Fusion protein capable of blocking PD-1/PD-L1 signaling conduction pathway and activating T cells and use thereof

A PD-L1 and fusion protein technology, applied in the field of medicine and biology, can solve the problems of complex and unpredictable effects of CD80 protein on T cell response

Active Publication Date: 2019-05-07
BEIJING BIYANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, it can be seen that the effect of CD80 protein on T cell response is complex and its effect cannot be predicted before actual testing (Sa

Method used

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  • Fusion protein capable of blocking PD-1/PD-L1 signaling conduction pathway and activating T cells and use thereof
  • Fusion protein capable of blocking PD-1/PD-L1 signaling conduction pathway and activating T cells and use thereof
  • Fusion protein capable of blocking PD-1/PD-L1 signaling conduction pathway and activating T cells and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Embodiment 1, the construction of the high-efficiency expression vector of glutamine synthetase comprising target gene

[0155] (1) Synthesis of the coding nucleotide of the anti-PD1 antibody BY18.1 as a control and construction of the expression vector

[0156] According to the amino acid sequence data of Nivolumab No. 9623 in the International Nonproprietary Name (INN) database, it was optimized to the following nucleotide sequence suitable for expression in Chinese hamster ovarian cancer cells (CHO), and commissioned by Shanghai Jierui Biotech Co., Ltd. Engineering Ltd. synthesized the nucleotide sequence. The anti-PD1 antibody produced after expression of said nucleotide sequence is denoted herein as antibody BY18.1.

[0157] Nucleotide sequence (SEQ ID NO: 72) of the light chain (BY18.1L) of the anti-PD1 antibody BY18.1:

[0158] CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTG...

Embodiment 2

[0210] Embodiment 2, expression and purification of fusion protein

[0211] (1) Transient expression of fusion protein

[0212] 293F (purchased from Invitrogen, catalog number: 11625-019) cells were suspended and cultured in serum-free CD 293 medium (purchased from Invitrogen, catalog number: 11913-019). Centrifuge the cell culture before transfection to obtain the cell pellet, suspend the cells with fresh serum-free CD 293 medium, and adjust the cell concentration to 1×10 6cells / ml. Place the cell suspension in shake flasks. Taking 100ml of cell suspension as an example, 250ug of DNA and polyethylene Add 500ug of polyethylenimine (PEI) (Sigma, catalog number: 408727) to 1ml of serum-free CD 293 culture medium and mix well. After standing at room temperature for 8 minutes, add the PEI / DNA suspension dropwise into 100ml of cell culture medium. suspension in a shaker flask. Mix gently and place in 5% CO 2 , Shaker culture at 37°C (120 rpm). After 5 days, the culture super...

Embodiment 3

[0219] Embodiment 3, use ELISA method to detect specific binding effect

[0220] Recombinant human CD28 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 50103-M08H), recombinant human PD-L1 (Beijing Baipu Saisi Biotechnology Co., Ltd., catalog number: PD1-H5229), and recombinant Human CTLA-4 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11159-H08H) was diluted to 0.5 μg / ml, 0.25 μg / ml and 1.0 μg / ml and coated with 96-well ELISA plate (Corning Company, Cat. No. :42592). Dilute the fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 purified in the above-mentioned Example 2(2) to 2000 μg / ml, then perform 3-fold serial dilutions, and dilute 15-16 pieces in total Gradient, for each concentration gradient to perform duplicate hole detection. Add 50 μl of the diluted sample to the 96-well plate coated with recombinant human CD28 or recombinant human PD-L1, and incubate at 37°C for 2 hours. After washing three time...

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Abstract

The present invention provides a fusion protein capable of blocking a PD-1/PD-L1 signaling conduction pathway and activating T cells. The fusion protein comprises (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) a CD80 extracellular domain (ECD). The present invention also provides a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector, and a use of the fusion protein in individual treating, preventing and/or diagnosingdiseases related with PD-1/PD-L1 signaling pathway activation and T cell function inhibition.

Description

technical field [0001] The present invention generally relates to the field of medical biotechnology. Specifically, the present invention relates to comprising (i) derived from anti-programmed death-1 (programmed death-1 (PD-1)) antibody and / or anti-programmed death-1 ligand 1 (programmed death-1 ligand (PD-1) -L1)) an antigen-binding fragment of an antibody; (ii) an immunoglobulin Fc domain; and (iii) a fusion protein of the CD80 extracellular domain (ECD), a polynucleotide encoding said fusion protein, comprising said polynuclear The carrier of the nucleotide, the host cell comprising the polynucleotide or the carrier, and the fusion protein are used in the treatment, prevention and / or diagnosis of individuals with activated PD-1 / PD-L1 signal transduction pathways and T cell functions. Use in inhibition-related diseases. Background technique [0002] Immune checkpoint (immune checkpoint) is a class of inhibitory signaling molecules in the immune system, which avoids tiss...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/395A61K38/17A61P35/00A61K51/10A61K101/02
CPCA61K38/17A61K39/00A61K39/395A61K51/10A61P35/00C07K19/00
Inventor 胡品良邹敬洪伟东何芸白洁宋凌云杨文第
Owner BEIJING BIYANG BIOTECH
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