A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency

Abstract

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.

Modeling Hepatocellular Toxicity of Enavatuzumab, a Humanized Anti-TweakR Antibody

“…Mimicking that function with a secondary antibody dramatically increased the level of cytokines released by enavatuzumab; this finding is consistent with increased functional activity of enavatuzumab and other TNFRSF agonist antibodies when crosslinked with a secondary antibody or Protein A (Chao et al, 2013;Salzmann et al, 2013;Chuntharapai et al, 2001;White et al, 2013;Li et al, 2006;Dhein et al, 1992;Chodorge et al, 2012). However, crosslinking the antibody did not increase ALT/AST release, which was only observed when immune cells were present.…”

A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis

“…We compared the flow cytometric on-bead binding signals of the anti-FasR variants with a range of affinities: Ep6b_B01, E09, E09_Y58S (with the K d values of 0.18 nM, 8.6 nM and 187 nM, respectively)37 and a non-binder CEA6 scFv40. Each scFv was displayed on the bead surface and subsequently incubated with 1 nM, 10 nM and 30 nM of the antibody-labelled FasR-Fc (Fig.…”

“…As a starting point for an affinity-maturation campaign, we chose the anti-Fas receptor scFv E09, which was originally selected from a naïve antibody phage library3637. The svFV E09 has previously been affinity matured by six rounds of ribosome display (RD)3637, enabling us to make comparisons to the current evolution method.…”

“…The svFV E09 has previously been affinity matured by six rounds of ribosome display (RD)3637, enabling us to make comparisons to the current evolution method. Four FACS-based screening rounds together with a phylogenetic analysis of the selected scFv mutants yielded two scFv variants with up to two orders of magnitude improved binding affinity for FasR.…”

Heartwood of Secang (CAESALPINIA SAPPAN L.) Ethanolic Extract Show Selective Cytotoxic Activities on T47D and Widr Cells But Not on Hela Cells

“…HeLa cells induce FasL expression via Corticotropin-releasing hormone (CRH) (Taliouri, et al, 2012). Higher-affinity antibodies (FasL) performed a significant decrease, rather than an increase, in agonist activity at the Fas receptor (Chodorge, et al, 2012). Compounds in SEE might be influence affinity of FasL and Fas receptor, but the mechanism must be explored in the next study.…”