr II and Pac I digested the plasmid to identify whether there...

Embodiment 2

[0063] Example 2 Using PD1-hFc as an antigen to immunize mice

[0064] Human PD1-hFc antigen was diluted to 50 μg / 75 μl with physiological saline, mixed with an equal volume of Freund’s complete adjuvant, and after complete phacoemulsification, 4-5 week-old Balb / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018) for subcutaneous multi-point injection. Three weeks later, the same amount of protein was also diluted to 75 μl and mixed with an equal volume of Freund's incomplete adjuvant. After phacoemulsification was complete, the mice were subcutaneously immunized at multiple points, and the immunization was repeated two weeks later. One week after the third immunization, the tails of all mice were cut to collect blood to separate serum, and the serum titer was detected by ELISA coated with human PD1-hFc antigen. For mice with serum antibody titer >10000, pulse immunization was performed one week after...

Embodiment 3

[0066] Embodiment 3 hybridoma fusion and screening

[0067] Splenocytes were collected three days after the mice were immunized for fusion.

[0068] Hybridoma sp2 / 0 cells in good growth state (from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were stored at 37°C, 5% CO 2 Cultured in an incubator, the medium was changed the day before fusion. The fusion and screening process is as follows: the mouse spleen is taken, ground and washed, and then counted. According to splenocytes:sp2 / 0 cells=10:1, the two kinds of cells were mixed and centrifuged at 1500rpm for 7 minutes. Wash away the supernatant. Add 1ml of PEG (1450) within 1 minute, shake gently for 90 seconds, add 5ml of serum-free DMEM culture solution (purchased from Gibco) within 2.5 minutes, and then add 5ml of serum-free culture solution to stop the reaction, and let it stand for 5 minutes. Centrifuge at 1280 rpm for 8 minutes. According to the number of 2 million sp2 / 0 ...

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Abstract

The invention specifically discloses a monoclonal antibody against human PD-1, and a preparation method and application thereof, belonging to the field of antibodies. The monoclonal antibody against human PD-1 in the invention has good biological activity, and can effectively bind to the extracellular region of a human PD-1 protein receptor, block PD-1 protein at a protein level and a cellular level, prevent binding of the PD- 1 protein to ligand PD-L1 and enhances immunity. The monoclonal antibody can be individually used or used in combination with other anti-tumor drugs for tumor immunotherapy and the diagnosis and screening of patients with PD-L1 positive tumors, and has good prospects in preparation of drugs for treating tumors, anti-autoimmune diseases and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-PD-1 monoclonal antibody and its preparation method and application. Background technique [0002] Human 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 st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00A61P37/02A61P31/00A61P37/06
CPCC07K16/2827A61K2039/505C07K2317/565C07K2317/92A61P31/00A61P35/00A61P37/02A61P37/04A61P37/06C12N5/10C12N15/85C07K16/2818C07K2317/76C07K2317/33C07K2317/52C07K2317/73C07K2317/24C07K2317/56
Inventor 赵杰高宏海郭锦林党尉朱玲巧
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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