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Vol. 43. Núm. S1.
Páginas S265 (Outubro 2021)
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Vol. 43. Núm. S1.
Páginas S265 (Outubro 2021)
Open Access
NK-SPECIFIC CAR DESIGN IMPROVES NK CYTOTOXICITY AGAINST TUMOR CELLS
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JTC Azevedoa, RN Silvestrea, DT Covasa,b, V Picanço-Castroa
a Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular, Centro Regional de Hemoterapia de Ribeirão Preto, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil
b Department of Medical Imaging, Hematology, and Clinical Oncology, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil
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Objectives

In recent years, studies with natural killer cells expressing CAR (CAR-NK) for cancer treatment have shown exciting results. Nevertheless, more studies focusing on the development of specific CAR molecules for NK cells are needed to generate CAR-NK cells with greater capacity for proliferation and activation and consequently improving their therapeutic efficacy. The aim of this study is to develop new combinations of CAR transmembrane region and intracellular domains to enhance CAR-NK signaling and cytotoxicity functions.

Methods

Eight lentiviral vectors containing IL-15 secreting CARs composed of an extracellular single-chain variable fragment (scFv) anti-CD19 followed by different combination of transmembrane region, activator receptors and co-receptors were designed. Four of them were constituted by CD8 transmembrane region in combination with CD3z, DAP12, 4-1BB and/or 2B4: CAR19-CD8-41BB-CD3z (Insert A), CAR19-CD8-41BB-DAP12 (Insert B), CAR19-CD8-2B4-CD3z (Insert C), CAR19-CD8-2B4-DAP12 (Insert D). The other 4 vectors were composed of NKG2D transmembrane region in combination with CD3z, DAP12, 4-1BB and/or 2B4: CAR19-NKG2D-41BB-CD3z (Insert E), CAR19-NKG2D-41BB-DAP12 (Insert F), CAR19-NKG2D-2B4-CD3z (Insert G), CAR19- NKG2D -2B4-DAP12 (Insert H). Then, NK92 cell line was transduced, CAR19+ cells were enriched using magnetic beads and CAR19+ expression on cell surface evaluated by flow cytometry. Lastly, we evaluated in vitro potential of CAR-NK92 cells to kill Raji CD19+ cancer cell lines stained with PKH67 cell linker at E:T ratio of 10:1 after 5 hours co-cultivation.

Results and discussion

The expression of CARs containing CD8 transmembrane region (Insert A, B, C and D) on cell surface ranged from 2-4% CAR on day 7 post-transduction and remained stable until 21 days post-transduction and, after two selections, NK92 cells expressing 70-90% of CAR were obtained. The expression of CARs containing NKG2D transmembrane region (Insert E, F, G and H) ranged from 0.15-0.38% on day 7 post-transduction, and it was unfeasible to select these cells. Next, the cytotoxicity of NK92-CAR cells containing CD8 transmembrane region was evaluated in vitro. All NK92 cells containing CAR were more efficient in killing target cells than NK92WT cells (% Cytotoxicity rate mean±SD: -1.80±6.32). In addition, NK92-CAR Insert B (68.53±3.72), C (87±2.77) and D (65.18±2.12) cells were more cytotoxic than NK92-CAR Insert A cells (42.22±7.23; p < 0.05), being the NK92-CAR Insert C cells the most cytotoxic of all CARs at 10:1 ratio (p < 0.05). It is important to note that CAR-Insert A is the CAR commonly used in CAR-T cell therapy, suggesting that the vector containing receptors and co-receptors specific for NK cells was able to improve their function.

Conclusion

Our results demonstrated that the transmembrane region affected CAR expression on cell surface, being the CD8 region more effective. Furthermore, NK92 cells expressing CAR specific for NK cells were more efficient in killing tumor target cells, and the NK92-CAR cell constituted by 2B4 and CD3z intracellular domain (Insert C) was especially effective. The therapeutic effectiveness of these cells will be evaluated in vivo.

Funding

Financially supported by FAPESP 2020/08279-8, FAPESP 2019/25309-0, CTC Center for Cell-based Therapy (FAPESP 2013/08135-2), and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).

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Hematology, Transfusion and Cell Therapy
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