Neutralizing Potency Measured in Cell-Based Assay

The TNF sensitive mouse fibroblast cell line L929s was utilized for measuring the anti-TNF activity of the selected Nanobodies™. L929 cells were grown until nearly confluent, plated out in 96-well microtiter plates at 5,000 cells per well, and incubated overnight. Actinomycin D was added to the cells at a final concentration of 1 µg/ml. Serial dilutions of the Nanobodies™ to be tested were mixed with a cytotoxic concentration of TNF (10 pM). After incubation for 30 min at 37°C, this mixture was added to the plated cells and incubated for 24 h at 37°C. Cell viability was determined by using the tetrazolium salt WST-1. Dose–response curves and IC₅₀ values (potency) were calculated with GraphPad Prism. The mean potencies for individual Nanobody™ constructs and benchmark anti-TNF biologics were calculated from a number of independent bioassays as well as the SD.