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Multifunctional fusion protein for Her2+tumor and application thereof

A fusion protein, multifunctional technology, applied in antitumor drugs, fusion polypeptides, immunoglobulins, etc., can solve the problems of CD47 cell clearance, low efficacy, lack of red blood cells, etc., to enhance the function of inhibiting tumor growth and increase immune response , the effect of a wide range of applications

Pending Publication Date: 2019-03-29
上海科弈药业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high affinity of the antibody, the immunosuppressive effect of the antibody will cause the elimination of other CD47-expressing cells, such as the lack of red blood cells, resulting in extreme anemia (eLife 2017; 6: e18173)
Due to the weak affinity of SIRPα-CD47 and the existence of cells expressing CD47 in large quantities, the effect of simply using SIRPα to shield CD47 is not ideal
[0004] So far, the single-function drugs targeting Her2-positive tumor cells have disadvantages such as low efficacy, and there is no clinically available protein drug that uses high-affinity fragments to increase the amount of protein around target cells to block low-affinity inhibitory signaling pathways

Method used

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  • Multifunctional fusion protein for Her2+tumor and application thereof
  • Multifunctional fusion protein for Her2+tumor and application thereof
  • Multifunctional fusion protein for Her2+tumor and application thereof

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 functional region amino acids of human SIRPα, anti-Her2scFv and IgG1Fc (positions 31-150 in the sequence shown in SEQ ID NO: 1, positions 1-119 in the sequence shown in SEQ ID NO: 2, positions in SEQ ID NO: 3 Bases corresponding to positions 1-107 in the shown sequence and positions 3-219 in the sequence shown in SEQ ID NO: 4 are synthesized with non-functional amino acid flexible fragments (sequence shown in SEQ ID NO: 5) or its repeat sequence) base-linked into a multifunctional fusion protein (SEQ ID NO: 6) gene, through restriction enzyme digestion and further cloning, and then transferred to the eukaryotic animal expression vector pcDNA3.1 (-). Finally, the fusion protein gene will be The vector was transfected into Chinese hamster ovary cells (CHO). The transfected cells were placed at 37°C, 5% CO 2 After culturing in an incubator, the su...

Embodiment 2

[0047] This example is an affinity test of the multifunctional recombinant fusion protein to Her2 positive tumor cells.

[0048] Determination of protein affinity curve for Her2+ cells: take Her2-positive SK-BR-3 tumor cells, 1×10 per well 4 Cells were placed in a 96-well plate in 0.1 ml of PBS, PE-labeled fusion proteins of different concentrations were added, mixed well, and left at room temperature for 0.5 hours. After the cells were collected, they were washed once with PBS, and finally PE fluorescence was measured and data analysis was performed by flow cytometry. image 3 Shown in , for Her2 positive cells, the fluorescence signal (MFI) of PE is positively correlated with the fusion protein concentration. This example proves that the fusion protein can bind to Her2-positive tumor cells, and proves that the fusion protein has good targeting to Her2.

Embodiment 3

[0050] This example shows the shielding effect of the multifunctional recombinant fusion protein on the CD47 signaling pathway.

[0051] Dilute 1 x 10 in 100 μl PBS 4 Her2-positive SK-BR-3 cells were transferred to a 96-well plate, and then different concentrations of fusion proteins were added, and left at room temperature for 20 minutes. After washing once, APC-labeled anti-human CD47 flow antibody was added for staining, and left at room temperature for 15 minutes. After washing, the fluorescence signal of APC and data analysis were measured by flow cytometer. Figure 4 Shown in is the signal intensity (APC fluorescence intensity) of CD47 antibody binding to Her2 positive cells analyzed by flow cytometry. It can be seen from the figure that the 200 ng / ml fusion protein almost completely shields the binding of CD47 and the antibody, that is, the fusion protein of the present invention can completely shield the CD47 signaling pathway, achieving the design purpose of this emb...

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Abstract

The invention relates to a multifunctional fusion protein for Her2+ tumor cells, wherein the multifunctional fusion protein includes a single-chain antibody variable region (scFv) specifically recognizing a tumor antigen Her2, an SIRP[alpha] extracellular functional region shielding a signal of a tumor immunosuppressive protein CD47, a high-affinity IgG1Fc binding to an Fc receptor, and non-functional amino acid fragments for connecting various functional regions. The invention also relates to preparation and an application of the fusion protein. The recombinant fusion protein not only can recognize Her2 positive tumor cells, but also can shield CD47 immunosuppressive signaling pathways. At the same time, the high-affinity Fc part can enhance the immune response of immune cells to tumors,and has good clinical prospects and wide application range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein for Her2+ tumors and its application in tumor treatment. Background technique [0002] Tumors are the result of uncontrolled cell growth due to genetic mutations in the body's cell division process. Breast cancer is the cancer with the highest incidence in women (Int J Cancer.2010, 127:2893–2917), and one-third of the patients are characterized by high expression of Her2 (ERBB2). Her2 is a member of the tyrosine kinase family, and its high expression can lead to excessive cell growth and tumor formation. Trastuzumab, an antibody against Her2, is the first antibody drug approved for the treatment of breast cancer, which can significantly increase the survival rate of Her2-positive patients, and the effective rate in clinical application is 11-26%. However, even if the initial phase is effective, most patients will relapse within one year (Oncogene. 2007, 26:3637-364...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/17A61P35/00
CPCA61K38/00A61P35/00C07K14/705C07K16/32C07K2319/30C12N15/85
Inventor 岳喜连张传能张朝宾徐圣涛吴国祥
Owner 上海科弈药业科技有限公司
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