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Recombinant human PD-1 antibody and application thereof

A PD-1, antibody technology, applied in recombinant DNA technology, applications, antibodies, etc., can solve the problem of different antigenic epitopes

Pending Publication Date: 2020-07-17
MABWELL (SHANGHAI) BIOSCIENCE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the antibodies reported so far can block the interaction between PD-1 and ligands, the epitopes they target are not exactly the same (Tan S, Zhang H, Chai Y, et al. Anunexpected N-terminal loop in PD-1 dominates binding by nivolumab. NatCommun.2017;8:14369)

Method used

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  • Recombinant human PD-1 antibody and application thereof
  • Recombinant human PD-1 antibody and application thereof
  • Recombinant human PD-1 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Screening, identification and antibody sequence determination of anti-human PD-1 antibody hybridoma cell lines

[0098] Immunization: Freund's adjuvant and water-soluble adjuvant were used for immunization of mice. Freund's adjuvant was used for intraperitoneal injection twice on day 0 and day 14, and water-soluble adjuvant was administered on day 0 and day 14. In 21 days, 8-10 week-old Balb / c mice were given two intramuscular immunization injections, and the immune antigen was: human PD-1 / mFc recombinant protein (serial number: NP_005009.2, 21aa-167aa, Lot: 2016.3. 16), recombinantly expressed in HEK293 cells by Beijing Kenuo Xincheng Technology Co., Ltd. The dose for the first immunization was 50 μg, and the dose for the second immunization was 25 μg. The mouse serum was taken before immunization as a negative control for detection, Freund's adjuvant was used on the 28th day after the initial immunization, and water-soluble adjuvant was used to collect b...

Embodiment 2

[0109] Example 2: Preparation of anti-human PD-1 chimeric antibody

[0110] The monoclonal antibody light chain variable region and heavy chain variable region genes obtained by cloning were introduced into restriction sites by PCR with the following primers (Table 3), and cloned into human-kappa light chain constant region and human IgG4 heavy chain constant region respectively. In the eukaryotic expression vector upstream of the region coding gene, the human-mouse chimeric light chain (pKN019-317L) and human-mouse chimeric heavy chain (pKN034-317H) expression plasmids were obtained, which were transformed into Escherichia coli for amplification, and a large amount of Plasmids containing the light and heavy chains of the human-mouse chimeric antibody were mixed with 293fectin and co-transfected into HEK293 cells. Cells were transfected for 5-6 days, and the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography column, and...

Embodiment 3

[0116] Example 3: Humanization of anti-human PD-1 monoclonal antibody and construction of stable cell lines

[0117]Firstly, the heavy chain sequence of the murine antibody was comprehensively analyzed to determine the complementarity determinant (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conservative three-dimensional conformation of the antibody. Then, according to the homology comparison results, the most similar human antibody template was found in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX), and VH3( 3-21) as the basic template, combined with the results of the full sequence blast, considering the frequency of amino acid occurrences (A49) of the rearranged antibody in a specific FR region, and performing CDR transplantation, considering that the FR3 region (S98) is close to the CDR3 region, no replacement According to the CDR3 sequence (Pdssgvay), JH4 (wgq...

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Abstract

The invention provides an antibody binding to programmed cell death protein 1 (PD-1), or a fragment of the antibody, and an application of the antibody or the fragment of the antibody to preventing ortreating of tumors or cancers. The antibody or the fragment of the antibody can effectively block interaction of PD-1 / PD-L1 and PD-1 / PD-L2, has a unique antigen binding epitope, and has unique properties in drug effect and safety.

Description

technical field [0001] The present invention relates to the field of antibody medicine, in particular, the present invention relates to an antibody against human PD-1 and its use for preparing medicine. Background technique [0002] Programmed cell death protein 1 (PD-1), also known as CD279, is a member of the T cell receptor CD28 family, expressed on the surface of a variety of immune cells, such as T cells, B cells, monocytes, etc., is An important immune checkpoint molecule that inhibits CD4+ and CD8+ T cell function in the tumor microenvironment. The key ligands of PD-1 include PD-L1 (B7-H1) and PD-L2 (B7-DC), which are expressed by immune cells and can also be induced on a variety of tissues, when PD-1 on T cells When combined with PD-L1 or PD-L2, T cells receive inhibitory signals, thereby inhibiting T cell proliferation and cytokine production, which will effectively reduce the immune response that T cells participate in. However, PD-L1 and PD-L2 are highly express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/85C12N5/10A61K39/395A61K45/06A61P35/00
CPCC07K16/2818A61K39/39566A61K45/06A61P35/00C07K2319/00A61K2039/505A61K39/00A61K2300/00C07K2317/24C07K2317/92C07K2317/33C07K2317/76C07K2317/77C07K2317/34C07K2317/55A61K47/6849
Inventor 张锦超王荣娟焦莎莎王双
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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