Fusion protein for restoring function of failing immune cell and application thereof

A technology of immune cells and fusion proteins, applied in the field of fusion proteins, can solve problems such as toxic side effects and off-target effects, achieve a wide range of applications, enhance the effect of inhibiting tumor growth, and have good clinical prospects

Active Publication Date: 2018-07-17
科弈(浙江)药业科技有限公司
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cytokines will activate all cells expressing their receptors, which may activate non-target cells and cause off-target effects. Clinical application will produce a series of toxic and side effects, which limits its clinical application (JI 2014, 192:5451 -5458), so the current clinical application range and efficacy of IL-2 are very limited (Immunity 2013, 38:13-25)
[0005] So far, there is no protein drug that can not only specifically recognize exhausted tumor-specific T cells, but also restore their function and expand their number

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fusion protein for restoring function of failing immune cell and application thereof
  • Fusion protein for restoring function of failing immune cell and application thereof
  • Fusion protein for restoring function of failing immune cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example is the gene construction and production purification of the recombinant fusion protein.

[0046] According to the C-terminal amino acid sequence of human PD-1 single-chain antibody (SEQ ID NO: 2) and the N-terminal amino acid sequence of interleukin 2 (SEQ ID NO: 3), through gene synthesis, enzyme digestion and further cloning, non-functional artificially constructed amino acids ( SEQ ID NO: 4) The two parts are connected to form the artificially constructed gene sequence of the fusion protein (SEQ ID NO: 5), and then transferred into the eukaryotic cell expression vector pcDNA3.1(-). Finally, the expression vector of the fusion protein was transfected into Chinese hamster ovary cells (CHO). Transfected cells were placed at 37°C, 5% CO 2 After culturing in an incubator, the supernatant was taken after 72 hours, and further purified by Protein A affinity chromatography. The final purified protein was a bifunctional recombinant fusion artificial protein (αPD...

Embodiment 2

[0048] This example is the activity and function determination of the bifunctional recombinant fusion protein on human PBMC cells cultured in vitro.

[0049] Human peripheral blood was separated and purified by lymphocyte density gradient centrifugation (Ficoll), and diluted with X-Vivo15 medium in a 24-well plate to a cell density of 5×10 6 / ml, the test protein was added to a final concentration of 200ng / ml. Then placed at 37°C, 5% CO 2 Cultured in the incubator for 72 hours, the cells were collected, stained with flow cytometry antibodies, and washed with flow cytometry for phenotypic determination and data analysis. figure 2 showed that compared with the control group without protein addition, PD-1 antibody (αPD1scFvFc) could not induce the expression of CD137 (0.06% vs 0.09%), while fusion protein (αPD1scFvFcIL2) treatment could significantly increase the expression of CD137 in CD8- and CD8+ cells Expression (0.96%). At the same time, if image 3 It showed that the P...

Embodiment 3

[0051] This example is the determination of the activity and function of the bifunctional recombinant fusion protein on human ascites immune cells.

[0052] The ascites of human lung cancer patients was taken, and the test protein was added to a 24-well plate to a final concentration of 200ng / 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 flow cytometry antibodies, and then perforin was used to break the cell membrane for internal staining of TNFα and IFNγ flow cytometry antibodies in CD8+ cells, washed and stained with flow cytometry antibodies instrument for phenotyping and data analysis. Such as Figure 4 showed that fusion protein (αPD1scFvFcIL2) treatment significantly increased the expression of TNFα in ascites PBMCs (13.0% vs 5.29%) compared with unfused PD-1 antibody (αPD1scFvFc).

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a fusion protein for restoring a function of a failing immune cell. The fusion protein comprises a functional area for recognizing the failing immune cell and a functional area for conducting activation and amplification on the failing immune cell, and the two functional areas are connected through a non-functional amino acid fragment with a certain length, wherein the amino acid sequence of the fusion protein is the sequence shown in SEQ ID NO:5 or the sequence with similar functions and a similarity of at least 90%. The invention further relates to the application ofthe fusion protein. By means of the fusion protein, the failing immune cell can be recognized while other immune cells can be activated, amplified and recognized, and the function of killing antigenpositive cells of the immune cell is restored. Compared with the prior art, the proliferative utility on a CD 8 effector cell is further improved, the activation of the effector cell and the expression of a costimulatory molecule are enhanced, the restriction of tumor growth and the function of virus infection control can be further enhanced, and the fusion protein has good clinical prospect and 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 exhausted immune cells and its preparation method and application. Background technique [0002] The occurrence of tumors is the result of gene mutations in the process of cell division in the body, and the growth of mutant cells loses regulatory control. If tumor cells cannot be cleared in a short period of time, continuous antigen stimulation will gradually cause functional exhaustion of tumor antigen-specific T cells (Nature Medicine 1999, 5:677-685), forming tolerance to tumors, and the immune system will respond to tumors. The cells become desensitized, causing further growth and spread of the tumor, forming cancer (PNAS. 2002, 99:12293-7). The occurrence of chronic diseases caused by viral infection is also the result of antigen-specific cell failure (Trends Immunol.2014, 35:51-60; Blood 2007, 109:4671-4678; Cell Death...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85A61K38/20A61K47/42A61P35/00A61P31/12
CPCC12N15/85C07K14/55C07K16/2818A61K38/00C07K2319/30
Inventor 岳喜连邱桂华张传能吴国祥
Owner 科弈(浙江)药业科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap