<p>DNAM1 and 2B4 Costimulatory Domains Enhance the Cytotoxicity of Anti-GPC3 Chimeric Antigen Receptor-Modified Natural Killer Cells Against Hepatocellular Cancer Cells in vitro</p>

Abstract

Purpose: Hepatocellular cancer (HCC) is the sixth most prevalent cancer and the third leading cause of cancer-related death worldwide. Cellular immunotherapy against glypican 3 (GPC3) has recently been used in the treatment of HCC, following the success of chimeric antigen receptor (CAR)-T therapy in treatment of B cell malignancy. However, CART cells are not "off-the-shelf" and always cause cytokine release syndrome, which can be eliminated by using natural killer (NK) cells as effector cells. Since a costimulatory signal is necessary for the activation, persistence, or cytotoxicity of CART cells, we speculated that the costimulatory signal is also required for CAR-NK cells in HCC treatment. Methods: Five anti-GPC3 CAR plasmids containing different costimulatory domains were constructed. They included Z (only the CD3ζ domain, no costimulatory domain), CD28.Z (T-cell costimulatory domain CD28), DNAM1/2B4.Z (NK-cell-associated costimulatory domain DNAM1 or 2B4), and DNAM1.2B4.Z (both NK-cell-associated costimulatory domains). Respective CAR-NK-92 cells were generated. The MTT viability assay was performed to evaluate the effect of the different costimulatory domains on CAR-NK-cell proliferation. The effect on persistence was analyzed using an apoptosis assay and flow cytometry. Special cytotoxicity against normal hepatocellular cells and GPC3 + malignant cells was investigated in vitro. The concentration of cytokines (TNF-α and IFN-γ) released by CAR-NK-92 cells was also measured by ELISA. Results: NK-cell-associated costimulatory signal was necessary for CAR-NK-92 cells. CAR-NK-92 cells with DNAM1 and/or 2B4 expanded more quickly and persisted with a lower apoptotic ratio, compared to the presence of CD28 or no costimulatory signal. All CAR-NK-92 cells showed special cellular cytotoxicity in vitro. CAR-NK-92 cells with NK-cell-associated costimulatory domains exhibited higher cytotoxic ability compared with those without any costimulatory domain or with T-cell costimulatory domain. CAR-NK-92 cells with both DNAM1 and 2B4 displayed the highest cytotoxicity. The cytokine release assay results were consistent with those of the cytotoxicity assay. Conclusion: We provided the first evidence supporting a strategy using DNAM1 and 2B4 costimulatory domains to generate anti-GPC3 CAR-NK-92 cells, which exhibits enhanced cytotoxicity against hepatocellular cancer cells in vitro.

CAR-NK Cells in the Treatment of Solid Tumors

“…A study found that CAR-NK cells containing NK-specific, DNAM1 and/or 2B4 co-stimulatory receptors demonstrate higher cytotoxicity against several hepatocellular cancer cell lines (HEpG2, Hep3B, L-02, Huh7) than CAR-T cell-derived constructs. The tested CAR structures were CD3ζ alone, CD28.CD3ζ (CAR-T cell derived), DNAM1.CD3ζ, 2B4.CD3ζ and DNAM1.2B4.CD3ζ (DNAM1 and 2B4 are both NK-cell-specific co-stimulatory receptors), which had been transferred into NK92 cells and directed against the GPC3 antigen [ 60 ]. In addition, CAR-T structures were also compared with CAR-NK structures against neoplasms originating from T lymphocytes.…”

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development

“…Consequently, based on signal activating potential, NK cell-specific co-stimulatory domains can be arranged in the order of DAP12 > CD3ζ > DAP10 [ 45 ]. Interestingly, a third generation of CAR NK cells containing DNAM1 and 2B4 has been shown to exhibit a much higher toxicity in hepatocellular carcinomas than CAR NK cells generated either with no co-stimulatory domain or with T cell-specific ones [ 63 ]. The upcoming fourth generation of armed CAR NK cells are engineered to co-express molecules such as cytokines with co-stimulatory domains to further improve functionality [ 8 ].…”

Advancement of chimeric antigen receptor-natural killer cells targeting hepatocellular carcinoma

“…Notably, the GPC3-CAR-NK-92 cells were cytotoxic to GFP3-negative HCC cells. Huang et al [ 40 ] made a further modification to GPC3-CAR-NK-92 cells by replacing the intracellular domains of CD28 and CD3ζ with the costimulatory domains of DNAM1 and 2B4. DNAM1, also known as CD226, is a costimulatory receptor in cytotoxic T cells, NK cells, and monocytes[ 41 ].…”