Kalyanasundaram Subramanian: No financial relationships to disclose
Objectives: The study aims to mechanistically elucidate the guide-effector relationship, where rapidly internalizing antigens, often in low abundance, induce the internalization of slowly internalizing yet highly expressed antigens. The objective is to identify parameters that govern neighboring antigen interactions, thereby establishing a mechanistic framework for designing bispecific antibody systems.
Methods: We developed an in vitro mechanistic model to understand the guide-effector relationship in bispecific antibody systems. The model consisted of the following processes: the binding of a bispecific antibody targeting EphA2 (fast-internalizing guide) and ALCAM (slow-internalizing effector), antigen turnover, receptor-antibody complex formation (dimers and trimers), and internalization of free and bound receptors. Surface antigen levels were tracked over time, and the simulated results compared with experimental data derived from literature1.
Results: Simulations predicted that the bispecific antibody (3F1/RYR, targeting ALCAM and EphA2) effectively removed ALCAM from the cell surface, with internalized receptor fraction at 60% in 1 hour, 86% in 4 hours, and ~99 % in 24 hours, closely matching experimental observations of 52%, 86%, and 90%, respectively. ALCAM removal occurred only with the bispecific antibody; neither the monoclonal anti-ALCAM antibody nor a control anti-EphA2 bispecific antibody achieved significant removal, consistent with observed data.
Simulations also revealed that bispecific antibody mediated percent surface internalization of ALCAM was dependent on the EphA2: ALCAM surface ratio. ALCAM disappearance from the surface increased with an increase in the ratio, a prediction that was verified by the experimental results. These findings demonstrate that having an increase in the number of guide receptors leads to greater internalization of the effector. Conversely, the presence of a slow internalizing receptor presents a significant drag on the internalization rate of the rapidly internalizing guide. At a EphA2: ALCAM ratio of 0.2, and with both the bispecific and anti-EphA2 antibodies tested at 100 nM, the bispecific antibody-mediated internalization of EphA2 was slower (~40% vs. 40 ± 14% observed) than the internalization mediated by the monospecific EphA2 antibody (~60% vs. 61 ± 14% observed).
Conclusions: This study provides a mechanistic understanding of the guide-effector relationship in bispecific antibody systems, showing how fast-internalizing antigens like EphA2 and slow-internalizing antigens like ALCAM influence each other. Applying this framework can offer valuable insights for designing effective bispecific antibodies to enhance targeted drug delivery.
Citations: [1] Lee NK, Su Y, Bidlingmaier S, Liu B. Manipulation of Cell-Type Selective Antibody Internalization by a Guide-Effector Bispecific Design. Mol Cancer Ther. 2019 Jun;18(6):1092-1103. doi: 10.1158/1535-7163.MCT-18-1313. Epub 2019 Apr 8. PMID: 30962321; PMCID: PMC6548581. [2] Walker-Daniels J, Riese DJ 2nd, Kinch MS. c-Cbl-dependent EphA2 protein degradation is induced by ligand binding. Mol Cancer Res. 2002 Nov;1(1):79-87. PMID: 12496371.
