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. 2025 Mar 21;5(3):477–496. doi: 10.1158/2767-9764.CRC-24-0478

Figure 5.

Figure 5

Dual FcγRIIIa and NKG2D activation by 23ME-01473, an Fc effector–enhanced anti-ULBP6/2/5 antibody, induces augmented antitumor immunity. A, IFNγ concentration of the supernatants of IL-2/IL-15–primed PBMCs cultured with plate-bound antibodies that activate FcγRIIIa (Fc-WT-anti-lysozyme antibody; X-lys-E+), NKG2D (Fc-Att-anti-lysozyme antibody fused to ULBP6-02; ULBP6-lys-E−), both receptors (Fc-WT-anti-lysozyme antibody with fused ULBP6-02; ULBP6-lys-E+), or neither receptor (Fc-Att-anti-lysozyme antibody; X-lys-E−) for 24 hours. Data represent mean ± SD of three technical replicates per condition from one of four biological replicates. B, FcγRIIIa activation as measured by RLU using a Promega ADCC assay with a luciferase effector cell line, COV644 cells, and 0.0001 to 10 nmol/L Fc-WT-anti-ULBP6/2/5 or 23ME-01473, or 10 nmol/L Fc-Att-anti-ULBP6/2/5 or Fc-enhanced isotype control, is depicted as a concentration-dependent response (left) or in response to 10 nmol/L of the indicated antibodies (right). Data represent mean ± SD of three technical replicates per condition from one of four biological replicates. C, IFNγ concentration of the supernatants of IL–2/IL-15–primed PBMCs cultured with COV644 cells, 50 nmol/L recombinant sULBP6-02, and 0.05–200 nmol/L 23ME-01473, or 200 nmol/L Fc-WT-anti-ULBP6/2/5 or Fc-enhanced isotype control, is depicted as a concentration-dependent response (left) or in response to 200 nmol/L of the indicated antibodies (right). Data represent mean ± SD of five technical replicates per condition from one of four biological replicates. D, Quantification of COV644-GFP cell growth, as measured by GFP area per well by an Incucyte live cell analysis system, in the presence of IL-2/IL-15–primed PBMCs, 50 nmol/L recombinant sULBP6-02, and 100 nmol/L 23ME-01473, Fc-Att-anti-ULBP6/2/5, or Fc-enhanced isotype control. Quantification is represented continuously over a 5-day time course (left) and at the end of the 5-day time point (right). Data represent mean ± SD of eight technical replicates per condition from one of four biological replicates. One-way ANOVA was used for statistical analyses. **, P ≤ 0.01; ****, P ≤ 0.0001. ns, not significant; RLU, relative light unit.