title: Maximizing the potency of an anti-TLR4 monoclonal antibody by exploiting proximity to Fcγ receptors.
abstract: 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 Fcγ receptors (FcγR), 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 FcγRI or FcγRIIA (via the Fc). The Kd of these interactions are in the nM range (3 nM of the Fv for TLR4 and 47 nM of the Fc for FcγRI). 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-FcγR interactions. Taken together, these results demonstrate an unforeseen level of contribution by FcγRs to an antibody's effectiveness when targeting a cell surface protein of relatively low abundance. These findings highlight an exploitable mechanism by which FcγR-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 FcγRs.
authors.affiliation: a NovImmune SA; Plan-les-Ouates , Switzerland.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Loyau
authors.forename: Pauline
authors.initials: P
authors.lastname: Malinge
authors.forename: Bruno
authors.initials: B
authors.lastname: Daubeuf
authors.forename: Limin
authors.initials: L
authors.lastname: Shang
authors.forename: Greg
authors.initials: G
authors.lastname: Elson
authors.forename: Marie
authors.initials: M
authors.lastname: Kosco-Vilbois
authors.forename: Nicolas
authors.initials: N
authors.lastname: Fischer
authors.forename: François
authors.initials: F
authors.lastname: Rousseau
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity / Q000276:immunology*; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism; D018448:Models, Immunological; D011485:Protein Binding / Q000276:immunology; D017452:Receptors, IgG / Q000276:immunology* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D051197:Toll-Like Receptor 4 / Q000276:immunology* / Q000378:metabolism; D020298:U937 Cells
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D017452:Receptors, IgG; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4
keywords: DAMP, damage-associated molecular pattern; Fc gamma receptors; Fc, fragment crystallizable; FcγR, Fc gamma receptor; Fv, fragment variable; IL, interleukin; IVIg, intravenous immunoglobulin; Ig, immunoglobulin; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; TLR4; affinity maturation; antibody; avidity; mAb, monoclonal antibody