Example 16
T1165.85.2.1 cells (R&D Systems) are a murine plasmacytoma cell line that proliferates in response to mouse, rat, or human IL-6. To measure antagonism from the EBI-029 Fab mutants, 25 μL of 2 ng/mL human IL-6 (R&D Systems 206-IL-010/CF) was mixed with 25 μL of each Fab variant at a range of concentrations in a 96 well plate and incubated at RT for 30 minutes. T1165 cells in log phase were pelleted and resuspended in assay media (90% RPMI 1640, 10% FBS, 2 mM L-glutamine, Pen-Strep) at 2×105 cells/mL. 50 L of cell suspension was added to each well of IL-6/Fab mixtures to bring the final IL-6 concentration to 0.5 ng/mL. The cells were incubated at 37° C./5% C02 for 72 hours. 100 μL of Cell-Titer Glo® reagent (Promega) was added to each well and incubated at RT for 10 minutes. Luminescence was measured on a SpectraMax M5 plate reader. All mutants showed significantly greater potency compared to the wt EBI-029 Fab with no measurable IL-6 signaling over the range of Fab concentrations tested (see