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Anti-immune checkpoint PD-L1 and PD-L2 tumor vaccines

An immune checkpoint and immune stimulation technology, applied in the field of bioengineering, can solve the problems of weak clinical efficacy

Active Publication Date: 2021-10-08
BEIJING POMONA BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, these tumor vaccines are aimed at tumor-associated antigens, and the clinical efficacy is weak, and further research and development are still needed to enhance the clinical efficacy

Method used

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  • Anti-immune checkpoint PD-L1 and PD-L2 tumor vaccines
  • Anti-immune checkpoint PD-L1 and PD-L2 tumor vaccines
  • Anti-immune checkpoint PD-L1 and PD-L2 tumor vaccines

Examples

Experimental program
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Effect test

Embodiment 1

[0095] In the following examples, the materials and methods used are as follows:

[0096] Preparation of dendritic (DC) cells

[0097]The method for isolating DC cells from mouse bone marrow is as follows: the bone marrow is flushed from the limbs of the mouse, passed through a nylon mesh, and red blood cells are removed with ammonium chloride. The cells were then fully rinsed with RPMI-1640 medium, and then cultured in 2.5ml RPMI-1640 medium containing 10% FBS, 20ng / ml recombinant mouse GM-CSF (rmGM-CSF) and 20ng / ml recombinant Mouse IL-4 (rmIL-4) (purchased from PeproTech, Inc., Rocky Hill, NJ). On day 2 and day 4 of the culture process, the cell supernatant was removed and replaced with fresh medium containing 20 ng / ml rmGM-CSF and 20 ng / ml rmIL-4. Cells were cultured in an incubator at 37°C, 5% CO2. At 48 hours of the culture process, non-adherent granulocytes were removed and fresh medium was replaced. After the cells were cultured for 7 days, by FACS analysis, more t...

Embodiment 2

[0102] Example 2 Construction of human fusion protein expression vector

[0103] Synthetic fusion gene 1: contains the complete Flagellin sequence, part of the human PD-L1 sequence or PD-L2 sequence (Accession number GenBank: AF177937.1), and the complete helper T cell PADRE epitope sequence and flanking cloning site sequence ( fusion gene-structure figure 1 shown) (synthesized by GENEWIZ, South Plainfield, NJ, USA). Synthetic fusion gene 2: contains part of human PD-L1 sequence or PD-L2 sequence (GenBank: AF177937.1) and complete helper T cell PADRE epitope sequence. These synthetic genes were digested by restriction endonucleases NdeI and XhoI (purchased from BoehringerMannheim) and cloned into the pET21a(+) expression vector (purchased from Novagen), identified by enzyme digestion and sequencing, and connected correctly without mutations. .

Embodiment 3

[0104] Example 3 Preparation and Purification of a Fusion Protein Containing Human PD-L1

[0105] In order to prepare and purify recombinant proteins, target recombinant plasmids were electrotransfected into Escherichia coli BL21(DE3) (Novagen) competent cells, after which Escherichia coli BL21(DE3) were inoculated on LB agar culture plates (containing 50 μg / ml ampicillin) Carry out expansion culture. The method of recombinant protein expression described below is one of a series of experiments under different experimental conditions.

[0106] In order to express the recombinant protein, 4YT medium containing ampicillin (containing 32g Bacto tryptone, 20g yeast extract and 5g NaCl / L, pH 7.2) was incubated with one clone, the incubation conditions were: shaking culture at 180rpm and 37°C 24h. Then IPTG (purchased from Sigma) was added to a final concentration of 1 mM, and culture was continued for 4-5 hours to express the recombinant protein. Finally, the cells were collecte...

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Abstract

The present invention proposes a recombinant protein and its application. The recombinant protein comprises: an immune checkpoint molecular fragment; a helper T cell epitope fragment; and an immunostimulatory molecular fragment. The recombinant protein stimulates the production of anti-immune checkpoint antibodies in vivo, mobilizes the existing spontaneously induced immune cell CTLs in the body, and stimulates the production of anti-immune checkpoint CTLs, thereby specifically killing tumor cells. The active immune killing effect on tumor cells caused by the recombinant protein proposed in the embodiment of the present invention is remarkable.

Description

technical field [0001] The present invention relates to the field of bioengineering, in particular, the present invention relates to recombinant protein and its application. Background technique [0002] Cancer is a disease in which cells proliferate uncontrollably due to genetic mutations in cells. It has become a major threat to human health and one of the main causes of human death. The World Health Organization (WHO) pointed out in the "Global Cancer Report 2014" that in 2012, the number of cancer patients and deaths worldwide increased rapidly, and nearly half of the new cancer cases occurred in Asia, most of which were in China. New cancer cases topped the list. According to the 2012 China Cancer Registration Annual Report, there are about 3.5 million new cancer cases in China every year, and about 2.5 million people die from it. Therefore, it is of great clinical value to find efficient and specific cancer treatment methods. [0003] Traditional tumor treatment me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N5/0784C07K16/28C07K16/12C07K16/06A61K38/17A61K38/16A61K39/00A61K39/395A61K35/15A61K48/00A61P35/00A61P31/14A61P31/20A61P31/18A61P31/22
CPCA61K35/15A61K38/00A61K39/0011A61K39/02A61K48/005A61K2039/505A61K2039/5154C07K14/195C07K14/705C07K16/12C07K16/28C07K16/30C07K2319/00C12N5/0639A61K39/395C07K19/00C12N15/70
Inventor 黄雪芬陈杰陈思毅
Owner BEIJING POMONA BIOLOGICAL TECH CO LTD
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