Mass photometry reveals stoichiometry and binding dynamics of bispecific tetravalent anti-VEGF-PD-1 antibody ivonescimab

Abstract Background The bispecific antibody ivonescimab targets programmed cell death protein 1 (PD-1) and vascular endothelial growth factor (VEGF). Recent clinical trials have shown it has greater efficacy against PD-L1 positive non-small cell lung cancer than pembrolizumab (Keytruda), a frequently prescribed anti-PD-1 monoclonal antibody. Ivonescimab binds to two VEGF and two PD-1 molecules, with complex formation through higher-order structure formation (or ‘daisy chain’ binding). However, the binding stoichiometries and interaction dynamics of ivonescimab with VEGF and PD-1 have not been characterized in depth. Methods We used mass photometry (MP) and kinetic modelling to analyze these interactions, quantifying the complexes formed and their affinities. Dissociation constants (K D ) for ivonescimab’s binding to VEGF and PD-1 were calculated from equilibrium counts and real-time measurements, respectively. Results VEGF drove oligomerization of ivonescimab, which bound VEGF predominantly in a 2:2 stoichiometry, with K D =0.08 nM. Higher-order oligomeric complexes, present only at low abundance, displayed markedly weaker affinities (3.17 nM; 1.29 nM). Ternary complexes of ivonescimab with its two targets consistently presented two PD-1 antigens for each ivonescimab molecule, with a 1.66 nM K D for the binding of the first PD-1 and the slightly stronger 0.89 nM for the second PD-1 molecule. Conclusions MP confirmed VEGF-induced ivonescimab oligomerization and revealed that dimers, not higher-order structures, were the most stable stoichiometry. MP enables detailed analysis of antibody–antigen interactions, even for bispecific antibodies that interact with antigens with complex stoichiometries. Statement of significance We report the first application of mass photometry to characterize the binding of the clinically promising bispecific antibody ivonescimab to its targets, VEGF and PD-1. The most stable assembly is a dimeric complex, not higher-order, as expected – advancing understanding of ivonescimab and demonstrating mass photometry’s value for complex biologics analysis.