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

Figure 4.

Figure 4

Neutralization of soluble ULBP6 by an anti-ULBP6/2/5 antibody promotes NK and CD8+ T cell activation and PBMC-mediated tumor killing. A, Crystal structure of 23ME-01473 Fab in complex with human ULBP6-02. The extracellular domain of ULBP6-02 is colored orange, glycan moieties from N-glycosylated asparagine are shown as sticks, and the coding single-nucleotide polymorphism regions are shown as green spheres. 23ME-01473 Fab is shown in surface representation with the light chain colored light purple and the heavy chain colored dark purple. B, Crystal structure of NKG2D in complex with human ULBP6-02 (PDB 4S0U). The two NKG2D protomers, NKG2D-A and NKG2D-B, of the dimer are shown in light and dark gray in surface representation, and ULBP6 is in the same orientation as in A, with its extracellular domain colored orange. C, Flow cytometric analysis of cell surface NKG2D expression on NK cells (CD45+ CD3– CD56+) or CD8+ T cells (CD45+ CD3+ CD56– CD8+) analyzed from IL-2/IL-15–primed PBMCs cultured with 50 nmol/L recombinant sULBP6-02 and 0.05–100 nmol/L Fc-Att-anti-ULBP6/2/5 or 100 nmol/L Fc-Att isotype control. Data represent mean ± SEM of three technical replicates per condition from one of four biological replicates. D, Flow cytometric analysis of granzyme B expression in NK cells (CD45+ CD3– CD56+) or CD8+ T cells (CD45+ CD3+ CD56– CD8+) analyzed from IL-2/IL-15–primed PBMCs cultured for 48 hours with COV644 cells, 50 nmol/L recombinant sULBP6-02, and 200 nmol/L Fc-Att-anti-ULBP6/2/5 or Fc-Att isotype control. Data represent mean ± SD of five technical replicates per condition from one of two biological replicates. The Mann–Whitney test was used for statistical analysis. E, Quantification of COV644-GFP cell growth, as measured by GFP area per well using 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 Fc-Att-anti-ULBP6/2/5 or Fc-Att 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. Welch’s t test was used for statistical analysis. **, P ≤ 0.01; ****, P ≤ 0.0001. HC, heavy chain; LC, light chain; MFI, mean fluorescence intensity; SNP, single-nucleotide polymorphism.