Abstact

Ozoralizumab (OZR) is a next-generation TNF inhibitor composed of two identical humanized anti-TNFalpha NANOBODY(R) molecules (TNF30s) recombinantly linked via one humanized anti-human serum albumin (HSA) NANOBODY(R) molecule (ALB8) and two peptide linkers. OZR is designed as a unique format to exert potent inhibitory effects against TNFalpha with long plasma half-life. However, the three-dimensional structure of OZR-TNFalpha-HSA complex has not yet been elucidated, and a complete understanding of its interaction mechanism with TNFalpha is yet to be gained. In this study, we successfully observed the formation of the OZR-TNFalpha-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 TNFalpha 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 TNFalpha 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 TNFalpha-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 TNFalpha inhibitors with enhanced and sustained efficacy.