Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc

Abstract

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the proinflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated "Fc-one/kappa" (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.

Selective Targeting of TNF Receptors as a Novel Therapeutic Approach

“…Atrosimab is an Fv-Fc fusion protein with approximately half the size of an antibody. The Fv fragment was generated from an alternative humanized version of H398, which was further affinity matured by CDR and random mutagenesis using phage display (Richter et al, 2019a). In order to force heterodimerization of the Fc region, a novel strategy was employed using CH3 domains engineered to comprise the CH1-CL interface of a Fab fragment.…”

“…In order to force heterodimerization of the Fc region, a novel strategy was employed using CH3 domains engineered to comprise the CH1-CL interface of a Fab fragment. This resulted in a monovalent antibody with improved binding and neutralizing activity compared to Atrosab (Richter et al, 2019a).…”

Inhibition of Tumor Cell Growth and Cancer Stem Cell Expansion by a Bispecific Antibody Targeting EGFR and HER3

“…Analysis of real-time binding kinetics were performed as previously described using the A-100 C-Fast or Cell-200 C-Fast biosensors (Attana; ref. 24). scDb hu225Â3-43-Fc was immobilized to the Attana LNB Carboxyl Sensor Chip (3623-3103, Attana).…”

The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation

“…This was attributed to a marginal agonistic response in a small concentration range observed in vitro due to bivalent TNFR1 binding of the full IgG molecule ( 14 ). Thus, we next developed Atrosimab, an optimized monovalent derivative of Atrosab, composed of affinity improved VH and VL domains fused to an effector-silenced heterodimerizing Fc region ( 19 ). In vitro , Atrosimab showed superior blocking activity compared to the parental bivalent molecule Atrosab ( 19 ).…”