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Engineered exosome in-situ nano vaccine as well as preparation method and application thereof

A nano-vaccine and exosome technology, applied in biochemical equipment and methods, chemical instruments and methods, and botanical equipment and methods, can solve the problems of poor efficacy of adjuvant radiotherapy and chemotherapy, and achieve the effect of inhibiting tumor growth

Pending Publication Date: 2022-04-29
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its special molecular phenotype, TNBC is not sensitive to hormone therapy or molecular targeted therapy, and the effect of conventional postoperative adjuvant chemotherapy and radiotherapy is poor

Method used

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  • Engineered exosome in-situ nano vaccine as well as preparation method and application thereof
  • Engineered exosome in-situ nano vaccine as well as preparation method and application thereof
  • Engineered exosome in-situ nano vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0038] Example 1: Preparation of α-LA engineered exosome in situ nanovaccine encapsulating ELANE and Hiltonol

[0039] A method for preparing an α-LA engineered exosome in situ nano-vaccine wrapping ELANE and Hiltonol, the method comprising the following steps:

[0040] 1. Construction of MDA-MB-231 cell line stably overexpressing α-LA

[0041]The full-length coding sequence of human α-LA (Accession: CR542017) was cloned from the total RNA of MDA-MB-231 cells (preserved in our laboratory), and cloned into the lentiviral expression vector pCDH-CMV-puro (JHepatol.2017Oct ; 67(4):739-748.) (preserved by our laboratory). Human 293T cells were inoculated in a 6cm dish and cultured for 24h, and then were mixed with pCDH-CMV-puro-α-LA, pMD2.G (J GeneMed.2018Jul; 20(7- 8):e3027.) (preserved in our laboratory) and psPAX2 (JGene Med.2018Jul; 20(7-8):e3027.) (preserved in our laboratory) plasmids were co-transfected into human 293T cells. Virus was harvested after 48 hours and titrate...

Embodiment 2

[0046] Example 2: In vitro biological function evaluation of nano-vaccine HELA-Exos

[0047] The in vitro biological function evaluation of nanovaccine HELA-Exos includes the following three parts:

[0048] 1. In vitro cell targeting ability evaluation of nano-vaccine HELA-Exos

[0049] HELA-Exos or Texs were labeled with DiI and incubated with a cell mixture of MDA-MB-231 cells and human peripheral blood mononuclear cells (PBMC) for 2 hours, or after MDA-MB-231 cells and human lung cancer cells A549 cells were plated, respectively Add DiI-labeled HELA-Exos or Texs, and evaluate the cell uptake efficiency by flow cytometry and fluorescent confocal experiments. The results are as follows: image 3 As shown in A-B, HELA-Exos significantly improved the targeting ability compared with Texs, and MDA-MB-231 cells took up more nano-vaccine HELA-Exos compared with PBMC and A549 cells, indicating that HELA-Exos has the effect on breast cancer cells Has excellent target specificity. ...

Embodiment 3

[0054] Example 3: In vivo anti-cancer effect and immune activation evaluation of nano-vaccine HELA-Exos

[0055] The in vivo anti-cancer effect and immune activation evaluation of nano-vaccine HELA-Exos includes the following three parts:

[0056] 1. Evaluation of tumor growth inhibition of nano-vaccine HELA-Exos in immunocompetent orthotopic mice

[0057] Immunocompetent Balb / c mice were orthotopically inoculated with the MDA-MB-231 cell line stably expressing luciferase, and the tumors grew to about 100 mm in size after about 21 days 3 DMEM basal medium, Hiltonol or HELA-Exos treatment was started at the beginning of the treatment, and the tumor volume was monitored. After 30 days of treatment, the mice were anesthetized for in vivo imaging, and then the mice were sacrificed. The result is as Figure 5 Shown, in vivo tumor imaging and tumor growth curve results Figure 5 A-C show that HELA-Exos treatment significantly inhibits tumor growth in mice. In addition, in HELA-E...

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Abstract

The invention discloses a recombinant vector for overexpressing alpha-LA, a cell line for overexpressing alpha-LA, an exosome, a nano vaccine prepared by using a breast cancer-derived exosome enriched with alpha-whey protein after engineering modification, and a preparation method and application of the nano vaccine. According to the nano vaccine, tumor tissue is specifically targeted through alpha-whey protein, tumor cells are induced to generate immunogenic cell death, tumor-related antigens, Hiltonol and injury-related molecular patterns (DAMPs) are released, 1-type conventional dendritic cells are activated in situ, then powerful tumor specific CD8 + T cell reaction is triggered, and effective anti-tumor activity is shown. The nano vaccine provided by the invention provides a new method for targeted killing of tumor cells, provides a new mode for in-situ activation of type 1 conventional dendritic cells, and provides a new idea for immunotherapy for improving breast cancer.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy drugs, and in particular relates to an engineered exosome in situ nano vaccine and its preparation method and application. Background technique [0002] As the most efficient antigen-presenting cells, dendritic cells (DCs) are central to the initiation and regulation of innate and adaptive immunity in the tumor microenvironment. Therefore, a variety of vaccines targeting DCs have been developed to improve cancer immunotherapy, and several clinical trials have been conducted, but it is still difficult to promote clinically. A kind of DCs vaccine that has been widely studied at present is a DCs nano-vaccine composed of antigen and adjuvant. This vaccine directly activates and mobilizes natural DCs subsets at multiple sites in the body, which is an important method to enhance the anti-tumor effect of DCs. However, due to the lack of targeting of adjuvants, it is easy to induce non-specif...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N5/10A61K39/00A61K39/39A61K47/46A61K38/48A61P35/00
CPCC12N15/86C12N5/0693C12N5/0631C07K14/47A61K39/0011A61K47/46A61K39/39A61P35/00A61K38/486C12N2740/15043C12N2510/00A61K2039/55577
Inventor 汪付兵黄兰祥袁纯辉荣媛
Owner WUHAN UNIV
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