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Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding. • 1D5-CAR targeting MICA/B α3 was designed based on structure. • 1D5-CAR-modified NK cells exhibited potent cytotoxicity. • 1D5-CAR didn't disturb NKG2D-MICA/B interaction. • 1D5-CAR could be combined with NKG2D-CAR. [ABSTRACT FROM AUTHOR]

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors. • A novel NK-tailored bispecific chimeric receptor PD1-DAP10/NKG2D is rationally designed with a structure-based approach. • Engineered NK92 cells can not only trigger apoptosis, but seemingly induce pyroptosis of SGC-7901 solid tumor cells. • Expressing PD1-DAP10/NKG2D mediates robust in vitro cytotoxicity and in vivo tumor control of NK92 cells. [ABSTRACT FROM AUTHOR]

• 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. [ABSTRACT FROM AUTHOR]