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216 Dual targeting of CAR-NK cells to PD-L1 and ErbB2 facilitates specific elimination of cancer cells of solid tumor origin and overcomes immune escape by antigen loss
  1. Jiri Eitler1,
  2. Kristin Freudenberg1,
  3. Paola Ortiz Montero1,
  4. Wiebke Rackwitz1,
  5. Winfried Wels2 and
  6. Torsten Tonn1
  1. 1Carl Gustav Carus, Dresden, Germany
  2. 2Goethe University, Frankfurt, Germany


Background Retargeting of natural killer (NK) cells with chimeric antigen receptors (CARs) can be a powerful approach to overcome NK-cell resistance of tumor cells. However, targeting a single tumor-associated antigen may be insufficient for some tumors to trigger effective NK-cell activation or result in the selection of antigen-loss variants and tumor immune escape.

Methods To overcome this hurdle, here we generated CAR-NK cells carrying two CARs that target the tumor-associated antigens PD-L1 and ErbB2 (HER2), respectively (figure 1A). NK-92 cells were transduced with lentiviral CAR constructs, and their cytotoxicity against cancer cell lines of different solid tumor origins was compared to that of parental NK-92 and corresponding single-target CAR variants.

Results Dual targeting significantly increased in vitro cytotoxicity against PD-L1 and ErbB2 double-positive tumor cell lines including breast, ovarian, pancreatic and gastric cancer cells when compared to single-target CAR variants (Figure 1B,C). These results were also confirmed with 3D spheroid tumor models. Off-target cytotoxicity was not observed. On a molecular level, this enhanced cell killing may be explained by synergistic activation of PLCγ and MAPK pathways. Incubation of cancer cells with IFN-γ further improved killing efficacy due to upregulation of PD-L1 expression. Furthermore, blocking experiments revealed that dual PD-L1/ErbB2-CAR NK-92 cells can overcome immune escape based on loss or inacessibility of a single target antigen.

Conclusions Altogether, we showed that dual targeting of PD-L1 and ErbB2 improves efficacy of CAR-NK cells against otherwise difficult to treat tumors, and counteracts potential resistance and immune escape mechanisms of cancer cells.

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