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抗Her2嵌合抗原受体自然杀伤细胞系NK92及其外泌体:生成、表征及对Her2阳性肿瘤细胞的选择性细胞毒性

Anti-Her2 CAR-NK92 Cells and Their Exosomes: Generation, Characterization, and Selective Cytotoxicity Against Her2-Positive Tumor Cells.

作者信息

Tîrziu Alexandru, Bojin Florina Maria, Gavriliuc Oana Isabella, Buzan Roxana Maria, Zbîrcea Lauriana Eunice, Grijincu Manuela, Păunescu Virgil

机构信息

Department of Functional Sciences, "Victor Babes" University of Medicine and Pharmacy, Tudor Vladimirescu Street, No. 14, 300174 Timisoara, Romania.

Center for Gene and Cellular Therapies in the Treatment of Cancer Timisoara-OncoGen, Clinical Emergency County Hospital "Pius Brinzeu" Timisoara, No. 156 Liviu Rebreanu, 300723 Timisoara, Romania.

出版信息

Int J Mol Sci. 2025 Aug 7;26(15):7648. doi: 10.3390/ijms26157648.


DOI:10.3390/ijms26157648
PMID:40806778
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12347400/
Abstract

Chimeric antigen receptor (CAR)-engineered NK cells are a promising approach for targeted immunotherapy in Her2-positive cancers. This study aimed to generate anti-Her2 CAR-NK92 cells, to evaluate their selective cytotoxicity against Her2-positive cancer cells, and to isolate and characterize their released exosomes. NK92 cells were electroporated with piggyBac transposon vectors encoding anti-Her2 CAR and the helper transposase. Puromycin selection was performed to enrich the transduced cells. CAR and GFP expression were assessed by flow cytometry, and exosomes were isolated and characterized in terms of protein cargo and surface protein expression. Cytotoxicity was evaluated using real-time cell analysis against Her2-positive SK-BR3 cells and Her2-negative MCF-7 cells. Electroporation did not significantly affect NK92 cell viability. Puromycin selection efficiently enriched for CAR-expressing cells, with GFP positivity reaching 99.8% and a 15-fold increase in CAR surface expression compared to wild-type cells. CAR-NK92 cells demonstrated robust, Her2-specific cytotoxicity in a E:T-dependent manner, with the greatest effect observed at a 10:1 effector-to-target ratio. Exosomes derived from CAR-NK92 cells contained CAR molecules and selectively targeted Her2-positive cells. Anti-Her2 CAR-NK92 cells and their exosomes exhibit potent and selective cytotoxicity against Her2-positive cancer cells, supporting their potential as innovative immunotherapeutic agents for solid tumors.

摘要

嵌合抗原受体(CAR)工程化的自然杀伤(NK)细胞是治疗Her2阳性癌症的一种有前景的靶向免疫疗法。本研究旨在生成抗Her2 CAR-NK92细胞,评估其对Her2阳性癌细胞的选择性细胞毒性,并分离和鉴定其释放的外泌体。用编码抗Her2 CAR的猪尾巴转座子载体和辅助转座酶对NK92细胞进行电穿孔。进行嘌呤霉素筛选以富集转导细胞。通过流式细胞术评估CAR和绿色荧光蛋白(GFP)的表达,并根据蛋白质含量和表面蛋白表达对外泌体进行分离和鉴定。使用实时细胞分析评估对Her2阳性SK-BR3细胞和Her2阴性MCF-7细胞的细胞毒性。电穿孔对NK92细胞活力无显著影响。嘌呤霉素筛选有效地富集了表达CAR的细胞,GFP阳性率达到99.8%,与野生型细胞相比,CAR表面表达增加了15倍。CAR-NK92细胞以效应细胞与靶细胞比例(E:T)依赖的方式表现出强大的、Her2特异性的细胞毒性,在效应细胞与靶细胞比例为10:1时观察到最大效应。源自CAR-NK92细胞的外泌体含有CAR分子,并选择性地靶向Her2阳性细胞。抗Her2 CAR-NK92细胞及其外泌体对Her2阳性癌细胞表现出强大的选择性细胞毒性,支持它们作为实体瘤创新免疫治疗药物的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/a50fcb201c21/ijms-26-07648-g014.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/74bad415e48b/ijms-26-07648-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/53f505f2688a/ijms-26-07648-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/1b31159cf405/ijms-26-07648-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/25b39a912cb5/ijms-26-07648-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/f9381f85a48f/ijms-26-07648-g011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/6ad50f73b1fd/ijms-26-07648-g012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/a438637551e9/ijms-26-07648-g013.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/a50fcb201c21/ijms-26-07648-g014.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/91074f9533c7/ijms-26-07648-g004.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/dbb7b07a6d24/ijms-26-07648-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/74bad415e48b/ijms-26-07648-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/53f505f2688a/ijms-26-07648-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/1b31159cf405/ijms-26-07648-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/25b39a912cb5/ijms-26-07648-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/f9381f85a48f/ijms-26-07648-g011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/6ad50f73b1fd/ijms-26-07648-g012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/a438637551e9/ijms-26-07648-g013.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4230/12347400/a50fcb201c21/ijms-26-07648-g014.jpg

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本文引用的文献

[1]
Safety and feasibility of 4-1BB co-stimulated CD19-specific CAR-NK cell therapy in refractory/relapsed large B cell lymphoma: a phase 1 trial.

Nat Cancer. 2025-4-18

[2]
Natural killer cell-based immunotherapy for cancer.

J Immunol. 2025-4-17

[3]
Advances in CAR T cell therapy: antigen selection, modifications, and current trials for solid tumors.

Front Immunol. 2025-1-6

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Next-Generation Immunotherapy: Advancing Clinical Applications in Cancer Treatment.

J Clin Med. 2024-10-30

[5]
Linker-specific monoclonal antibodies present a simple and reliable detection method for scFv-based CAR NK cells.

Mol Ther Methods Clin Dev. 2024-8-22

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CAR-T cell-derived exosomes: a new perspective for cancer therapy.

Stem Cell Res Ther. 2024-6-18

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CAR-based immunotherapy for breast cancer: peculiarities, ongoing investigations, and future strategies.

Front Immunol. 2024

[10]
Emerging Strategies to Overcome Current CAR-T Therapy Dilemmas - Exosomes Derived from CAR-T Cells.

Int J Nanomedicine. 2024

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