Genetically engineered antigen presenting extracellular vesicle as well as preparation method and application thereof

A genetic engineering and antigen technology, applied in the field of genetically engineered antigen-presenting extracellular vesicles and their preparation, to achieve the effect of inhibiting tumor metastasis, facilitating clinical transformation, and enhancing tumor immunotherapy

Pending Publication Date: 2022-07-29
苏州歆牧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the combination of dendritic cells with CAR-T therapy and immune

Method used

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  • Genetically engineered antigen presenting extracellular vesicle as well as preparation method and application thereof
  • Genetically engineered antigen presenting extracellular vesicle as well as preparation method and application thereof
  • Genetically engineered antigen presenting extracellular vesicle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction of a dendritic cell tumor vaccine (Engineered DCs, eDCs) that highly expresses anti-CD19 scFv and PD-1 and knocks down PD-L1

[0070] (1) Isolation and culture of mouse bone marrow dendritic cells (BMDCs): Isolate mouse bone marrow cells from tibia and femur, add 20ng / mL GM-CSF and 10ng / mL to RPMI 1640 medium containing 10% fetal bovine serum IL-4 is cultured for 6-7 days, which is dendritic cells.

[0071] (2) Construction of dendritic cells highly expressing anti-CD19 scFv (HM852952) and PD-1 (NM_008798.3): After the formation of mouse bone marrow-derived dendritic cells (BMDCs), lentivirus-mediated gene transfer The staining method made BMDCs highly express anti-CD19 scFv and PD-1, and immunofluorescence ( figure 1 ) and flow cytometry ( figure 2 ) results showed that anti-CD19 scFv and PD-1 were highly expressed on BMDCs, indicating that BMDCs with high expression of anti-CD19 scFv and PD-1 were successfully established.

[0072](3) Constr...

Embodiment 2

[0075] Example 2Extraction and characterization of Engineered DC-derived extracellular vesicles (eDC-EVs)

[0076] (1) The BMDCs loaded with tumor-associated antigens and highly expressed anti-CD19 scFv and PD-1 and knocked down PD-L1 were replaced with RPMI 1640 medium without exosome serum for 48 hours, and the medium supernatant was collected;

[0077] (2) The supernatant of the medium was centrifuged at 300g for 10min at 4°C to remove the precipitation, 1000g for 10min to remove the precipitation, 2000g for 20min to remove the precipitation, 10000g for 30min to remove the precipitation, 100000g for 70min, and the supernatant was discarded. Add PBS to suspend and precipitate, namely the obtained genetically engineered DC-derived extracellular vesicles (eDC-EVs);

[0078] (3) Using transmission electron microscopy and DLS to analyze the morphology and particle size of the extracellular vesicles obtained in step (2) ( Image 6 , Figure 7 ), Zeta potential -20mV or so ( F...

Embodiment 3e

[0080] Example 3 The ability of eDC-EVs to interact with CD19 on PD-L1 and CT26 cells in vitro and the effect on the anti-tumor efficiency of T cells

[0081] (1) eDC-EVs and PD-L1 + Cellular interactions: CT26 colon cancer cells, bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) express PD-L1 under the induction of 20 ng / mL IFN-γ, and Cy5. The cells expressing PD-L1 were co-incubated for 24h, and the co-localization of Cy5.5 and PD-L1 was observed by Confocal Microscopy. The results showed ( Figure 11 ), PD-1 on extracellular vesicles has a strong interaction with PD-L1.

[0082] (2) Interaction between eDC-EVs and human-CD19 expressed on CT26 colon cancer cells: CT26 colon cancer cells highly expressed human-CD19 under the mediation of lentivirus encoding human-CD19 gene, and then interacted with Cy5.5-labeled The eDC-EVs were co-incubated for 24 hours, and the co-localization of Cy5.5 and CD19 was observed by Confocal Microscopy. The results showed ( ...

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Abstract

The invention discloses a genetically engineered antigen presenting extracellular vesicle, the antigen presenting extracellular vesicle loads a tumor associated antigen and overexpresses an immunomodulatory molecule and a single-chain antibody at the same time, and a vaccine preparation for tumor treatment is prepared based on the antigen presenting extracellular vesicle. Particularly, the compound has outstanding performance in inhibition of tumor metastasis, and has huge potential in serving as a new-generation drug loading platform. The genetically engineered extracellular vesicles constructed by the invention not only maintain the antigen presentation capability, but also target tumor antigens, and regulate solid tumor immune microenvironment so as to effectively activate T cells in tumors to exert anti-tumor activity, and finally realize tumor killing and metastasis inhibition functions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetically engineered antigen-presenting extracellular vesicle and a preparation method and application thereof. Background technique [0002] Cancer immunotherapy targeting the immune system has achieved clinical success in a variety of cancers. However, for solid tumors, the relatively low response rate of immunotherapy remains a challenge. For example, chimeric antigen receptor (CAR) T cells, due to their relatively large size, cannot effectively penetrate the tumor tissue through the vascular endothelium. Solid tumors cannot be effectively treated. Immune checkpoint blockade (ICB) antibodies have relatively low tumor targeting effects, lack of tumor accumulation, and have a relatively low response rate in some solid tumors. [0003] Nanoscale extracellular vesicles (EVs) are membranous vesicles secreted by cells and released into the extracellular matrix, with a diameter of ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/13C12N15/12C12N15/28C12N15/867A61K39/00A61P35/00A61P35/04
CPCC12N5/0639C12N5/0636C12N5/0645C07K14/70596C07K14/7151C07K14/70503C07K16/32C07K16/2803C07K16/3084C07K16/18C07K16/3007C07K16/2863C12N15/86A61K39/0011A61P35/00A61P35/04C07K2317/622C07K2317/56C12N2740/15043C12N2510/00A61K2039/5158
Inventor 汪超许方
Owner 苏州歆牧生物科技有限公司
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