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Double-targeted fusion protein of target programmed death-1 (PD-1) or target programmed death-1 ligand (PD-L1) and a target vascular endothelial cell growth factor (VEGF) family, and application of double-targeted fusion protein

A PD-L1 and PD-1 technology, applied in the field of medicine and biology, can solve problems such as pain, difficulty in finding chemically and physically stable preparation conditions, and inconvenient compliance for patients

Active Publication Date: 2019-04-05
BEIJING BIYANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although two injections allow flexibility in dosing volume and timing, it creates inconvenience and pain for patients
Also, although combination formulations may offer some flexibility in dosing, it is often difficult to find formulation conditions that allow for chemical and physical stability of the two proteins in solution due to the differing molecular characteristics of the two proteins
In addition, co-administration and combination formulation of two different drug therapies may add additional costs to patients and / or payers

Method used

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  • Double-targeted fusion protein of target programmed death-1 (PD-1) or target programmed death-1 ligand (PD-L1) and a target vascular endothelial cell growth factor (VEGF) family, and application of double-targeted fusion protein
  • Double-targeted fusion protein of target programmed death-1 (PD-1) or target programmed death-1 ligand (PD-L1) and a target vascular endothelial cell growth factor (VEGF) family, and application of double-targeted fusion protein
  • Double-targeted fusion protein of target programmed death-1 (PD-1) or target programmed death-1 ligand (PD-L1) and a target vascular endothelial cell growth factor (VEGF) family, and application of double-targeted fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Embodiment 1, the construction of the high-efficiency expression vector of glutamine synthetase comprising target gene

[0124] (1) Synthesis of the coding nucleotide of the anti-PD1 antibody BY18.1 as a control and construction of the expression vector

[0125] 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.

[0126] Nucleotide sequence (SEQ ID NO: 66) of the light chain (BY18.1L) of the anti-PD1 antibody BY18.1:

[0127]CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGT...

Embodiment 2

[0239] Embodiment 2, expression and purification of fusion protein

[0240] (1) Transient expression of fusion protein

[0241] 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 6 cells / ml. Place the cell suspension in shake flasks. Taking 100ml of cell suspension as an example, 250ug of the recombinant expression vector plasmid DNA prepared in Example 1 and 500ug of polyethyleneimine (PEI) (Sigma, catalog number: 408727) were added to 1ml of serum-free CD 293 culture medium and mixed Evenly, after standing at room temperature for 8 minutes, the PEI / DNA suspension was added dropwise to the shake flask with 100ml of cell suspension. Mix gently and place in 5% CO 2 , Shaker cultu...

Embodiment 3

[0248] Example 3, using the ELISA method to detect the binding of the fusion protein of the present invention to human PD-1 and recombinant human VEGF-A

[0249] Antigen PD-1 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H) and antigen VEGF 165 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11066-HNAH) diluted to 0.5 μg / ml and 0.02 μg / ml and respectively coated with 96-well ELISA plates (purchased from Corning Company, catalog number: 42592). Dilute the dual-targeting fusion protein purified in Example 2 (2) above to 5 μg / ml, then perform 3-fold serial dilutions, dilute 9 gradients in total, and perform duplicate well detection for each concentration gradient. Add 50 μl of the diluted sample to the antigen PD-1 or antigen VEGF respectively 165 Incubate the coated 96-well plate at 37°C for 2 hours. After washing three times, horseradish peroxidase-labeled goat anti-human secondary antibody (product of Beiji...

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Abstract

The invention provides a double-targeted fusion protein of target programmed death-1 (PD-1) or target programmed death-1 ligand (PD-L1) and a target vascular endothelial cell growth factor (VEGF) family. The double-targeted fusion protein comprises (i) an anti-PD-1 antibody or an anti-PD-L1 antibody and (ii) a VEGF family inhibiting domain (VID) effectively connected to the C end of each of two heavy chains of the anti-PD-1 antibody or the anti-PD-L1 antibody. The invention further provides polynucleotide coding the double-targeted fusion protein, a carrier containing the polynucleotide, a host cell containing the polynucleotide or the carrier, and application of the double-targeted fusion protein to therapy, prevention and / or diagnosis of diseases related to PD-1 activity, PD-L1 activityand VEGF family activity in an individual.

Description

technical field [0001] The present invention generally relates to the field of medical biotechnology. Specifically, the present invention relates to targeting programmed death protein-1 (programmed death-1 (PD-1)) or programmed death protein ligand 1 (programmed death-1 ligand (PD-L1)), and targeting vascular endothelial Cell growth factor (Vascular Endothelial Cell Growth Factor (VEGF)) family double-targeting fusion protein, polynucleotide encoding said dual-targeting fusion protein, vector comprising said polynucleotide, comprising said multi-core The host cell of the nucleotide or carrier, and the use of the dual targeting fusion protein in treating, preventing and / or diagnosing diseases related to PD-1 or PD-L1 activity and VEGF family activity in individuals. Background technique [0002] Immune checkpoint (immune checkpoint) is a class of inhibitory signaling molecules in the immune system, which avoids tissue damage by regulating the persistence and intensity of imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63G01N33/68G01N33/574A61K39/395A61K38/18A61P35/00
CPCA61K38/00C07K14/71C07K16/2818C07K16/2827C07K2317/51C07K2319/00G01N33/57407G01N33/6872A61K38/18A61K39/395A61P35/00C07K19/00C12N15/62C12N15/63G01N33/574G01N33/68
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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