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Development of c?MET?specific chimeric antigen receptor?engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

ABSTRACT: In recent years, cellular immunotherapy has served an important role in the combined treatment of hepatocellular carcinoma. The possibility of specific cell therapies for the treatment of solid tumours has been further explored following the success of chimeric antigen receptor (CAR)?T cell therapy in the treatment of haematological tumours. The present study aimed to evaluate the specificity and efficiency of c?MET?targeted CAR?NK cell immunotherapy on human liver cancer in vitro. A CAR structure that targeted and recognised a c?MET antigen was constructed. c?MET?CAR was transferred into primary NK cells using lentiviral infection. c?MET?positive HepG2 cells were used as an in vitro study model. The cytotoxicity assay results revealed that c?MET?CAR?NK cells exhibited more specific cytotoxicity for HepG2 cells with high c?MET expression compared with the lung cancer cell line H1299, which has low levels of c?MET expression. The results of the present study demonstrated that c?MET may be a specific and effective target for human liver cancer cell CAR?NK immunotherapy. Based on these results, CAR?NK cell?based immunotherapy may provide a potential biotherapeutic approach for liver cancer treatment in the future.

SUBMITTER: Liu B

PROVIDER: S-EPMC6691195 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Development of c‑MET‑specific chimeric antigen receptor‑engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

Liu Bing B Liu Zheng-Zhi ZZ Zhou Mei-Ling ML Lin Jian-Wei JW Chen Xue-Mei XM Li Zhu Z Gao Wen-Bin WB Yu Zhen-Dong ZD Liu Tao T

Molecular medicine reports 20190725 3

In recent years, cellular immunotherapy has served an important role in the combined treatment of hepatocellular carcinoma. The possibility of specific cell therapies for the treatment of solid tumours has been further explored following the success of chimeric antigen receptor (CAR)‑T cell therapy in the treatment of haematological tumours. The present study aimed to evaluate the specificity and efficiency of c‑MET‑targeted CAR‑NK cell immunotherapy on human liver cancer in vitro. A CAR structu ...[more]

PMID: 31524233

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Project description:Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.

Project description:BackgroundChimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering.MethodsTwo second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5+ malignant cell lines, primary CD5+ malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5+ T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo.ResultsBoth BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5+ malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK.ConclusionsAnti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

Dataset Information

Development of c?MET?specific chimeric antigen receptor?engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

Publications

Development of c‑MET‑specific chimeric antigen receptor‑engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

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