Figure 4.

In Vitro Characterization of Tumor-Targeted MRP1-ICOS Bi-specific Aptamer

(A) Binding of the bi-specific aptamer to ICOS recombinant protein by ³²P blotting. (B) Binding of the bi-specific aptamer labeled with ³²P to B16-MRP1 cells. (C and D) T cell activation was determined as proliferation using CFSE dilution (C) or IFN-γ secretion measured by ELISA (D). (E) Assay to elucidate that both parts of the bi-specific aptamer are functional concurrently. B16-MRP1 cells were incubated with the MRP1-ICOS bi-specific aptamer and washed twice to remove the unbound aptamer fraction. Aptamer-coated tumor cells were incubated with anti-CD3-stimulated T lymphocytes. (F) Activation of T lymphocytes cocultured with tumor cells as described in (E) determined by ELISA of IFN-γ production in the cell supernatant. (All data are expressed as the mean ± SEM of experimental triplicates. The experiments were repeated twice, with similar results.) **p < 0.01, ***p < 0.005.