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Fusion protein for restoring functions of exhaustion immune cells and application thereof

A technology of immune cells and fusion proteins, applied in the field of fusion proteins, can solve problems such as specific cells that cannot be targeted for exhaustion, achieve good clinical prospects, enhance the effect of inhibiting tumor growth and restoring functions

Active Publication Date: 2017-08-22
科弈(浙江)药业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Likewise, T cells are activated by the activating ligands 4-1BBL and OX-40L, but neither of these effects is directed against exhausted specific cells (J.Leukoc.Biol. 21011, 89:989–999)
[0006] So far, there is no protein drug that can not only specifically recognize exhausted specific T cells, but also restore their function and expand their number

Method used

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  • Fusion protein for restoring functions of exhaustion immune cells and application thereof
  • Fusion protein for restoring functions of exhaustion immune cells and application thereof
  • Fusion protein for restoring functions of exhaustion immune cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example is the gene construction, production and purification of recombinant fusion protein.

[0044] According to the C-terminal of human PD-1 antibody heavy chain (SEQ ID NO: 6) and the N-terminal amino acid sequence of interleukin 2 (SEQ ID NO: 14), through gene synthesis, restriction digestion and further cloning, the two parts were passed through 17 non- Functional amino acids (SGGGGSGGGGSGGGGSG) are connected to form the fusion protein heavy chain construction gene (SEQ ID NO: 23), and then transferred into the eukaryotic animal expression vector pcDNA3.1. Through gene synthesis, restriction digestion and further cloning, the light chain (SEQ ID NO: 7) gene of the PD-1 antibody was transferred into the eukaryotic expression vector pcDNA3.1. Finally, the heavy chain expression vector and light chain expression vector of the fusion protein were simultaneously transfected into Chinese hamster ovary cells (CHO). Place the transfected cells at 37℃, 5% CO 2 After cul...

Embodiment 2

[0046] This example is the determination of the activity and function of the bifunctional recombinant fusion protein on human PBMC cells cultured in vitro.

[0047] Human peripheral blood was separated and purified by lymphocyte density gradient centrifugation (Ficoll). The cell density was diluted to 5×10 in X-Vivo15 medium in a 24-well plate. 6 / ml, add test protein to a final concentration of 2μg / ml. Incubate at 37°C for 30 minutes. After centrifugation, change the X-Vivo15 medium to the final cell density of 5x10 5 / ml. Then put it at 37℃, 5% CO 2 After culturing in the incubator for 72 hours, they were taken out. After the cells were collected, they were stained with CD8 and PD-1 antibodies, and phenotype determination and data analysis were performed by flow cytometry. figure 2 Shows that the unfused PD-1 antibody ( figure 2 Part 2) and Interleukin 2 ( figure 2 Part 3) compared to fusion protein treatment ( figure 2 Part 1) can significantly increase the proportion of CD...

Embodiment 3

[0049] This example shows the effect of bifunctional recombinant fusion protein on effector cells in vivo.

[0050] Small black mice were injected with fusion protein (4μg / mouse / day) or interleukin-2 (40μg / mouse / day) or PBS (control group) through the abdominal cavity for 3 consecutive days. On the 4th day, peripheral blood was taken from the tail and used anti-mouse CD8 And NK1.1 flow cytometry antibody staining and testing, data analysis such as Figure 4 . With the control group ( Figure 4 Part 1, 4.0%) and Interleukin 2 group ( Figure 4 Compared with part 2, 15.3%), the recombinant fusion protein significantly increased the proportion of NK cells in lymphocytes ( Figure 4 Part 3, 27.0%). At the same time, in the CD8+ T cell part, and the control group ( Figure 4 Part 1, 15.5%) Compared to native interleukin 2 did not increase the proportion of CD8+ cells ( Figure 4 Part 2, 15.1%), and the proportion of CD8+ T cells in peripheral blood of mice treated with fusion pr...

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Abstract

The invention relates to a fusion protein for restoring functions of debilitating immune cells. The fusion protein comprises a functional zone for identifying exhaustion immune cells and a functional zone for activating and amplifying the exhaustion immune cells. The two functional zones are connected through a non-functional amino acid fragment. According to the identification functional zone, an immune checkpoint specific antibody is utilized for identifying a phenotypic receptor of the exhaustion immune cells. According to the activation and amplification functional zone, cytokine or functional analogy mutant, or a ligand of surface receptor or functional analogy mutant, or an activated antibody is adopted for activation of the exhaustion immune cells. The invention also relates to application of the fusion protein. Tests show that the prepared fusion protein can identify exhaustion immune cells and also can activate and amplify the identified immune cells, restore the immune cells' function of killing antigen positive cells and enhance the functions of inhibiting tumor growth and controlling virus infection, and has a good clinical prospect and a wide application range.

Description

Technical field [0001] The invention relates to the technical field of fusion proteins, in particular to a fusion protein for restoring the function of debilitating immune cells and its application. Background technique [0002] Cancer is the most serious disease that is common and threatens people's lives and quality of life. Chronic diseases caused by virus infection are even more difficult problems that plague the world. Current clinically applied drugs for cancer have various shortcomings. For example, chemotherapy drugs have large side effects, targeted drugs are prone to drug resistance (Curr Pharm Des. 2010, 16:3-10), and immune checkpoint inhibitors have low efficiency ( N Engl J Med 2012, 366: 2443-2454), Chimeric antigen receptor T (Car-T) cell therapy has the disadvantages of cytokine storm and high recurrence rate (Curr Opin Pediatr. 2017, 29:27-33) . There are hundreds of millions of people infected with HBV (hepatitis B virus) in China (APJCP, 2011, 12:1405-1408)....

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85A61K38/17A61K38/20A61P35/00A61P31/12
CPCC12N15/85C07K14/55C07K16/2827A61K38/00C07K2319/00C07K2319/33C07K2319/30A61K2039/505C07K16/2818
Inventor 岳喜连刘根桃吴国祥
Owner 科弈(浙江)药业科技有限公司
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