Cryo-EM elucidates the interaction mechanism of ozoralizumab, a humanized anti-TNFα NANOBODY® compound

Ozoralizumab (OZR) is a next-generation TNF inhibitor composed of two identical humanized anti-TNFα NANOBODY® molecules (TNF30s) recombinantly linked via one humanized anti-human serum albumin (HSA) NANOBODY® molecule (ALB8) and two peptide linkers. OZR is designed as a unique format to exert potent inhibitory effects against TNFα with long plasma half-life. However, the three-dimensional structure of OZR-TNFα-HSA complex has not yet been elucidated, and a complete understanding of its interaction mechanism with TNFα is yet to be gained. In this study, we successfully observed the formation of the OZR-TNFα-HSA ternary complex by single-particle cryo-electron microscopy. The single-particle analysis revealed that the two TNF30 molecules of OZR simultaneously bind bivalently to TNFα in a 1:1-bivalent binding mode, while the ALB8 molecule binds to HSA, forming a ternary complex. Thus, OZR exhibits a binding mode significantly different from that of other IgG-type TNFα inhibitors. Furthermore, surface plasmon resonance (SPR) analysis demonstrated that the 1:1-bivalent binding mode confers an exceptionally slow dissociation rate, thereby contributing to the potent TNFα-neutralizing activity of OZR. These findings not only lend support to the favorable clinical efficacy of OZR from a structural standpoint but also lay the foundation for the rational design and development of next-generation TNFα inhibitors with enhanced and sustained efficacy.