Figure 8.

ICOSL vIgD proteins can be formatted to provide localized T cell costimulation. (A) Schematic diagram of a tumor localizing fusion protein consisting of an ICOSL vIgD costimulatory domain (N-terminal), a variant NKp30 domain to localize the protein to B7H6 expressing tumors, and an antibody Fc domain. (B–D) ICOSL-NKp30 vIgD-Fc fusion proteins can provide T cell costimulatory signals that are dependent on the presence of B7H6. Plates were coated with 40 nM recombinant B7H6-Fc protein and 10 nM anti-CD3. CFSE-labeled primary human T cells were added with titrated concentrations of the variant ICOSL-Fc alone (orange circle), the variant NKp30-Fc alone (purple circle), or an ICOSL-NKp30-Fc variant fusion protein (green circles) for 3 days. Cells were analyzed for proliferation by CFSE dilution in (B) human CD4 or (C) human CD8 T cells. (D) Supernatants were collected and assessed for IFNγ production by ELISA. (E) Variant ICOSL-NKp30-Fc fusion proteins can also be used to localize T cell costimulatory signals to B7H6 positive cells. K562 cells that express B7H6 were plated with CFSE-labeled human T cells and varying concentrations of a control Fc-protein (red circles), the variant NKp30-Fc protein alone (purple circles), the variant ICOSL-Fc fusion protein alone (orange circles), a wild type NKp30-ICOSL-Fc fusion protein (blue circles) or an ICOSL-NKp30-Fc fusion variant (green circles). Results shown are representative of at least two experiments, and individual points represent mean values of triplicate wells ±s.d.