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. 2024 Feb 27;12(2):e008155. doi: 10.1136/jitc-2023-008155

Figure 3.

Figure 3

CAR-mediated targeting overcomes immune escape caused by ICAM-1 downregulation. (A) CAR expression on NK-92/5.28.z cells (left) was detected by flow cytometry using human recombinant ErbB2-Fc protein followed by anti-Fc secondary antibody. Parental NK-92 cells (right) were included for comparison. Filled gray areas indicate negative controls only stained with secondary antibody. Representative data from at least three independent experiments are shown. (B) Specific cytotoxicity of NK-92/5.28.z against MDA-MB-453 cells was measured using a Europium-based assay after 2 hours of co-culture at an effector to target ratio of 10:1. (C) Specific CAR-mediated cytotoxicity of NK-92/5.28.z against MDA-MB-453 ICAM-1 KO, NT and WT cells (left) and against WT cells following incubation of NK-92/5.28.z cells with LFA-1 blocking antibody or control IgG for 60 min prior to co-culture (right). (D) Natural cytotoxicity of NK-92/5.28.z against K562 ICAM-1 KO, NT and WT cells (left) and against WT cells following incubation of NK-92/5.28.z cells with LFA-1 blocking antibody or control IgG for 60 min prior to co-culture (right). (E) Flow cytometric analysis of ICAM-1 expression on MCF-7 breast and BxPc3 pancreatic cancer cells. Blue lines indicate isotype controls and gray lines indicate unstained controls. (F) Specific cytotoxicity of haNK cells in combination with trastuzumab or CAR-engineered NK-92/5.28.z cells against MCF-7 and BxPc3 cells. NK cells were pretreated with LFA-1 blocking antibody or control IgG as indicated. Cytotoxicity data were pooled from at least three independent experiments. Mean values±SEM are shown. (G) Primary NK cells modified with an ErbB2-specific CAR or combined with trastuzumab were co-cultured with MDA-MB-453 ICAM-1 KO or WT cells for 2 hours and specific cytotoxicity was measured using a Europium-based assay. Representative data from three independent experiments using three different donors are depicted. Mean values±SEM are shown. CAR, chimeric antigen receptor; haNK, high-affinity FcγRIIIa-modified NK-92 cells; KO, knockout; NK, natural killer; NT, non-targeting; WT, wild type.