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Fusion protein and applications thereof

A fusion protein, binding region technology, applied in the direction of immunoglobulin, immunoglobulin superfamily, animal/human protein, etc., can solve problems such as the application of restriction marker genes

Active Publication Date: 2018-04-10
CARSGEN THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These shortcomings limit the application of these marker genes

Method used

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  • Fusion protein and applications thereof
  • Fusion protein and applications thereof
  • Fusion protein and applications thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0124] Example 1. Expression of fusion protein FR806

[0125] In this embodiment, eGFP is selected as a fluorescent marker for analysis and testing, and eGFP is enhanced green fluorescent protein. Select F2A as the self-cleaving sequence. F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") from foot-and-mouth disease virus, which has the "self-cleaving" function of 2A; The partial amino acid sequence (SEQ ID NO:32) of type 1 (FOLR1) and the partial sequence (SEQ ID NO:28) of EGFR were expressed as fusion protein FR806 (SEQ ID NO:44), and the signal peptide of FOLR1 was selected as the signal peptide. The following genetic engineering operations were performed using standard methods known to those skilled in the art. The preparation method of the nucleotide (SEQ ID NO:1) of eGFP-F2A-FR806 is as follows:

[0126] SEQ ID NO:1

[0127] (eGFP is shown in bold, F2A is shown underlined, FR SP (folate receptor signal peptide) is shown in bold and underlined, epitope 80...

Embodiment 2

[0161] Synthesis and titration of embodiment 2, CH12-biotin

[0162] The CH12 antibody was labeled with biotin. Dilute CH12 antibody to 2.5mg / ml, PBS pH7.4, labeling volume 1.6ml; take 1mg of Sulfo-NHS-LC-Biotin (Thermo Company), add 180ul of ultrapure water to dissolve; take 79ul of Biotin and add to 1.6 ml of CH12 antibody, react overnight. Use PD-10 desalting column (GE company, USA) to desalt, replace in PBS 5% glycerol buffer solution, obtain CH12-Biotin, OD280 / 1.45 concentration is 0.77mg / ml.

[0163] Dilute CH12-biotin to different concentrations (100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0 μg / ml) with PBS containing 1% FBS, respectively, and express eGFP-F2A -The T cells of FR806 were incubated for 45 minutes, then washed with PBS, the secondary antibody was diluted with PE-SA (ebioscience company) 1:300 medium, and after adding the resuspended cells, incubated for 45 minutes. After washing twice with PBS, flow cytometry analysis, the results are as follo...

Embodiment 3

[0164] Example 3. Using CH12-biotin to sort FR806 positive T cells

[0165] Take 1×10 7 T cells expressing eGFP-F2A-FR806 were washed with PBS, incubated with CH12-biotin (10 μg / ml, diluted in PBS containing 1% FBS) for 45 min at 4°C, then washed with PBS, and anti-Biotin sorting magnetic beads (purchased from Meitian Nii Company), according to the steps given by the sorting magnetic bead product, the T cells of FR806 were sorted out. Take an appropriate amount of cells before and after sorting, and perform flow cytometry analysis. The results are as follows: Figure 4 As shown, T cells expressing FR806 can be effectively sorted out by anti-Biotin sorting magnetic beads after being combined with CH12-biotin, and the sorting positive rate reaches 95%.

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Abstract

The present invention provides a fusion protein represented by a formula I, and an immune effector cell Z-A-L-BI expressing the fusion protein on the surface, wherein Z is an optional signal peptide,A is an antibody binding region, L is an optional linker part, and B is an endocytosis functional region. The present invention further provides an immune effector cell expressing a chimeric antigen receptor and the fusion protein, wherein the fusion protein contains the antibody binding region and the endocytosis functional region. According to the present invention, the immune effector cells canperform the killing by using the antibody-conjugated drug of the antibody binding region, and has excellent differential toxicity; and the fusion protein can mediate endocytosis, such that the antibody-conjugated drug is endocytosed into the cell after the fusion protein is bound to the antibody-coupled drug to kill the cell in the cell so as to provide the significant killing capacity.

Description

technical field [0001] The present invention relates to the field of immunotherapy. More specifically, the present invention relates to fusion proteins for controlling chimeric antigen receptor immune effector cells or TCR-T cells and applications thereof. Background technique [0002] In recent years, adoptive cell therapy (ACT) for malignant tumors, such as CAR-T and TCR-T, has made considerable progress, among which the development of CAR-T therapy is the most significant. [0003] However, with the development of clinical trials of CAR-T cell therapy, many serious side effects have also appeared, such as cytokine storm, off-target effects, etc. When serious adverse reactions occur, if CAR-T cells cannot be inhibited in time, it will lead to Severe adverse reactions even endanger the lives of patients. Therefore, when using CAR-T therapy, it is necessary to introduce a safety switch at the same time. When a patient has a serious adverse reaction after using CAR-T cells,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C12N15/62G01N33/569C07K16/28C07K16/30C07K16/32A61K47/68
CPCC07K14/70532C07K14/70578C07K14/70596C07K14/82C07K16/2803C07K16/2818C07K16/2827C07K16/2863C07K16/2878C07K16/2887C07K16/303C07K16/32C12N5/0636G01N33/56966C07K2319/02A61P35/00G01N33/56972A61K47/6903A61K47/6949C07K14/705C07K14/7051C07K14/71C07K2317/34C07K2319/01C07K2317/622C07K2319/03C07K2319/33A61K39/001112A61K2039/5156A61K2039/5158C12N2510/00A61K2039/572A61K2300/00C07K19/00C12N5/10C12N15/62C12N15/85G01N33/537G01N33/547G01N33/577C07K16/30
Inventor 李宗海吴秀奇王华茂蒋华石必枝
Owner CARSGEN THERAPEUTICS
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