Fluorescence cross-correlation spectroscopy quantifies affinity, cooperativity, and kinetic stability in ternary protein complexes

Many biological processes and emerging therapeutic modalities rely on higher-order protein complexes whose properties cannot be predicted from their constituent binary interactions. However, methods for directly quantifying affinity, cooperativity, and kinetic stability within such assemblies remain limited. Here, we establish fluorescence cross-correlation spectroscopy (FCCS) as a solution-phase approach for characterizing multicomponent protein interactions and apply it to the clinically important HER2-targeting antibodies trastuzumab and pertuzumab. Using fluorescently labelled HER2, trastuzumab, and pertuzumab, we quantified binary binding affinities, directly measured ternary complex formation, and characterized the dissociation kinetics of binary and ternary complexes. FCCS measurements revealed positive cooperativity in the formation of the HER2-trastuzumab-pertuzumab ternary complex, while dissociation experiments demonstrated that the ternary complex is kinetically more stable than the corresponding binary interactions. Together, these findings provide direct solution-phase evidence that cooperative interactions stabilize the HER2-trastuzumab-pertuzumab complex, offering a molecular explanation for the enhanced efficacy of dual HER2 targeting in cancer therapy. More broadly, this work demonstrates that FCCS can robustly quantify affinity, cooperativity, and kinetic stability of multicomponent protein complexes using a commercially available platform. We provide a broadly accessible framework for studying higher-order protein interactions and supporting the development of next-generation multispecific and combination therapeutics. Significance StatementMany proteins function as part of multicomponent complexes, yet most experimental methods characterize interactions only one pair at a time, or indirectly through secondary reporters, or necessitating saturation of binary interactions first. We show that fluorescence cross-correlation spectroscopy can directly quantify how multiple binding partners interact simultaneously by measuring affinity, cooperativity, and kinetic stability in solution. Applying this approach to the clinically important HER2-targeting antibodies trastuzumab and pertuzumab reveals cooperative stabilization of their ternary complex. Because the measurements are performed on a commercially available instrument and require no surface immobilization, this broadly accessible method should facilitate mechanistic studies of complex biomolecular interactions and aid the development of multispecific and combination therapeutics.