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Human IL2 (interleukin 2) and anti-EpCAM (epithelial cell adhesion molecule) single-chain antibody fusion protein and application thereof

A technology of fusion protein and epithelial cells, which is applied in the fields of genetic engineering, medicine and biomedical medicine, can solve the problems of drug entry, restriction of macromolecular antibody drugs and tumor immune cell tumor killing effect, difficulty of entry of immune cells and macromolecular drugs, etc. , to achieve the effects of reducing toxicity, promoting recognition and signal transduction, and better molecular penetration

Inactive Publication Date: 2018-09-07
ZHEJIANG K2 ONCOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, due to the problem of drug entry in solid tumors, it is difficult for immune cells and macromolecular drugs to enter the tumor, which limits the tumor killing effect of macromolecular antibody drugs and tumor immune cells

Method used

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  • Human IL2 (interleukin 2) and anti-EpCAM (epithelial cell adhesion molecule) single-chain antibody fusion protein and application thereof
  • Human IL2 (interleukin 2) and anti-EpCAM (epithelial cell adhesion molecule) single-chain antibody fusion protein and application thereof
  • Human IL2 (interleukin 2) and anti-EpCAM (epithelial cell adhesion molecule) single-chain antibody fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of fusion protein of anti-EpCAM single chain antibody and IL-2

[0050] The fusion protein EPIL2 of anti-EpCAM single-chain antibody and IL-2 comprises from N-terminal to C-terminal sequence: 1) IL-2 or IL-2 mutant; 2) linker; 3) anti-EpCAM single-chain antibody. The amino acid sequence of IL-2 or IL-2 mutant is shown in SEQ ID NO.2, 4, 6 and 8; The amino acid sequence of linker is shown in SEQ ID NO.10; The amino acid sequence of anti-EpCAM single chain antibody is shown in Shown in SEQID NO.11, 13.

[0051] The DNA sequence of the fusion protein of the anti-EpCAM single-chain antibody and IL-2 is synthesized by the method of full-sequence gene synthesis (SEQ ID NO.16 and 18). After the synthetic sequence is inserted into the T vector, it is digested with HindIII and NheI pTT5 plasmid vector and pEASY-T1 positive clone loaded with target sequence. Recover the long fragment of the vector and the target fragment excised from the T vector, connec...

Embodiment 2

[0058] Example 2: Expression and purification of fusion protein of anti-EpCAM single chain antibody and IL-2

[0059] The low-endotoxin large-scale plasmid transfected HEK293 cells by liposome method, the specific steps are: inoculate 6x10 cells in 20ml DMEM (+10% FBS+antibiotics) medium 6 HEK293 cells. Cells were cultured at 37°C, 175rpm, 5% CO2 for 3 days. Take the cell count, when the cell density is about 2-3x10 6When cells / ml, dilute the treated cell density to 1x106 cells / ml with fresh DMEM (+10% FBS, no antibiotics) medium, and the volume of each bottle of cell liquid is 20mL, then tighten the bottle mouth and put it in a shaker to continue culturing. Transfection can be performed after 2-4 hours. In a sterile test tube, 500 microliters of DMEM was used to dilute 20 micrograms of plasmid, and in another test tube, 50ul of lipofectamine 2000 (brand: invitrogen product number: #11668-027) was added, and after standing for 5 minutes at room temperature, the plasmid was ...

Embodiment 3

[0062] Example 3: Detection of the affinity activity of the fusion protein of anti-EpCAM single chain antibody and IL-2

[0063] 1. Qualitative verification of the ability of the fusion proteins EPIL-2-2303301 and EPIL-2-2303407 to bind to cellular EpCAM:

[0064] skov3 cells were used for staining to test the affinity of EPIL-2-2303301 and EPIL-2-2303407 to EpCAM protein. The skov3 cells were grown adherently in a 24-well plate for 24 hours, fixed with 4% PFA (paraformaldehyde) for 10 minutes, added 5% BSA to block non-specific antigens, added anti-human IL-2 primary antibody and incubated for 1 hour, After washing with PBST for 3 times, the secondary antibody was added, and after incubation for 1 hour, DAB (diaminobenzidine) was added to develop the color. After washing with PBS, photographs were taken under a bright-field microscope. It can be seen that EPIL-2-2303301 and EPIL-2-2303407 can form strong cell staining, which qualitatively proves their ability to bind EpCAM a...

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Abstract

The invention relates to an anti-EpCAM (epithelial cell adhesion molecule) single-chain antibody conjugated and genetic engineering optimized IL2 (interleukin 2) fusion protein. The molecular weight is about 46 Kda, an IgG structure is replaced with the single-chain antibody, the molecular structure is smaller, and steric hindrance is reduced, so that the protein has more flexible space structureand higher biological activity titer. The fusion protein has the affinity as high as that of EpCAM protein and stronger T cell proliferation promoting capability; the fusion protein can be crosslinkedwith the EpCAMs on tumor cell surfaces and interleukin receptors on T cell surfaces, promote recognition of immune cells and target molecule expression cells and signal transduction, trigger recognition and killing of tumor cells by the immune cells and can further activate T cell proliferation promoting interleukin receptor signals, and further has better lymphocyte tumor infiltration promotingcapability. The fusion protein can serve as a new-generation biological drug to be applied to treatment of tumor diseases or serve as a tool protein for in-vitro detection.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering, medicine and biomedicine, and specifically relates to a fusion protein of human interleukin (IL-2) and anti-human epithelial cell adhesion molecule (EpCAM) single-chain antibody, and a nucleic acid molecule encoding the fusion protein, comprising The pharmaceutical composition and kit of the fusion protein, and the application of the fusion protein in the treatment of tumor diseases. Background technique [0002] Human interleukin 2 (IL-2) is a cytokine secreted by helper T lymphocytes, mainly produced by activated Th1 cells, and plays an important role in the normal regulation and defense mechanism of the immune system of the body. Central to the regulation of responses. IL-2 does not directly kill tumor cells, but indirectly exerts anti-tumor effects by stimulating and activating effector cells. Effector cells involved in tumor immunity mainly include T lymphocytes, natural kille...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62G01N33/68C07K14/55G01N33/574C12N5/0783C12N15/26A61K38/20A61K39/395A61P35/00
CPCA61K38/00A61P35/00C07K14/55C07K16/30C07K2317/622C07K2319/00C12N5/0636C12N2501/2302C12N2501/58G01N33/57419G01N33/6869A61K2039/505C07K2317/92G01N2333/55
Inventor 孙志坚康平郭子贞张铭浩
Owner ZHEJIANG K2 ONCOLOGY CO LTD
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