Figure 9.

Engineered ICOSL costimulatory vIgDs can be fused to an antibody, retain binding, and provide localized costimulatory signals. (A) Schematic diagram of V-mAb fusion proteins. Blue structures represent heavy and light chains of the monoclonal antibody, solid red structures represent the IgV domain of an ICOSL vIgD, and open red structures are the IgC domain of an ICOSL vIgD. ICOSL-trastuzumab V-mAbs retain binding to (B) HER2, (C) CD28, and (D) ICOS. HEK-293 HER2 transfectants were stained with titrated amounts of indicated V-mAbs and analyzed by flow cytometry. (E) ICOSL-trastuzumab V-mAbs retain costimulatory activity. Titrated amounts of indicated V-mAbs were coated to plates with a fixed concentration of anti-CD3 antibody (10 nM) and incubated for 3 days with CFSE labeled T cells. Graph shows the percentage of CD8+ T cells that divided vs. protein concentrations. (F) ICOSL-trastuzumab V-mAbs costimulate primary human T cells in the presence of a HER2+ tumor cell line. NCI-N87 (HER2+) human gastric carcinoma cells were transduced with anti-CD3 single chain Fv (OKT3). Primary human T cells were plated with OKT3 expressing tumor cells at an E:T ratio of 1:4 and assayed 72 h later for IFN-γ production. (G,H) ICOSL-trastuzumab V-mAb driven T cell costimulation is dependent on HER2 expression. Primary human T cells were transduced with lentiviral vector encoding the HLA-A^*0201 restricted TCR directed against the viral oncoprotein human papillomavirus type 16 (HPV-16) E6. Parental T2 cells (G) or HER2-transduced T2 cells (H) were pulsed with 1 ng ml⁻¹ E6 peptide (29–38) for 90 min then plated with E6 TCR transduced T cells at an E:T ratio of 1:3. ICOSL-trastuzumab V-mAbs were added at the indicated concentrations and supernatants were harvested for assessment of IFNγ after 24 h. Localization of the ICOSL vIgD on HER2+ targets with the ICOSL-trastuzumab V-mAb significantly increased IFNγ production over trastuzumab alone.