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VEGFR2-targeting metastatic cancer vaccine

A metastatic cancer, specific technology, applied in the field of cancer immunotherapy and prevention, can solve the problems of limited immune response, ineffective induction, and limited vaccine effectiveness.

Active Publication Date: 2020-07-24
NEWISH TECH (BEIJING) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, their research content is only applicable to people who carry the two subtypes of HLA-A2 and A24. Since the T cell immune response is HLA-restricted and peptide immunity generally cannot effectively induce antibody-mediated humoral immune response, HLA- A2 and HLA-A24 restricted VEGFR immunogenic peptides may have no effect on other HLA subtype populations
Moreover, the degree of immune response that can be induced by the immune peptides without epitope optimization is limited, which may limit the effectiveness of this type of vaccine

Method used

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  • VEGFR2-targeting metastatic cancer vaccine
  • VEGFR2-targeting metastatic cancer vaccine
  • VEGFR2-targeting metastatic cancer vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Design of Fusion Protein Vaccine Antigen and Construction and Preparation of Expression Plasmids in Mammalian Cells and Insect Bad Cells

[0061] (1) Construction of fusion gene mammalian cell expression vector:

[0062] Construct the fusion protein XCL1-VEGFR2 according to the amino acid sequence of mouse VEGFR2 (SEQ ID No.1 and SEQ ID No.2) and mouse XCL1 (SEQ ID No.4) protein, in order to promote the nucleic acid that expresses fusion protein to translate out in vivo The fusion protein can be effectively secreted to the outside of the cell and chemoattract MHC-II+CD11c+CD8a+antigen cross-presenting DC cells. We retained the secretion signal peptide of the XCL1 protein (SEQ ID No.5), and at the same time, VEGFR2 mediated cell cross-presentation The membrane-localized signal peptide was removed (SEQ ID No. 6). Amino acid sequences of the final fusion protein (SEQ ID No.7 and SEQ ID No.8). For a single VEGFR2 protein, the secretion signal peptide of the XCL1...

Embodiment 2

[0070] Example 2 Prediction of three-dimensional structure of fusion protein

[0071] According to the nucleotide sequence of the fusion protein, we used the http: / / raptorx.uchicago.edu / website to predict the three-dimensional structure of the XCL1 and VEGFR2 extracellular region fusion protein. XCL1 is a chemokine, and its normal chemotactic function needs to maintain a complete spatial structure. In order to ensure that XCL1 still retains its own spatial structure after the fusion of the extracellular region of XCL1 and VEGFR2, we predicted the three-dimensional structure of the fused amino acid sequence on the http: / / raptorx.uchicago.edu / website, and the results are as follows figure 2 It was shown that after the fusion of XCL1 and VEGFR2, the two still retain their original spatial structure, which does not affect the chemotactic function of XCL1.

Embodiment 3

[0072] Example 3 Detection of expression effect of mammalian cell expression vector encoding XCL1-VEGFR2 fusion protein

[0073] 24 hours before transfection, seed 1*10 cells in a 6-well cell culture plate 6 A HEK293T cell, and the transfection experiment was started when the cell density reached 70%-80%. Before transfection, pre-warm the cell culture medium and serum-free Opti-MEM medium in a 37° water bath. During transfection, add 5 μg of empty vector (Vector), separate VEGFR2 expression vector, XCL1-VEGFR2 wild-type and mutant fusion gene plasmids and 20 μL of PEI transfection reagent to 200 μL of serum-free Opti-MEM, mix well , let stand at room temperature for 10 minutes. Replace the cells to be transfected with fresh medium, gently add the above transfection system, and shake gently. Return the cells to the cell culture incubator for 6 hours before changing the medium. After 48 hours of transfection, the cells were harvested and Western Blot was used to detect the e...

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Abstract

The invention relates to the fields of immunotherapy and prevention of cancer, in particular to a VEGFR2 (KDR)-targeting metastatic cancer vaccine. Through fusion expression of VEGFR2 protein promoting generation of tumor angiogenesis with a DC cell ligand XCL1, the efficiency of cytophagy, processing and presentation of the VEGFR2 protein by DC cells is improved, and the effect of inhibiting tumor growth is improved. Experiments prove that the protein for nucleic acid vaccine expression can effectively bind the DC cells, induce specific T-cell reaction of VEGR2 and significantly inhibit growth of tumor highly expressing VEGFR2 in multiple models.

Description

technical field [0001] The invention relates to the field of immunotherapy and prevention of cancer, in particular to a metastatic cancer vaccine targeting VEGFR2. Background technique [0002] Tumor metastasis is the main cause of cancer death, more than 90% of cancer mortality is due to metastasis, and tumor angiogenesis is essential for tumor growth and metastasis (Valastyan and Weinberg). Tumor blood vessels provide sufficient oxygen and nutrients for tumor growth and metastasis. The formation of new blood vessels is a necessary step in the formation of tumor blood vessels. The neovascularization theory states that tumors induce the formation of new blood vessels from pre-existing vessels. Based on this theory, researchers have developed a series of drugs targeting tumor neovascularization. At present, the anti-angiogenic drugs approved by FDA are mainly divided into three categories, monoclonal antibodies, receptor tyrosine kinase inhibitors and fusion polypeptides. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K35/15A61K39/00A61P35/00A61P35/04
CPCA61K35/15A61P35/00A61P35/04A61K39/00113C07K14/521C07K14/71C07K2319/00
Inventor 齐海龙王晓芳严小娥李日勇罗天明谢皇帆刘德芳郭潇孙忠杰
Owner NEWISH TECH (BEIJING) CO LTD
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