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Anti-human PD-1 monoclonal antibody 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 good biological activity

Inactive Publication Date: 2017-07-07
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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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

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  • Anti-human PD-1 monoclonal antibody and preparation method and application thereof

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preparation example Construction

[0046] The preparation method of the anti-human PD-1 monoclonal antibody disclosed in the present invention comprises: cultivating the above-mentioned host cells under expression conditions, thereby expressing the anti-human PD-1 monoclonal antibody; isolating and purifying the anti-human PD-1 1 monoclonal antibody. Using the above method, the recombinant protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.

[0047] The anti-human PD-1 monoclonal antibody disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer and pH change can be used to wash the antibody. Monoclonal antibody bound to an affinity column. The inventors of the present invention conducted a detection experiment on the obtained anti-human PD-1 monoclonal antibody, and the experimental results showed that the ...

Embodiment 1

[0048] Example 1 Preparation of PD-1 antigen

[0049] In order to produce anti-human PD-1 (hPD-1) antibodies, the cDNA encoding the open reading frame of the full-length amino acid sequence of human PD-1 was obtained by PCR method, and the obtained cDNA was subcloned into the vector pcDNA3.1 (Invitrogen, Carlsbad, CA) to obtain hPD-1 vector. Wherein the structure of the vector pcDNA3.1 is as follows figure 1 As shown, the cDNA sequence is shown in SEQ ID NO: 11, and the GenBank number of the sequence is: BC074740.2.

[0050] The resulting hPD-1 vector was stably transfected into CHO-K1 cells (purchased from Life technologies), and the expression of PD-1 was monitored by flow cytometry (FACS), and the CHO-K1 expressing human PD-1 on its membrane was isolated cloned and named it CHO-hPD1, please refer to the relevant cell technology manual for the specific operation method.

[0051] Mice were immunized by gene gun with Helios gene gun (BioRad) and DNA-coated gold bullets (Bio...

Embodiment 2

[0054] Preparation, screening and library building of embodiment 2 hybridomas

[0055] Four days after the last immunization, the mice were sacrificed according to the literature (Steenbakkers et al., 1992, J.Immunol.Meth.152:69-77; Steenbakkers et al., 1994, Mol.Biol.Rep.19:125-134 ), preparing a population of splenocytes depleted of red blood cells, and fusing the resulting splenocytes. 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 taken at 37°C and 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...

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Abstract

The present invention belongs to the field of antibodies, and more specifically, the present invention discloses an anti-human PD-1 monoclonal antibody and a preparation method and application thereof. The anti-human PD-1 monoclonal antibody of the present invention has good biological activity, can effectively bind the extracellular region of the human PD-1 protein receptor, and can effectively block PD-1 protein at the protein level and cellular level, preventing PD-1 protein 1 protein binds to the ligands PD-L1 and PD-L2, and effectively enhances immune function. The monoclonal antibody can be used alone or in combination with other anti-tumor drugs in tumor immunotherapy and diagnosis and screening of PD-L1 positive tumor patients, and also has a good prospect for preparing drugs for treating tumors and anti-autoimmune diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-human 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, CancerImmunotherapy, 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 studi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12P21/08A61K39/395A61P35/00A61P37/02A61P31/00A61P37/06
CPCC07K16/2818C07K2317/56C07K2317/565
Inventor 李彩辉张彦李丽君朱晓丹耿妍
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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