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Humanized antibody or antibody fragment for PD-L1 extracellular fragments and application thereof, nucleotide sequence and carrier

A technology of nucleotide sequences and antibody fragments, applied in the direction of antibodies, medical preparations of non-active ingredients, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., to enhance the ability and specificity of secretion High, enhanced immune response effect

Active Publication Date: 2017-03-15
武汉科源安博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no humanized PD-L1 monoclonal antibody that is effective in the treatment of cancer in the Chinese market

Method used

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  • Humanized antibody or antibody fragment for PD-L1 extracellular fragments and application thereof, nucleotide sequence and carrier
  • Humanized antibody or antibody fragment for PD-L1 extracellular fragments and application thereof, nucleotide sequence and carrier
  • Humanized antibody or antibody fragment for PD-L1 extracellular fragments and application thereof, nucleotide sequence and carrier

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preparation example Construction

[0045] The preparation method of the antibody or antibody fragment provided by the present invention is specifically as follows:

[0046] First, using recombinant DNA technology and gene transfection methods known in the art, or obtaining the antibody or antibody fragment of the present invention in a host cell transfectoma;

[0047] Secondly, after obtaining the DNA sequence of the monoclonal antibody or antibody fragment of the present invention, obtain the heavy chain variable region sequence and light chain variable region sequence of the monoclonal antibody by means of gene synthesis;

[0048] Then, the two sequences were inserted into two pFUSE-IgG1 antibody expression vectors by standard molecular cloning techniques, and the two obtained vectors were simultaneously transfected into 293F cells and cultured in suspension;

[0049] Finally, the supernatant was collected, separated and purified with Protein A to obtain the antibody or antibody fragment of the present invent...

experiment example 1

[0123] Experimental example 1 Screening of anti-PD-L1 antibody

[0124] 1. Construction of the antibody library: human peripheral blood mononuclear lymphocytes were used as the cell source, and primers were designed (for primer sequences, refer to Antibody Engineering Methods and Protocols, Chapter 2, Section 5, Second Edition, and Chapter 2, Section 5 was selected. All primers) to amplify the heavy chain variable region and light chain variable region of the antibody, and link the heavy chain variable region and light chain variable region with Linker by overlapping extension PCR to obtain a full-length PCR product. Digest the PCR product and pCGMT phagemid vector with SfiI, electrotransform the ligated transformation product into XL1-blue competent cells, then add 3ml of SOC medium to the competent cells, incubate at 30°C for 1 hour, then add the final concentration Ampicillin at 50 μg / ml, tetracycline at 10 μg / ml in 20 ml, cultured with shaking at 37° C. for 2 hours. Then ...

experiment example 2

[0134] Experimental Example 2 Construction of pFUSE-IgG1 expression vector for anti-PD-L1 full-length antibody

[0135] The full-length nucleotide coding sequence of the heavy chain variable region of the antibody screened in Experimental Example 1 was synthesized (GenScript), and according to the instructions of the pFUSE heavy chain expression vector (Cat. No. pFUSEss-CHIg-hG1), during synthesis, the Restriction endonucleases BamH1 and BglII restriction sites were added to both ends, and then the fragment was inserted into the pFUSE expression vector by the method of enzyme digestion and ligation, thereby obtaining the pFUSE-IgG1 expression vector of the full-length antibody heavy chain of the present invention. Also directly synthesized according to the light chain variable region sequence, with EcoRI and AvrII restriction sites on both ends, and inserted into the light chain pFUSE expression vector (Invivogen Company, pFUSE2ss-CLIg-hl2) by restriction enzyme connection meth...

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Abstract

The invention discloses a humanized antibody or antibody fragment for PD-L1 extracellular fragments and application thereof, a nucleotide sequence and a carrier, belongs to the technical field of antibody humanization. The antibody or the antibody fragment comprises a heavy chain and a light chain. Each of the heavy chain and the light chain comprises a variable region. Each variable region comprises complementary determining regions. The complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain are represented by HCDR1, HCDR2 and HCDR3 respectively. The complementary determining regions CDR1, CDR2 and CDR3 of the light chain are represented by LCDR1, LCDR2 and LCDR3 respectively. The antibody or the antibody fragment can improve immunity systems and promote lymphocyte to secrete interleukin-2, and is applied to preparation of diagnostic reagents and anti-tumor, anti-inflammation and anti-contagion drugs.

Description

technical field [0001] The present invention relates to an antibody against human programmed death receptor ligand 1 (PD-L1), in particular to a human antibody or antibody fragment and its use, nucleotide sequence and carrier against the extracellular portion of PD-L1. Background technique [0002] Programmed death-ligand 1 (PD-L1) plays an immunosuppressive role during chronic infectious disease, pregnancy, tissue transplantation, autoimmune disease and cancer. PD-L1 regulates the immune system's response by binding to the programmed death receptor 1 (PD-1). PD-1 is mainly expressed on the surface of T cells, B cells and monocytes. PD-L1 can also downregulate T cell function by interacting with its additional receptor B7-1. Activation of the PD-L1 / PD-1 signaling pathway can down-regulate the activity of T cells and the secretion of cytokines, and inhibit the anti-cancer activity of the immune system. [0003] PD-L1 is highly expressed in many cancer tissues, such as blad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K47/68A61P35/00A61P35/02A61P29/00A61P31/00A61P33/00
CPCC07K16/2827C07K2317/24C07K2317/56C07K2317/565C07K2317/70C07K2317/92
Inventor 易庆华
Owner 武汉科源安博生物技术有限公司
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