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Cell membrane nano-vesicle wrapping immunosuppressant and over-expressing PD-L1 and preparation method and application thereof

A PD-L1 and immunosuppressant technology, applied in the field of biomedicine, can solve problems such as gastrointestinal intolerance, achieve the effect of inhibiting immune rejection in vivo, improving efficacy, and good biocompatibility

Active Publication Date: 2020-11-13
中山大学深圳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to use the immune checkpoint of the PD-1 / PD-L1 biological axis to treat immune rejection in organ transplantation is still rarely reported.
[0004] Rapamycin (RAPA), an immunosuppressant targeting the mTOR pathway in late stages of T cell differentiation and proliferation, however, mTOR inhibitors have a variety of other cellular targets in addition to lymphocytes, clinically cause myeloid Suppression and gastrointestinal intolerance and other serious adverse reactions, so it is urgent to find a method that can reduce the dosage, side effects and improve the efficacy of immunosuppressants such as rapamycin

Method used

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  • Cell membrane nano-vesicle wrapping immunosuppressant and over-expressing PD-L1 and preparation method and application thereof
  • Cell membrane nano-vesicle wrapping immunosuppressant and over-expressing PD-L1 and preparation method and application thereof
  • Cell membrane nano-vesicle wrapping immunosuppressant and over-expressing PD-L1 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of Cell Membrane Nanovesicles Overexpressing PD-L1 Protein

[0046] 1. Construct a 293T cell line capable of stably overexpressing PD-L1 protein and OFP tagged protein on the cell membrane

[0047] (1) 950 μL of 1× HBS buffer, 10 μL of an aqueous solution of a mixed plasmid with a concentration of 1 μg / μL, the mixed plasmid is a second-generation lentiviral packaging vector pSPAX2 plasmid (geneseed ), GSF1) and pMD2.G plasmid (geneseed (geneseed), GSF2), 10 μg of human PDCD1 gene lentiviral ORFcDNA expression virus plasmid (Sino Biological Inc, HG10377-UT); mix well, and drop Inject 50 μL of CaCl with a concentration of 2.5 mol / L 2 Aqueous solution, mixed evenly, and placed at room temperature in the dark for 20-30 minutes to obtain the virus packaging mixture;

[0048] (2) will be cultured with 3×10 6 The 10% FBS DMEM medium of 293T cells was replaced with a new identical medium, and then the virus packaging mixture was added to the medium, an...

Embodiment 2

[0063] Example 2 In vitro biological behavior and in vivo distribution of PD-L1 NVs

[0064] Since hyperactivated T cells are negatively regulated through the PD-L1 / PD-1 pathway, we wanted to investigate whether the PD-L1 protein on NVs interacts with the corresponding ligand PD-1 on target cells in vitro. Here, we incubated PD-L1-OFPNVs with corresponding IL-2-stimulated Jurkat T cells or bone marrow-derived dendritic cells (dc), and the specific experimental steps are as follows:

[0065] Nanovesicle cell binding assay: Jurkat T cells were incubated with PD-L1 NVs (50 μg / ml, protein weight) for 30 minutes, and centrifuged with Ceoporep-4 to make slides (China Yingtai). Wheat germ agglutinin (WGA) and Alexa-Fluor 488 conjugate were added to stain the cell membrane for 10 min, and HEK 293T cells were incubated with PD-L1-OFP NVs (50 μg / mL, protein amount) for 30 min. On this basis, wheat germ agglutinin (WGA) and Alexa-Fluor 488 conjugate were added to stain the cell membrane...

Embodiment 3

[0068] Example 3 RAPA@PD-L1 NVs preparation and in vitro culture test

[0069] In this example, we verified whether PD-L1 NVs could serve as a targeted drug delivery system to deliver small doses of rapamycin (RAPA) to effector T cells to enhance immune suppression. According to the mechanism of rapamycin inhibition of mTOR feedback AKT / mTOR / p70S6K pathway and T cell proliferation ( Figure 4 A), rapamycin can inhibit the phosphorylation of mTOR upstream protein AKT and downstream S6 ribosomal protein. Therefore, we prepared rapamycin-enveloped vesicles (RAPA@PD-L1 NVs) by electroporation, carried out encapsulation experiments between rapamycin and PD-L1 NVs, and then determined the in vitro activity of rapamycin. Release. The specific test steps are as follows:

[0070] RAPA loading (RAPA@PD-L1 NVs preparation): 6 mg (protein weight) of purified vesicles (PD-L1 NVs) and 1 mg RAPA (100 mg / mL diluted in PBS, pH 10) were electroporated in 1 mL at 4 °C buffer (1.15mM potassiu...

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Abstract

The invention discloses a cell membrane nano-vesicle wrapping an immunosuppressant and overexpressing PD-L1 and a preparation method and application thereof. The cell membrane nano-vesicle is composedof a biological cell membrane, PD-L1 protein is expressed on the surface of the cell membrane, and the immunosuppressant is wrapped in the cell membrane nano-vesicle. PD-L1 protein can be expressed on the surface of a cell membrane; the PD-L1 protein can be combined with PD-1 on the surface of a T cell and activate a PD-1 / PD-L1 immune checkpoint signal axis; meanwhile, the PD-L1 cell membrane nano-vesicle is used as a targeted drug delivery system for loading the immunosuppressant, and the wrapped immunosuppressant is brought to effector T cells expressed by PD-1 to play a role so that the transmission of immunosuppressive drugs to target tissues is enhanced; through combined application of PD-1 / PD-L1 and an immunosuppressant double immune tolerance mechanism to T cells, in-vivo immunological rejection is significantly inhibited, the immunotherapy effect is improved, meanwhile, the dosage of the immunosuppressant is reduced, and the toxicity of the immunosuppressant is reduced.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, more specifically, to a cell membrane nanovesicle that encapsulates an immunosuppressant and overexpresses PD-L1, and a preparation method and application thereof. Background technique [0002] The use of transplanted healthy tissue to replace missing organ tissue has always been one of the goals of the medical community. Organ transplantation is almost the only hope for a complete cure for patients with organ failure, but often because the recipients reject foreign individuals due to their own immune reactions. Therefore, immune rejection is the biggest obstacle to the development of organ transplantation. Using certain immunosuppressants or intervening in the process of immune regulation can reduce the immune ability of individuals after organ transplantation, prolong the time that the graft stays in the body, and increase the number of organs. Or the survival rate after tissue tran...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K47/46A61K31/436A61P37/06C07K14/705C12N15/867C12N5/10
CPCA61K9/1271A61K47/46A61K31/436A61P37/06C07K14/70532C12N15/86C12N2740/15043
Inventor 陈红波杨敏程芳徐占雪颜海兰贺超
Owner 中山大学深圳
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