Modified immune cells and application thereof

A technology of immune cells and cells, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problem of low killing activity, achieve the effects of promoting aggregation, expanding the scope of application, and promoting infiltration

Active Publication Date: 2020-03-10
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention provides genetically modified immune cells, aiming to enhance the accumulation of modified immune cells in solid tumors, and to solve the defect of low killing activity of modified immune cells such as CAR-T cells in solid tumors

Method used

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  • Modified immune cells and application thereof
  • Modified immune cells and application thereof
  • Modified immune cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Bioinformatics screening of chemokines highly expressed in solid tumors.

[0078] The Cancer Genome Altas (TCGA), initiated by the NCI in the United States, aims to deepen human understanding of the molecular mechanisms of cancer through high-throughput sequencing technology. TCGA is a world-renowned cancer database, including 33 cancer types and more than 11,000 clinical samples. Screen the expression level of chemokine CCL20 in various tumors in TCGA through the analysis tool of GEPIA website, such as figure 1 Shown, CCL20 in cervical squamous and adenocarcinoma, colon cancer, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal clear cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian Carcinoma, pancreatic cancer, rectal cancer, gastric cancer, endometrial cancer, uterine sarcoma showed overexpression levels.

[0079] Collect the tumor tissue and paracancerous tissue of lung cancer...

Embodiment 2

[0082] Construction of the expression system.

[0083] The expression system consists of a CAR-expressing vector and a CCR6-expressing vector; CAR contains a leader sequence, an antigen-recognizing scFv, a hinge and a transmembrane domain, an intracellular co-stimulatory signal, and an intracellular activation signal CD3ζ; the carrier is a lentiviral vector FUW (See Chimeric antigen receptor T (CAR-T) cells expanded with IL-7 / IL-15mediate superior antitumor effects, Protein&Cell, June 2019).

[0084] Artificially synthesize the coding gene of CAR and CCR6, and add XbaI restriction site at the front of the sequence, add FseI restriction site at the end of the sequence, and connect it into the cloning plasmid; use XbaI and FseI to digest the lentivirus Expression plasmid and cloning plasmid containing the target gene, followed by agarose gel electrophoresis and gel cutting to recover the linearized lentiviral expression plasmid and target gene fragment; use T4 ligase to connect ...

Embodiment 3

[0098] Isolation, culture and infection of peripheral blood T cells from healthy people.

[0099] The peripheral blood of healthy people was collected, and the mononuclear cells were separated by density gradient centrifugation, and then T cells were enriched using Miltenyi's T cell enrichment kit, and inoculated in Lonza's X-VIVO 15 medium And add 100 UI / mL IL-2, and then add Thermo Fisher's CD3 / CD28 Dynabeads according to the instructions to activate and expand T cells.

[0100] After T cells were activated for 36 h, taking the target EGFR and CCL20-CCR6 system of lung adenocarcinoma as an example, the CAR targeting EGFR and the chemokine receptor CCR6 lentiviral particles were added at a multiplicity of infection ratio of 10 for infection. After 1 week, the infection efficiency of T cells was detected by flow cytometry, as shown in Figure 5 As shown, T cells were successfully infected with CAR and CCR6, and the proportion of T cells positive for both CAR and CCR6 reached ...

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Abstract

The invention belongs to the field of cell therapy, and particularly relates to modified immune cells. Through gene modification of immune cells such as T cells, tumor infiltration T cells, NK cells,NKT cells and macrophages, homing and aggregation at solid tumors and infiltration of the solid tumors are enhanced, so that the treatment effect of the immune cells on the solid tumors is improved.

Description

technical field [0001] The invention belongs to the field of immune cell therapy. Specifically, the present invention relates to genetically modified immune cells to enhance homing and aggregation in solid tumors, thereby enhancing the therapeutic effect of immune cells such as CAR-T cells on solid tumors. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly, especially adoptive cell therapy (ACT), which means that immune cells such as T cells and NK cells are isolated from patients, modified and expanded in vitro, and then reintroduced into patients for tumor treatment. method of treatment. In 2013, the immunotherapy of tumors was rated as the first among the "Top Ten Breakthroughs" by Science Impurities. [0003] CAR-T and TCR-T are important components of adoptive cell therapy, especially CAR-T therapy, which has made remarkable achievements in the treatment of blood tumors and achieved a high remission rate. A typical CAR structure c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/62C12N15/12A61K39/00A61P35/00
CPCC12N5/0636C12N5/0646C12N5/0645C12N15/86C07K16/2863C07K14/70521C07K14/7051C07K14/523A61K39/0011A61K39/001104A61K39/001142A61P35/00C12N2510/00C12N2800/107C12N2740/15043C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74A61K2039/5156A61K2039/5158
Inventor 赵同标靳利远朱颖婕曹磊曹佳妮李晓燕
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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