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Fusion protein for EGFR+ tumors and application thereof

A fusion protein, tumor technology, applied in the biological field, can solve the problem of low efficacy, achieve the effects of wide application range, increase immune response, and good clinical prospects

Inactive Publication Date: 2019-05-10
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

[0005] So far, 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, and drugs with a single function targeting EGFR-positive tumor cells have the disadvantage of low curative effect

Method used

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  • Fusion protein for EGFR+ tumors and application thereof
  • Fusion protein for EGFR+ tumors and application thereof
  • Fusion protein for EGFR+ tumors 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 functional region amino acids of human SIRPα, anti-EGFR Fab and IgG1Fc (positions 31-150 in the sequence shown in SEQ ID NO: 1, positions 1-118 in the sequence shown in SEQ ID NO: 2, and positions in SEQ ID NO: The bases corresponding to the 1-107 positions in the sequence shown in 3 and the 3-219 positions in the sequence shown in SEQ ID NO: 4 are synthesized with non-functional amino acid flexible fragments (shown in SEQ ID NO: 5) sequence or its repeated sequence) to form a multifunctional fusion protein (SEQ ID NO: 6) gene, which is digested and further cloned, and then transferred to the eukaryotic expression vector pcDNA3.1 (-). Finally, the fusion protein will be contained The gene carrier 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 supern...

Embodiment 2

[0048] This example is an affinity test of the multifunctional recombinant fusion protein to EGFR positive tumor cells.

[0049] After staining the HCC827 tumor cell line with antibodies against human EGFR and CD47, the high expression of EGFR and CD47 was verified by flow cytometry ( figure 2 ).

[0050] Determination of the affinity curve of the protein to EGFR cells: take EGFR-positive HCC827 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 EGFR positive cells, the fluorescence signal (MFI) of PE is positively correlated with the fusion protein concentration, and the calculated IC50 value is 245ng / ml. This example proves that the fusion p...

Embodiment 3

[0052] This example shows the shielding effect of the multifunctional recombinant fusion protein on the CD47 signaling pathway.

[0053] Dilute 1 x 10 in 100 μl PBS 4 EGFR-positive HCC827 cells were transferred to a 96-well plate, and then different concentrations of fusion proteins were added, placed at room temperature for 20 minutes, washed once and stained with APC-labeled anti-human CD47 flow antibody, placed at room temperature for 15 minutes, washed with flow Cytometer was used to measure APC fluorescence signal and data analysis. Figure 4 Shown in is the signal intensity (APC fluorescence intensity) of CD47 antibody binding to EGFR positive cells analyzed by flow cytometry. It can be seen from the figure that the shielding EC50 of the fusion protein CD47 antibody is 13ng / ml. It can be seen that the fusion protein of the present invention can completely shield the CD47 signaling pathway.

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Abstract

The invention relates to a multifunctional fusion protein for EGFR+ tumor cells. The protein comprises an antibody function domain which specifically recognizes tumor antigen EGFR, a SIRP alpha extracellular function domain which blocks a tumor immunosuppressive antigen CD47 signal, a high affinity IgG1Fc which binds to an Fc receptor and a non-functional amino acid fragment for being connected with the function domains; the invention also relates to the preparation and application of the above fusion protein. The recombinant fusion protein both can recognize EGFR-positive tumor cells and block a CD47 immunosuppressive signal pathway, a high-affinity Fc portion can enhance the immune response of immune cells to tumors, and the protein has good clinical prospects and wide range of application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein for EGFR-positive tumors and its application in treating cancer. Background technique [0002] The spread and metastasis of tumor cells are the characteristics of malignant cancer (Nat.Rev.Cancer 2003, 3:362–374), and the overexpression of EGF and its receptor EGFR on tumor cells is one of the main reasons (J.Cell.Physiol . 2008, 214:559–567). The activation of EGF / EGFR signaling pathway can stimulate the growth of tumor cells, and EGFR is highly expressed in most tumor cells (Gastric Cancer 2012, 15:252-64). Antibodies targeting EGFR can promote the endocytosis of EGFR, reduce cell growth signals, and then inhibit tumor growth, spread and migration. Cetuximab is a therapeutic monoclonal antibody against EGFR, which can bind to EGFR and EGFRvIII with high affinity (Radiotherapy and Oncology 2009, 92: 393–398), causing the body’s primary and secondary immune respons...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K38/16A61K47/42A61P35/00
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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