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Tumor-targeted delivery system for in-vivo in-situ induction of CAR-T cells and application of tumor-targeted delivery system

A delivery system and cell technology, applied in the field of immuno-oncology, can solve problems that do not involve solid tumor treatment

Active Publication Date: 2021-09-14
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the way NPs deliver CAR genes has good application prospects, but it has not yet been involved in the treatment of solid tumors

Method used

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  • Tumor-targeted delivery system for in-vivo in-situ induction of CAR-T cells and application of tumor-targeted delivery system
  • Tumor-targeted delivery system for in-vivo in-situ induction of CAR-T cells and application of tumor-targeted delivery system
  • Tumor-targeted delivery system for in-vivo in-situ induction of CAR-T cells and application of tumor-targeted delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Delivery system for in situ induction of CAR-T cells targeting tumors

[0040] 1. Preparation of PBAE / pDNA

[0041] Dilute PBAE, CD123 gene and CRISPR plasmids (purchased by the company) in 25mM sodium acetate solution respectively, add the PBAE solution dropwise to the same volume of CRISPR plasmid solution and mix, then add the CD123 gene plasmid and mix well, let it stand for 15 minutes at room temperature, and automatically Assemble and construct PBAE / pDNA nanoparticles (such as figure 1 shown), the ratio of CD123 gene and CRISPR plasmid is 1:1, and the ratio of PBAE and CD123 gene plasmid is 30:1.

[0042] 2. Preparation of platelet membrane-wrapped PBAE / pDNA nanocarriers

[0043] After C57BL / 6J mice were anesthetized, the living heart was taken into an anticoagulant tube, added platelet separation solution, centrifuged at 300g for 15 minutes, the first layer of plasma layer was taken into a sterile centrifuge tube, and the same volume of sample diluent...

Embodiment 2

[0045] Characterization and Identification of Embodiment 2 Nanoparticles

[0046] Utilize transmission electron microscope to observe the form of the nano-carrier prepared in embodiment 1, draw 5ul nano-carrier and dilute 3 times with deionized water, then draw a small amount of diluted sample and drip on the copper grid, the result is as follows figure 2 As shown, the particle size of nanometer is 150-200nm.

[0047] Utilize the dynamic light scattering laser nanometer particle size analyzer to measure the size and zeta potential of nanoparticles, take 1mL samples of nanocarriers respectively and put them into the particle size analyzer sample cell and the potential measurement sample cell, the results are as follows image 3 with Figure 4 As shown, the main particle size of nanoparticles is distributed at 220nm, and the Zeta potential of the last wrapped nanometer is -18mV.

Embodiment 3

[0048] Example 3 Efficiency Detection of In vitro Transfection of Plasmids into T Cells by Nanoparticles

[0049] After the pure pDNA, PBAE / pDNA and platelet membrane-wrapped PBAE / pDNA groups were cultured with mouse spleen-derived T cells (PBMC), the transfection efficiency was observed with a confocal microscope at 20 min and 120 min after transfection. The result is as Figure 5 As shown, the platelet membrane-wrapped PBAE / pDNA nanocarrier has a good transfection efficiency compared with the plasmid pDNA group alone.

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Abstract

The invention discloses a tumor-targeted delivery system for in-vivo in-situ induction of CAR-T cells and application of the tumor-targeted delivery system, and belongs to the technical field of immunooncology. The tumor-targeted delivery system comprises nanoparticles and a nano-carrier formed by wrapping the nanoparticles with a platelet membrane, wherein the nanoparticles are formed by assembling a cationic polymer, CAR gene plasmids and a CRISPR system, and the CRISPR system is delivered to a tumor through the nano-carrier to achieve in-situ editing of the T cells. The tumor-targeted delivery system achieves in-situ editing of the T cells in the solid tumor, improves the tumor microenvironment, enhances the proliferation and durability of the CAR-T cells, and has higher safety, simple process and low cost compared with traditional CAR-T.

Description

technical field [0001] The invention relates to the technical field of immuno-oncology, in particular to a delivery system for in situ induction of CAR-T cells targeting tumors in vivo and its application. Background technique [0002] In recent years, CAR-T cell immunotherapy (that is, chimeric antigen receptor T cell immunotherapy) has shown good targeting, lethality and durability in clinical trials, and has made breakthroughs in the treatment of hematological tumors. research hotspot. In August 2017, Novartis’s Kymrial was approved by the US FDA, marking the real entry of CAR-T cell therapy into clinical application. However, CAR-T therapy is not effective in solid tumors such as lymphoma, mainly due to: 1) the lack of specific antigens on the surface of tumor cells. Solid tumors lack tumor-associated antigens like CD19 that specifically exist in hematological tumors, resulting in the molecular targets of CAR-T cells appearing on the surface of cancer cells and normal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K48/00A61P35/00
CPCA61K48/0025A61K48/005A61K39/001129A61P35/00
Inventor 吴小艳胡振华
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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