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Multifunctional fusion protein and application thereof

A fusion protein, multi-functional technology, applied in the field of fusion proteins, can solve the problems of low curative effect, achieve the effects of improving curative effect, improving inhibition and clearance, and wide application range

Inactive Publication Date: 2018-03-30
JIANGSU CTL BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, there is no clinically available protein drug that can specifically block multiple immunosuppressive signaling pathways, and drugs targeting a single pathway have the disadvantage of low efficacy

Method used

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  • Multifunctional fusion protein and application thereof
  • Multifunctional fusion protein and application thereof
  • Multifunctional fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This example is the gene construction and production purification of the recombinant fusion protein.

[0041] According to the functional region bases of human SIRPα, IgG1Fc and PD-1, the multifunctional fusion protein (SEQ ID NO: 5) gene is formed by gene synthesis and non-functional amino acid flexible fragment bases, and then transferred into the eukaryotic expression vector pcDNA3 .1. Through gene digestion and further cloning, the multifunctional fusion protein gene is transferred into a eukaryotic animal expression vector. Finally, the vector of the fusion protein was transfected into Chinese hamster ovary cells (CHO). Transfected cells were placed at 37°C, 5% CO 2 Cultured in an incubator, the supernatant was taken after 72 hours, and further purified by ProteinA affinity chromatography, and the final purified protein was a multifunctional recombinant fusion protein. The molecular weight of the purified protein is confirmed by electrophoresis detection, which ...

Embodiment 2

[0043] This example is the test of killing tumor cells in the ascites of patients with colorectal cancer by the multifunctional recombinant fusion protein.

[0044] Ascites of patients with colorectal cancer containing tumor cells and immune cells were collected and divided into two groups with 1ml per well and placed in a 24-well plate. The control group was added with PBS, and the experimental group was added with fusion protein to a final concentration of 1 μg / ml. After mixing, place at 37°C, 5% CO 2 Incubate for 48 hours in the incubator. After the cells were collected, they were washed once with PBS, and then stained with flow cytometry antibodies against immune cells (CD45) and tumor cells (EpCAM) for 20 minutes. After washing, flow cytometry was used for measurement and data analysis. figure 2 showed that compared with ascites of patients without fusion protein, fusion protein treatment can significantly reduce the proportion of tumor cells in ascites (0.23% vs 0.064%...

Embodiment 3

[0046] This example shows that the multifunctional recombinant fusion protein promotes the phagocytosis of tumor cells by macrophages in vitro.

[0047] 1x105 macrophages diluted in 100 μl DMEM medium were transferred to a 96-well plate, placed in a 37°C, 5% CO2 incubator for 2 hours, and then 2x105 tumor cells labeled with CFSE in 100 μl DMEM medium were added. Add fusion protein to a final concentration of 5 μg / ml as the fusion protein group, and wells without fusion protein as the control group. After continuing to culture for 2 hours, all the cells were collected, centrifuged, added anti-CD14 loss antibody, stained for 20 minutes, washed and analyzed by flow cytometry. image 3 The middle indicates that adding the fusion protein promotes the phagocytosis of H358 tumor cells. Compared with the control group, the proportion of CD14 cells containing CFSE tumors increased from 32% in the control group to 90.9% in the fusion protein group.

[0048] The above method was used to...

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Abstract

The invention relates to multifunctional fusion protein and an application thereof. The multifunctional fusion protein comprises: a. a functional area for identifying positive tumor cells CD47: an extracellular part of SIRP alpha, b. a functional area for identifying positive tumor cells PD-L1: an extracellular part of PD-1 and c. a functional area for being bonded to immune cells: a high-affinityhuman IgG1Fc part. The fusion protein disclosed by the invention can meet the needs of patients on immunotherapy of tumors; the recombinant fusion protein can identify positive tumor cells (CD47 andPD-L1) and can also be bonded to immune effector cells with Fc receptors; and due to clinical application of the fusion protein, the functions of inhibiting tumor growth and controlling viral infection can be enhanced, so that the fusion protein has very good clinical prospects and extensive range of application.

Description

[technical field] [0001] The invention relates to the technical field of fusion proteins, in particular to a multifunctional fusion protein and its application in cancer treatment. [Background technique] [0002] Cancer is the most serious disease that is common and threatens people's life and quality of life. There are various deficiencies in the current clinically applied drugs for cancer, such as large side effects of chemotherapy drugs, and targeted drugs are prone to drug resistance (Curr PharmDes.2010, 16: 3-10), the clinical efficacy of immune checkpoint inhibitors alone is low (NEM 2012, 366:2443-2454), and 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). Therefore, it is the most urgent subject and demand in the medical and health field in my country and even in the world to discover new drugs for treating cancer with high efficiency and low toxicity, reduce mortal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/17A61K47/68A61P35/00A61P35/02
CPCC07K14/70521C07K14/70596C07K2319/30A61K38/00
Inventor 岳喜连施炜星杨春霞陈瑛唐涛
Owner JIANGSU CTL BIOLOGICAL TECH CO LTD
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