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Macrophage exclusive chimeric antigen receptor, controllable polarized mononuclear/macrophage for expressing macrophage exclusive chimeric antigen receptor and preparation method and application thereof

A chimeric antigen receptor and macrophage technology, applied in the field of controllable polarized monocyte/macrophage and its preparation, can solve the problems of time-consuming and labor-intensive, difficult transformation, loss of immune function, etc.

Active Publication Date: 2021-09-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, adoptive transplantation of macrophages has the following problems: 1. M1 macrophages with cellular immune function are easily polarized to the M2 state under the influence of the tumor microenvironment, and then lose their immune function and promote tumor growth. 2. The traditional method of reinfusion of autologous immune cells for patients faces problems such as less source of cells, low separation efficiency, difficulty in transformation, time-consuming and labor-intensive, etc., and primary macrophages are difficult to pass through the way of lentivirus infection. genetic engineering

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  • Macrophage exclusive chimeric antigen receptor, controllable polarized mononuclear/macrophage for expressing macrophage exclusive chimeric antigen receptor and preparation method and application thereof
  • Macrophage exclusive chimeric antigen receptor, controllable polarized mononuclear/macrophage for expressing macrophage exclusive chimeric antigen receptor and preparation method and application thereof
  • Macrophage exclusive chimeric antigen receptor, controllable polarized mononuclear/macrophage for expressing macrophage exclusive chimeric antigen receptor and preparation method and application thereof

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preparation example Construction

[0076] In a fourth aspect, the present invention provides a method for preparing macrophages, comprising the following steps: constructing a lentiviral expression system containing the gene expression sequence of the chimeric antigen receptor, and using the lentiviral expression system to express the chimeric antigen receptor The gene expression sequence of the body is integrated into the pluripotent stem cells, and the macrophages are prepared after induction and differentiation;

[0077] Preferably, the pluripotent stem cells are firstly induced to differentiate into monocytes, and then differentiated into macrophages.

[0078] The inventors have found that primary macrophages are difficult to be infected by lentiviruses, and difficult to be edited and engineered. Based on this, the present invention provides a method for preparing the above-mentioned macrophages, which can be prepared by the method of the present invention Macrophages can overexpress chimeric antigen recept...

Embodiment 1

[0089] A GBM cell line expressing EGFRvIII antigen was constructed.

[0090] Obtain the expression sequence of GBM-specific membrane protein EGFRvIII, and clone it into the lentiviral expression plasmid Lenti-EF1A-PGK-Puro by means of molecular cloning, and construct the obtained EGFRvIII lentiviral expression system, as shown in figure 1 Shown in A. The GBM-specific membrane protein EGFRvIII was expressed in the GBM cell lines U87MG and LN-229 with the help of a lentiviral expression system, and U87MG stably expressing the membrane protein was obtained EGFRvIII cell line.

[0091] Western Blotting was used to detect the expression levels of total EGFR and EGFRvIII in U87MG cells after lentivirus infection, and the experimental steps were as follows:

[0092] 1. Preparation of Cellular Total Protein Samples

[0093] (1) Inoculate wild-type U87MG cells and U87MG cells overexpressing EGFRvIII through lentivirus into 6CM cell culture dishes, and incubate at 37°C / 5%CO 2 Cultur...

Embodiment 2

[0129] Construct CAR-iMAC, and explore its targeted killing function on GBM cell lines through in vitro experiments.

[0130] 1. The method of constructing iPSCs induced to differentiate into iMAC

[0131] 1) Reprogramming PBMCs into iPSCs

[0132] PBMCs isolated from fresh blood have the advantages of large quantity and easy acquisition, and are excellent materials for inducing iPSCs. After the PBMCs were isolated, the expression vectors expressing five transcription factors, OCT3 / 4, SOX2, KLF4, L-MYC, and LIN28A, were transfected into PBMCs by electroporation to induce their reprogramming, and finally iPSCs were obtained.

[0133] 2) Inducing the differentiation of iPSCs into monocytes (iMONO) and M1 macrophages (iMAC) based on the formation of mimic ligand (EB), the experimental steps are as follows:

[0134] 1. EB formation (day 0). When iPSCs have grown to cover 60-80% of the dish, they are digested with versene into single cells or smaller cell aggregates. After cent...

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Abstract

The invention provides a macrophage exclusive chimeric antigen receptor, a controllable polarized mononuclear / macrophage for expressing the macrophage exclusive chimeric antigen receptor, and a preparation method and application thereof, and relates to the field of biotechnology. The invention provides a chimeric antigen receptor which comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular activation domain which are sequentially connected, and the extracellular antigen binding domain comprises signal peptide and / or scFv and can specifically recognize cell membrane surface protein EGFRvIII specifically expressed by GBM; the transmembrane domain comprises CD8alpha which linking the extracellular antigen binding domain and the intracellular activation domain; an intracellular activation domain comprises TIR, CD3ZETA or GM-CSFRalpha / beta and promotes polarization of macrophages to M1, the chimeric antigen receptor is introduced into the macrophages, the macrophages are endowed with a targeted killing effect on GBM, and the M1 polarization state of the macrophages is effectively promoted and maintained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a macrophage-specific chimeric antigen receptor, a controllable polarized monocyte / macrophage expressing the receptor, and a preparation method and application thereof. Background technique [0002] Glioblastoma (GBM), thought to arise from glial progenitor cells, is the most prevalent and aggressive primary brain tumor ever discovered. Its incidence accounts for 12%-15% of intracranial tumors and 50%-60% of astrocytic tumors. The median survival of patients with this tumor is about 15 months, and the 5-year survival rate is less than 10%. Therefore, this tumor poses a great threat to human life and health. Although the current treatment methods for GBM include surgery, radiotherapy, and chemotherapy, but because the tumor infiltrates and grows deep into the brain lobe, and has the characteristics of rapid growth and easy recurrence, the effect is minimal and it is easy to cause di...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10C12N15/867A61K39/00A61P35/00
CPCC07K14/7051C07K16/2863C07K16/40C07K16/32C12N5/0645C12N15/86A61K39/0011A61P35/00C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2506/115C12N2740/15043A61K2039/80
Inventor 张进雷安华田琳
Owner ZHEJIANG UNIV
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