Cancer targeting enhanced anti-tumor fusion protein, preparation method and use thereof

A fusion protein and enhanced technology, applied in the field of fusion proteins, can solve the problems to be improved, and achieve the effect of accelerating cancer cell apoptosis and obvious killing effect.

Active Publication Date: 2019-10-08
孙嘉琳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The activity of the above B7.1, B7.2 or superantigens in inhibiting tumors still needs to be improved

Method used

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  • Cancer targeting enhanced anti-tumor fusion protein, preparation method and use thereof
  • Cancer targeting enhanced anti-tumor fusion protein, preparation method and use thereof
  • Cancer targeting enhanced anti-tumor fusion protein, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction of vectors expressing various proteins

[0070] The expression plasmid adopted is pET22b (Novagen Company), which contains various restriction enzyme cutting sites (BamHI-EcoRI-SacI-SalI-HindIII-NotI-XhoI) for the insertion of DNA fragments encoding foreign proteins, and outside expression The C-terminus of the source protein has a His-tag (containing 6 His) for protein purification.

[0071] DNA fragments were synthesized by TAKARA Company and Shanghai Sangon Bioengineering Company.

[0072] The nucleic acid sequence of the fragmented Trastuzumab single-chain antibody is shown by SEQ ID No. 11, and VL and VH are linked by linker peptide 2 (SEQ ID No. 43).

[0073] Fragment Trastuzumab single-chain antibody-B7.1 fusion protein is connected with a connecting peptide 2 nucleic acid sequence shown in SEQ ID No.43 behind the Trastuzumab nucleic acid sequence shown in SEQ ID No.11, which is connected with a linker peptide 2 nucleic acid sequence shown...

Embodiment 2

[0135] Example 2 Expression, denaturation and renaturation and purification of various proteins

[0136] various expression plasmids

[0137] pET22b-Trastuzumab, pET22b-Trastuzumab-B7.1, pET22b-Trastuzumab-B7.2, pET22b-Trastuzumab-SEA, pET22b-Trastuzumab-B7.1-B7.2, pET22b-Trastuzumab-B7.1-B7.2- SEA, pET22b-Rituximab, pET22b-Rituximab-B7.1, pET22b-Rituximab-B7.2, pET22b-Rituximab-SEA, pET22b-Rituximab-B7.1-B7.2, pET22b-Rituximab-B7.1-B7. 2-SEA, pET22b-Cetuximab, pET22b-Cetuximab-B7.1, pET22b-Cetuximab-B7.2, pET22b-Cetuximab-SEA, pET22b-Cetuximab-B7.1-B7.2, pET22b-Cetuximab-B7.1- B7.2-SEA, pET22b-panitumumab, pET22b-panitumumab-B7.1, pET22b-panitumumab-B7.2, pET22b-panitumumab-SEA, pET22b-panitumumab-B7.1-B7.2, pET22b-panitumumab-B7. 1-B7.2-SEA, pET22b-Ramucirumab, pET22b-Ramucirumab-B7.1, pET22b-Ramucirumab-B7.2, pET22b-Ramucirumab-SEA, pET22b-Ramucirumab-B7.1-B7.2 and pET22b-Ramucirumab-B7.2 and pET22b-Ramucirumab-B7.2 B7.1-B7.2-SEA were transformed into Escherichia coli BL...

Embodiment 3

[0139] Example 3 Preparation of mouse sarcoma cell line S180 expressing human HER2 (ErbB-2)

[0140] According to the GenBank database (NM_004448.3), the human HER2 (ErbB-2) gene fragment was inserted into the pEGFP-N1 plasmid (Clontech Company), and the plasmid DNA (ug) of pEGFP-N1-HER2 (erbB-2) was mixed with the transfection reagent Lipofectamine2000 (Invitrogen Company) (ul) was mixed at a ratio of 1:2, and then the mixture was added to culture in a 24-well plate containing mouse sarcoma cell S180 (1×10 5 cells / well), on the 2nd day, the culture medium containing G418 antibiotic (concentration: 500ug / ml) was used to continue culturing and screening (limiting dilution method). Mouse anti-human HER2 (ErbB-2) antibody (Abcam Company) confirmed that the monoclonal cell line expresses human HER2 (ErbB-2), so that the S180 / HER2 (ErbB-2) monoclonal cell line was obtained.

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Abstract

The invention discloses a cancer targeting enhanced anti-tumor fusion protein, a preparation method and use thereof. The cancer targeting enhanced anti-tumor fusion protein comprises: a) an antibody and b) the fusion protein that induces and activates T cells. The components of the cancer targeting enhanced anti-tumor fusion protein of the invention have the synergistic effect, and produces a superimposed amplification effect, which shows that the biological activity far exceeds a single molecule; the fusion protein is capable of inducing and activating T cells to kill tumors and enhancing therole of cytokines secreted by T cells, the experiments show that the cancer targeting enhanced anti-tumor fusion protein of the present invention enhances the secretion amount of interferon-gamma(IFN-gamma) and granzyme,the apoptosis of cancer cells can be accelerated, and the fusion protein has obvious killing effect on various cancers and solid tumors.

Description

technical field [0001] The invention relates to a fusion protein, in particular to a cancer-targeting enhanced anti-tumor fusion protein, its preparation method and application. Background technique [0002] Antibody technology has been widely used in targeted therapy of various diseases, especially tumor therapy, which reduces the side effects of chemical drugs. Antibody drugs that have been marketed include: [0003] Trastuzumab (Herceptin) antibody against HER2 (ErbB2) target, which is applied to the treatment of breast cancer (Proc Natl Acad Sci USA, 89, 4285-4289, 1992; Clin Cancer Res, 12, 904-916, 2006; Cancer Res, 69 , 9330-9336, 2009); [0004] Rituximab (Rituxan) antibody targeting CD20, which is used in the treatment of B-cell-derived lymphoma (Cancer Res, 68, 2400-2408, 2008; Cell Physiol Biochem, 32, 645-654, 2013); [0005] Cetuximab (Erbitux) antibody against the EGFR target, which is used in the treatment of colon cancer (Clin Cancer Res, 1, 1311-1318, 199...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/395A61P35/00
CPCC07K16/32C07K16/2887C07K16/2863C07K14/70532A61P35/00C07K2319/55A61K2039/505
Inventor 孙嘉琳
Owner 孙嘉琳
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