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Multifunctional fusion protein for type III epidermal growth factor receptor deletion-mutant receptor (EGFRvIII), and application of multifunctional fusion protein

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

Inactive Publication Date: 2018-11-13
SHANGHAI BIOMED UNION BIOTECHNOLOGY CO LTD
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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 parts to increase the amount of protein around target cells to block low-affinity inhibitory signaling pathways, and single-function drugs have the disadvantage of low curative effect, especially for EGFRvIII-positive tumors cell drug

Method used

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  • Multifunctional fusion protein for type III epidermal growth factor receptor deletion-mutant receptor (EGFRvIII), and application of multifunctional fusion protein
  • Multifunctional fusion protein for type III epidermal growth factor receptor deletion-mutant receptor (EGFRvIII), and application of multifunctional fusion protein
  • Multifunctional fusion protein for type III epidermal growth factor receptor deletion-mutant receptor (EGFRvIII), and application of multifunctional fusion protein

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-EGFRvIII scFv and IgG1Fc (31-150 positions in the sequence shown in SEQ ID NO: 1, 1-242 positions in the sequence shown in SEQ ID NO: 2, SEQ ID NO: The base corresponding to the 3-219 position in the sequence shown in 3) is synthesized with a non-functional amino acid flexible fragment (the sequence shown in SEQ ID NO: 4 or a mutant containing at least 90% of the same sequence as the above-mentioned position) through gene synthesis or its repeating sequence) to form a multifunctional fusion protein (SEQID NO: 5) gene, which is digested and further cloned, and then transformed into the eukaryotic expression vector pcDNA3.1(-). Finally, the vector containing the fusion protein gene was transfected into Chinese hamster ovary cells (CHO). Transfected cells were placed at 37°C, 5% CO 2 Cultured in ...

Embodiment 2

[0048] This example is an affinity test of the multifunctional recombinant fusion protein on EGFRvIII negative and positive tumor cells.

[0049] EGFRvIII-negative and positive MDA tumor cells were collected and divided into two groups, 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 the flow cytometer was used for measurement and data analysis. figure 2 It shows that for EGFRvIII-positive cells, the fluorescence signal (MFI) of PE is positively correlated with the fusion protein concentration, but is poorly correlated with EGFRvIII-negative cells, even when the protein concentration is 10000ng / ml, EGFRvIII-negative cells have no obvious fluorescence signal . This example proves that the fusion protein can selectively bind to EGFRvIII-positive tumor cells,...

Embodiment 3

[0051] This example shows the shielding effect of the multifunctional recombinant fusion protein on the CD47 signaling pathway.

[0052] Dilute 1 x 10 in 100 μl PBS 4 Transfer EGFRvIII positive cells to 96-well plate, then add PBS or SIRPα-Fc protein or fusion protein respectively to a final concentration of 5 μg / ml, let stand at room temperature for 20 minutes, then add APC-labeled anti-CD47 flow antibody staining, room temperature After standing for 15 minutes, after washing, the flow cytometer was used for measurement and data analysis. image 3 Shown in is the signal intensity (APC fluorescence intensity) of CD47 antibody binding to EGFRvIII positive cells analyzed by flow cytometry. It can be seen from the figure that SIRPα-Fc has almost no shielding effect on CD47 signal, while the fusion protein has more than 90% shielding effect on CD47 antibody binding, which proves that CD47 ligand (SIRPα) has a weak shielding effect on CD47, while this The inventive fusion protein...

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Abstract

The invention relates to a multifunctional fusion protein for the type III epidermal growth factor receptor deletion-mutant receptor (EGFRvIII). The multifunctional fusion protein comprises an antibody functional area for specifically recognizing the tumor antigen EGFRvIII, a SIRP alpha extracellular functional area for shielding a tumor surface immunosuppressive protein CD47, a high-affinity human antibody IgG1Fc for recognizing an Fc receptor, and non-functional amino acid fragments for linking the functional areas; the invention also relates to preparation and application of the fusion protein. The fusion protein provided by the invention can meet the needs of tumor immunotherapy for patients; the recombinant fusion protein not only can recognize EGFRvIII-positive tumor cells, but alsocan shield a CD47 immunosuppressive signaling pathway; furthermore, the high-affinity Fc part can increase the immune response of immune cells to tumor cells; therefore, the multifunctional fusion protein can enhance the function of inhibiting tumor growth after being clinically applied, and has a good clinical prospect and a wide application scope.

Description

technical field [0001] The invention relates to the technical field of fusion proteins, in particular to a fusion protein targeting EGFRvIII and its application in cancer treatment drugs. Background technique [0002] The occurrence of tumors is due to gene mutations in the process of cell division in the body, and the growth of mutant cells loses regulatory control. Tumor cells can maintain uncontrolled growth through excessive activation of the EGFR signaling pathway, and EGFR mutation is the most common way to maintain growth signal activation. EGFRvIII mutation is caused by the deletion of EGFR exon 2-7, and the signaling pathway is always in an abnormally activated state. The appearance of this phenotype is positively correlated with the malignancy of the tumor (Cancer Res (2003) 63: 6962-70). Studies have shown that up to 30% of malignant brain tumors are caused by EGFRvIII, and EGFR drugs are not sensitive to this phenotype of tumors, and have no clinical effect. Si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85A61K39/395A61P35/00A61K38/17A61K35/15A61K35/17
CPCA61K35/15A61K35/17A61K38/1774A61K39/39558A61P35/00C07K14/70503C07K16/2863C07K2317/622C12N15/85A61K2300/00
Inventor 岳喜连丘桂华张传能张朝宾吴国祥
Owner SHANGHAI BIOMED UNION BIOTECHNOLOGY CO LTD
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