Structure-based rational design of a novel chimeric PD1-NKG2D receptor for natural killer cells

[Display omitted] •Precise 3D structure modeling of transmembrane proteins is developed.•NK cell-tailored chimeric receptors are rationally designed with NKG2D signaling.•PNBB-NK92 cells demonstrate in vitro cytotoxicity against various tumor cells. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have the potential to provide the potential for the implementation of allogeneic “off-the-shelf” cellular therapy against cancers. Currently, most CARs are not optimized for NK cells, so new NK-tailored CARs are needed. Here, a major activating receptor of NK cells, NKG2D was harnessed to design different chimeric receptors that mediate strong NK cell signaling. In these NKG2D signaling-based chimeric receptors, the extracellular domain of inhibitory receptor PD-1 was employed to reverse the immune escape mediated by PD-1 ligands in the solid tumors. To achieve the rational design of chimeric PD1-NKG2D receptors, we developed a transmembrane protein tertiary structure prediction program (PredMP & I-TASSER) and optimized the conformation of the PD-1 ectodomain by genetically altering the sequences encoding the hinge and intracellular domain. Finally, we identified a chimeric PD1-NKG2D receptor containing NKG2D hinge region and 4-1BB co-stimulatory domain to exhibit stable surface expression and mediate in vitro cytotoxicity of NK92 cells against various tumor cells. This strategy now provides a promising approach for the computer-aided design (CAD) of potent NK cell-tailored chimeric receptors with NKG2D signaling.