|Table of Contents|

Experimental study on inhibitory effect of TGF-βRⅡ receptor knockout CLDN18.2 CAR T cells on gastroesophageal junction carcinoma

Journal Of Modern Oncology[ISSN:1672-4992/CN:61-1415/R]

Issue:
2023 18
Page:
3347-3352
Research Field:
Publishing date:

Info

Title:
Experimental study on inhibitory effect of TGF-βRⅡ receptor knockout CLDN18.2 CAR T cells on gastroesophageal junction carcinoma
Author(s):
LI Shun; FAN Hua; YANG Yongxu; CAI Yingbin
Endoscopic Diagnosis and Treatment Center,Cancer Hospital Affiliated to Xinjiang Medical University,Xinjiang Urumqi 830011,China.
Keywords:
gastroesophageal junction carcinoma; chimeric antigen receptor T cells; CRISPR/Cas9; TGF-βRⅡ
PACS:
R735.2
DOI:
10.3969/j.issn.1672-4992.2023.18.003
Abstract:
Objective:To construct knockout TGF-βRⅡ targeting CLDN18.2 CAR T cells and explore its therapeutic potential in gastroesophageal junction carcinoma (GEJ).Methods:TGF-βRⅡ in T cells was knocked out by CRISPR/Cas9 and CLDN18.2 CAR T cells were constructed by lentivirus infection.Flow cytometry was used to detect the positive rate of CAR T cells,the expression of CLDN18.2 in esophageal adenocarcinoma cell lines,the phosphorylation level of Smad2/3 in CAR T cells,and the expression level of PD-1.The expression of TGF-βRⅡ in CAR T cells was detected by immunofluorescence assay.LDH release assay was used to detect cytotoxicity of target cells.Tumor inhibition ability was detected by tumor transplantation in nude mice.ELISA assay was used to detect the release of cytokines in tumor tissues.Results:CLDN18.2 CAR T cells with TGF-βRⅡ knockout (18.2BBZ/TGFBRKO) and the second generation CLDN18.2 CAR T cells (18.2BBZ) were successfully constructed.18.2BBZ and 18.2BBZ/TGFBRKO showed dose-dependent cytotoxicity to CLDN18.2+ tumor cells in vitro.TGF-β1 could not phosphorylate Smad2/3 of 18.2BBZ/TGFBRKO.In the presence of TGF-β1,the expression level of PD-1 of 18.2BBZ/TGFBRKO after co-incubation with tumor cells was significantly lower than that of 18.2BBZ (P＜0.001).18.2BBZ/TGFBRKO showed stronger inhibitory effect on transplanted tumor in nude mice than 18.2BBZ (P＜0.000 1) and maintained stronger cytokine secretion level in tumor tissues (P＜0.01).Conclusion:CRISPR/Cas9 knockout of TGF-βRⅡ in CLDN18.2 CAR T cells can effectively reduce T cell exhaustion and produce stronger antitumor activity and increase the secretion of pro-inflammatory cytokines in mouse transplanted tumor models.

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Memo:
新疆维吾尔自治区自然科学基金项目（编号：2022D01C299）
Last Update: 1900-01-01