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Multifunctional fusion protein for CD19<+> tumors and application of protein

A fusion protein and multi-functional technology, applied in the direction of fusion polypeptide, antineoplastic drugs, immunoglobulin, etc., can solve the problems of lack of red blood cells, low curative effect, side effects, etc., achieve enhanced inhibition of tumor growth, wide application range, and increase immune response Effect

Pending Publication Date: 2019-08-16
科弈(浙江)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, since CD47 is generally expressed in red blood cells and platelets, simply shielding CD47 / SIRPα will not only cause serious side effects (Blood.2006; 107(6): 2548–56), but also require a larger dose of antibody drugs to achieve shielding purpose (J Exp Med 2001, 193:855–862)
At the same time, due to the high affinity of the antibody, the immunosuppressive effect of the antibody alone will cause the elimination of normal cells expressing CD47, such as the lack of red blood cells, resulting in extreme anemia (eLife 2017; 6: e18173)
Due to the weak affinity between SIRPα and CD47 and the existence of a large number of cells expressing CD47 in the body, the effect of simply using SIRPα to shield CD47 is not ideal, and the clinical effect is also poor.
[0003] 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 CD19-positive tumor cells have the disadvantage of low efficacy

Method used

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  • Multifunctional fusion protein for CD19&lt;+&gt; tumors and application of protein
  • Multifunctional fusion protein for CD19&lt;+&gt; tumors and application of protein
  • Multifunctional fusion protein for CD19&lt;+&gt; tumors and application of protein

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-CD19Fab and IgG1Fc (positions 31-150 in the sequence shown in SEQ ID NO: 1, positions 1-121 in the sequence shown in SEQ ID NO: 2, positions in SEQ ID NO: 3 Bases corresponding to positions 1-114 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 Cultured in an incubator, the supernatan...

Embodiment 2

[0046] This example is an affinity test of the multifunctional recombinant fusion protein to CD19 positive tumor cells.

[0047] Determination of protein affinity curve for CD19 cells: take CD19 positive Raji tumor cell line, 1×10 per well 4 Cells were placed in 96-well plates in 0.1 ml of PBS, PE-labeled fusion proteins and CD19 antibody proteins of different concentrations were added, mixed well, and placed at 4°C 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. figure 2 showed that for CD19-positive cells, the fluorescence signal (MFI) of PE was positively correlated with the concentration of the fusion protein, and the fusion protein had a similar affinity curve with CD19. This example proves that the fusion protein can bind to CD19-positive tumor cells, and proves that the fusion protein has good targeting to CD19.

Embodiment 3

[0049] This example shows the shielding effect of the multifunctional recombinant fusion protein on the CD47 signaling pathway.

[0050] Dilute 1 x 10 in 100 μl PBS 4 CD19-positive Raji cells were transferred to a 96-well plate, 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. image 3 Shown in is the signal intensity (APC fluorescence intensity) of CD47 antibody binding to CD19 positive cells analyzed by flow cytometry. It can be seen from the figure that the shielding of the fusion protein to the CD47 antibody presents a concentration gradient. 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 fusion protein for CD19<+> tumors. The fusion protein comprises an antibody functional area, an SIRP (signal regulatory protein) alpha extracellular functional area, high-affinity IgG1Fc combined with an Fc receptor and a non-functional amino acid fragment, wherein the antibody functional area specifically recognizes CD19 antigens, the SIRP alpha extracellular functionalarea shields tumor immune suppression antigen CD47 signals, and the non-functional amino acid fragment is used for connecting the functional areas. The invention further relates to preparation and application of the fusion protein. The recombinant fusion protein can recognize CD19 positive tumor cells and can also shield a CD47 immune suppression signal channel, the high-affinity Fc portion can enhance the immune reaction of immune cells for the tumors, and the fusion protein has an excellent clinical prospect and a wide application range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multifunctional fusion protein targeting CD19-positive tumors and its application in treating cancer. Background technique [0002] Various tumors evade immune control through activation of the CD47 / SIRPα signaling pathway (Cancer Res. 2011; 71(4): 1374–84). Tumor cells with high expression of CD47 can inhibit the phagocytosis of SIRPα-expressing macrophages on tumors, thereby reducing the function of antigen-presenting cells and affecting the body's secondary immune activity against tumors (Nat Med.2015; 21(10): 1209– 15), so the density of tumor CD47 expression is negatively correlated with the clinical outcome of cancer patients (Cell. 2009; 138(2): 286-99). Shielding the CD47 / SIRPα signaling pathway with an antibody drug against CD47 can increase the phagocytosis of tumors by macrophages (Cell. 2010; 142(5): 699-713). However, since CD47 is generally expressed in red blood ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85A61K38/17A61K47/68A61P35/00
CPCC07K16/2803C07K14/70503C12N15/85A61P35/00C07K2319/30C07K2317/622A61K38/00
Inventor 岳喜连张传能张朝宾徐圣涛吴国祥
Owner 科弈(浙江)药业科技有限公司
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