EGFP-Wnt2 fusion protein antigen, Wnt2 monoclonal antibody and application of Wnt2 monoclonal antibody

A monoclonal antibody and fusion protein technology, applied in the field of biomedicine, can solve problems such as complex mechanisms, achieve efficient clearance, obvious anti-tumor immunotherapy effect, and enhance the effect of anti-tumor immune response

Active Publication Date: 2020-09-04
杭州科兴生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CAFs have been proven to regulate the immune escape of tumor cells, due to the complex mechanism of tumor cell immune tolerance, the molecular regulatory network in which CAFs participate still needs to be further explored and elucidated.

Method used

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  • EGFP-Wnt2 fusion protein antigen, Wnt2 monoclonal antibody and application of Wnt2 monoclonal antibody
  • EGFP-Wnt2 fusion protein antigen, Wnt2 monoclonal antibody and application of Wnt2 monoclonal antibody
  • EGFP-Wnt2 fusion protein antigen, Wnt2 monoclonal antibody and application of Wnt2 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Correlation between the expression of Wnt2 and the components of tumor immune microenvironment

[0061] In this example, 50 patients with esophageal squamous cell carcinoma were collected. Firstly, Wnt2 and Vimentin co-positive (Wnt2+ / Vimentin+) CAFs, FoxP3 and CD4 co-positive (FoxP3+ / CD4+) Treg cells were detected by immunofluorescence method , and the distribution of IFN-γ and CD8+ co-positive (IFN-γ+CD8+) T cells in the tumor tissue of patients with esophageal squamous cell carcinoma, the results are as follows figure 1 shown.

[0062] Then, the TissueTAXS panoramic imaging system software was used to analyze the number of Wnt2+ / Vimentin+CAFs in each tumor tissue, the ratio of Treg to CD4+T cells (Treg / CD4+T), and the ratio of IFN-γ+CD8+T cells to CD8 The ratio of +T cells (IFN-γ+CD8+T / CD8+T) was finally analyzed by statistical methods, and the results were obtained: the number of Wnt2+ / Vimentin+CAFs was positively correlated with the ratio of Treg / CD4+T c...

Embodiment 2

[0063] Example 2 Wnt2 antibody pharmaceutical composition prepared by human wild-type Wnt2 gene

[0064] 1. Preparation of recombinant human wild-type Wnt2 protein

[0065] The fusion gene of the EGFP gene and the human wild-type Wnt2 gene (NM_003391.2) was constructed downstream of the CMV promoter of the lentiviral expression vector pLV-Puro by restriction enzymes EcoRI and NotI to obtain the plasmid pCMV-EGFP-Wnt2 , and a 6×His tag was attached to the carboxyl terminus of Wnt2 for nickel ion affinity column purification. Co-transfect pCMV-Wnt2 with plasmids pH1 and pH2 into lentiviral packaging line cell 293V, and after 5 passages or more than 20 days of culture to obtain cells stably expressing the target protein, use 5-10 μg / ml puromycin, 10% FBS After 3-5 days, use high-sugar DMEM medium containing 2.5 μg / ml puromycin and 10% FBS to maintain the culture; after proper expansion, use 10 3 The cell density of 1 cell / ml was inoculated in 6-hole cell culture plate, 2ml / well...

Embodiment 3

[0136] Example 3 Wnt2 secreted by mCAFs regulates the effect of T cells through DC cells

[0137] 1. Establishment of C57 mouse esophageal carcinoma in situ model

[0138] Methods: The carcinogen 4-nitroquinoline-1-oxide (4NQO) was used to induce primary esophageal cancer in C57BL / 6 mice by drinking water. After the mice drank 4NQO solution (100ug / ml) continuously for 16 weeks, they were changed to drink sterile water for 12-16 weeks; this method can induce the formation of esophageal carcinoma in situ in some mice.

[0139] 2. Primary isolation, identification and culture of mCAFs and mEC-1

[0140] Methods: The esophageal carcinoma in situ tissue of C57 mice was removed, and the tumor was cut into small pieces with clean surgical instruments. The chopped tumor tissue was digested with 1 mg / ml collagenase at 37°C for 30 minutes, and then processed with a 70um mesh sieve. Filtration; the single cell suspension obtained after filtration was centrifuged and washed, then resusp...

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Abstract

The invention relates to the technical field of biomedicine, in particular to an EGFP-Wnt2 fusion protein antigen, a Wnt2 monoclonal antibody and an application of a Wnt2 monoclonal antibody. The EGFP-Wnt2 fusion protein antigen is mainly a fusion protein of EGFP and a full-length amino acid sequence of a humanized Wnt2 signal-erasing peptide. The EGFP-Wnt2 fusion protein antigen comprises an EGFPamino acid sequence, a TEV restriction enzyme cutting site and a FLAG tag sequence, and the Wnt2 signal-erasing peptide full-length amino acid sequence, a 6* His tag sequence and an anti-Wnt2 monoclonal antibody can be used for inhibiting immune escape and growth of tumor cells. When the Wnt2 monoclonal antibody disclosed by the invention is combined with the human Wnt2 antigen secreted by cells,the promotion of the cell on the formation of an inhibitory immune microenvironment in a tumor is antagonized, and the immune escape and growth of the tumor cell are inhibited. The Wnt2 monoclonal antibody provided by the invention can be used in immune treatment of solid tumors such as esophageal squamous carcinoma, gastric cancer, pancreatic cancer, colorectal colon cancer, lung cancer, breastcancer, glioma, liver cancer and the like.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application of EGFP-Wnt2 fusion protein antigen, Wnt2 monoclonal antibody and Wnt2 monoclonal antibody. Background technique [0002] Immune escape of solid tumor cells is one of the hotspots and difficulties in clinical tumor immunotherapy and basic research of tumor immunology. At present, PD-1 / PD-L1 blocking antibodies have shown good potential in early clinical trials for the treatment of solid tumors. However, immunotherapy is often subject to the immune microenvironment of solid tumors and its complex immune escape regulation, resulting in an overall response rate Not high or even resistant. In-depth exploration of the molecular mechanism of tumor immune escape and the search for new immunotherapy targets will provide very important guiding significance for the development of new and efficient tumor immunotherapy. [0003] At present, biomedical researchers are pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C07K16/22C12N15/13A61K39/395A61P35/00
CPCC07K14/43595C07K16/22A61P35/00C07K2319/43C07K2319/21C07K2319/00C07K2317/56A61K2039/505
Inventor 付利黄土雄范春雷武虎陈喆匡红刘美星
Owner 杭州科兴生物科技有限公司
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