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), and has shown promise against PD-L1 positive non-small cell lung cancer in clinical trials. While the stoichiometries and dynamics of ivonescimab’s interactions with VEGF and PD-1 are not fully understood, VEGF binding is thought to promote higher-order structure formation.
Methods
We used mass photometry (MP) and kinetic modelling to analyze these interactions, quantifying the complexes formed and their affinities. Dissociation constants (KD) 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 KD=0.08 nM. Higher-order oligomeric complexes, present only at low abundance, displayed markedly weaker affinities (1.29 nM; 3.17 nM). Ternary complexes of ivonescimab with its two targets consistently presented two PD-1 antigens for each ivonescimab molecule, with near-identical KDs for the binding of the first (2.37 nM) and second (2.36 nM) PD-1 molecules.
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.
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