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A fusion protein and its application for restoring the function of exhausted immune cells

A technology of immune cells and fusion proteins, applied in the field of fusion proteins, can solve the problems of specific cells that cannot be targeted at exhaustion, achieve good clinical prospects, wide application range, and enhance the effect of controlling viral infection

Active Publication Date: 2018-11-13
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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  • A fusion protein and its application for restoring the function of exhausted immune cells
  • A fusion protein and its application for restoring the function of exhausted immune cells
  • A fusion protein and its application for restoring the function of exhausted immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0044] According to the amino acid sequence of the C-terminal of the human PD-1 antibody heavy chain (SEQ ID NO: 6) and the N-terminal of interleukin 2 (SEQ ID NO: 14), through gene synthesis, enzyme digestion and further cloning, the two parts were passed through 17 non- Functional amino acids (SGGGGSGGGGSGGGGSG) were connected to form the fusion protein heavy chain construct gene (SEQ ID NO: 23), and then transferred into the eukaryotic expression vector pcDNA3.1. Through gene synthesis, enzyme 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). Transfected cells were placed at 37°C, 5% CO 2 Culture...

Embodiment 2

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

[0047] Human peripheral blood was isolated and purified by lymphocyte density gradient centrifugation (Ficoll), and the cell density was diluted to 5×10 in X-Vivo15 medium in a 24-well plate. 6 / ml, add the test protein to a final concentration of 2 μg / ml. Incubate at 37°C for 30 minutes, replace the X-Vivo15 medium after centrifugation, until the final cell density is 5x10 5 / ml. Then placed at 37°C, 5% CO 2 After being cultured in the incubator for 72 hours, the cells were collected and stained with CD8 and PD-1 antibodies, and flow cytometry was used for phenotypic determination and data analysis. figure 2 Shown in , with unfused PD-1 antibody ( figure 2 Part 2 of ) and interleukin 2 ( figure 2 Part 3 of ) compared to fusion protein treatment ( figure 2 Part 1 of ) can significantly increase the proportion of CD8+ c...

Embodiment 3

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

[0050] Black mice were injected with fusion protein (4 μg / mouse / day) or interleukin 2 (40 μg / mouse / day) or PBS (control group) through the abdominal cavity for 3 consecutive days, and peripheral blood was collected from the tail on the 4th day, and treated with anti-mouse CD8 and NK1.1 flow cytometry antibody staining detection, data analysis such as Figure 4 . with the control group ( Figure 4 Part 1, 4.0%) and interleukin 2 group ( Figure 4 Compared with the second part of , 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 part of CD8+T cells, compared with the control group ( Figure 4 Part 1 of , 15.5%), native interleukin 2 did not increase the proportion of CD8+ cells ( Figure 4 Part 2 of the fusion protein, 15.1%), while the proportion of CD8+T ...

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Abstract

The present invention relates to a fusion protein that restores the function of exhausted immune cells. The fusion protein includes a functional region that recognizes exhausted immune cells and a functional region that activates and expands exhausted immune cells. The two functional regions pass through a certain length of non-functional amino acid fragments connect. The recognition domain is the use of immune checkpoint-specific antibodies to recognize the phenotypic receptors of exhausted immune cells; the activation and expansion of the functional domain uses cytokines or functionally similar mutants, or ligands or functionally similar mutants of surface receptors, or Activating antibodies to activate exhausted immune cells; it also involves the use of this fusion protein. Tests have shown that the fusion protein prepared by the present invention can not only recognize exhausted immune cells, but also activate and expand the recognized immune cells, restore the function of immune cells to kill antigen-positive cells, and enhance the functions of inhibiting tumor growth and controlling viral infection. It has a good clinical prospect and a wide range of applications.

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 exhausted immune cells and its application. Background technique [0002] Cancer is the most common and serious disease that threatens people's lives and quality of life. Chronic diseases caused by viral infections are even more difficult problems that plague the world. There are various deficiencies in the current clinical application of drugs for cancer, such as large side effects of chemotherapy drugs, drug resistance of targeted drugs (Curr Pharm Des.2010, 16:3-10), and low efficiency of immune checkpoint inhibitors ( 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) . The number of people infected with HBV (hepatitis B virus) in China reaches hundreds of millions (APJCP, 2011, 12:1405-14...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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