Fig. 2.

In vitro characterization of five F8-4-1BBL formats. (A) Binding to 4-1BB was measured by flow cytometry with the murine cytotoxic T cell line CTLL-2, which expresses 4-1BB. (B) Binding of the F8 moiety to the EDA-positive ectodomain of fibronectin was measured by surface plasmon resonance on chips coated with recombinant EDA. (C) Biological activity was tested using an NF-κB reporter cell line that secretes luciferase on activation of the NF-κB pathway by signaling through 4-1BB. The assay was performed both with and without EDA immobilized on solid support (n = 3). Curve fitting was done using the agonist vs. response (three-parameter) fit of GraphPad Prism 7.0. Data represent mean ± SD.