Maximizing the potency of an anti-TLR4 monoclonal antibody by exploiting proximity to Fcγ receptors

In order to treat Toll like receptor 4 (TLR4)-mediated diseases, we generated a potent antagonistic antibody directed against human TLR4, Hu 15C1. This antibody's potency can be modulated by engaging not only TLR4 but also Fc gamma receptors (Fc gamma R), a mechanism that is driven by avidity and not cell signaling. Here, using various formats of the antibody, we further dissect the relative contributions of the Fv and Fc portions of Hu 15C1, discovering that the relationship to potency of the different antibody arms is not linear. First, as could be anticipated, we observed that Hu 15C1 co-engages up to 3 receptors on the same plasma membrane, i.e., 2 TLR4 molecules (via its variable regions) and either Fc gamma RI or Fc gamma RIIA (via the Fc). The K-d of these interactions are in the nM range (3 nM of the Fv for TLR4 and 47 nM of the Fc for Fc gamma RI). However, unexpectedly, neutralization experiments revealed that, due to the low level of cell surface TLR4 expression, the avidity afforded by engagement through 2 Fv arms was significantly limited. In contrast, the antibody's neutralization capacity increases by 3 logs when able to exploit Fc-Fc gamma R interactions. Taken together, these results demonstrate an unforeseen level of contribution by Fc gamma Rs to an antibody's effectiveness when targeting a cell surface protein of relatively low abundance. These (f)indings highlight an exploitable mechanism by which Fc gamma R-bearing cells may be more powerfully targeted, envisioned to be broadly applicable to other reagents aimed at neutralizing cell surface targets on cells co- expressing Fc gamma Rs.