Dual density-dependent molecular AND gates: ultraselective bispecific targeting via avidity

About This Grant

Project Summary Selective therapeutic delivery enables precision medicine. Overexpressed biomarkers provide delivery targets, yet moderate expression in healthy tissue results in on-target, off-disease toxicity thereby limiting the therapeutic window. Exquisite cellular targeting remains a challenge. Strategies to increase disease-specific delivery are sorely needed. To increase cell specificity, we will [1] advance from targeting biomarker presence to targeting high biomarker density, and [2] require the presence of two distinct biomarkers rather than one to reduce on- target / off-disease opportunities. These stringency factors will be achieved through low-affinity, high-avidity, cooperative binding via controlled heterobivalency (i.e., dual density-dependent molecular AND gate binders). While density-dependent binding has been achieved in several cases, more rigorous and systematic evaluation is needed to elucidate design principles and empower precise and tunable control to maximize selectivity. Thus, increased insight and efficient modular engineering will be especially valuable in rationally tailoring the engineered avidity and cooperativity in bispecific systems that are needed to achieve ultraselectivity. We pursue an innovative strategy that leverages modular synthetic miniproteins, systematic engineering, and assembly of molecular components through an integrated experimental/computational approach. Objective: Engineer targeting ligands that selectively bind cells expressing high levels of two antigens — i.e. dual density- dependent molecular AND gates — via engineered bispecific avidity. We pursue [1] specific application of bispecificity to engineer a Trop2/EGFR density-dependent bispecific targeting agent, with physiological validation in a murine tumor model; [2] broader elucidation of the molecular factors — both ligand and target — that dictate performance; and [3] establishment of a generalizable engineering platform for efficient generation of dual density-dependent molecular AND gates. Aim 1: Engineer dual density-dependent Trop2/EGFR AND gate via bispecific avidity. We will engineer a bispecific density-dependent binder to Trop2 and EGFR by modulating affinity, valency, and epitope, and the length, rigidity, and orientation of the linker. The benchmark for success is a ligand with a sub-nM EC50 for binding cells with dual high expression and ≥100:1 binding differential relative to cells with single-high density or dual-moderate density. Physiological targeting will be evaluated in murine xenograft tumor models via PET imaging and excised tissue gamma counting. Aim 2: Elucidate factors that drive dual density dependence in bispecific molecules. We will more broadly evaluate binding with a set of systematically varying ligands and receptors experimentally via flow cytometry and computationally via a mechanistic mathematical model. The impact of molecular design parameters and target settings on dual density dependence and binding selectivity will be quantified across multiple biomarkers for generality. Resolution of model mechanisms with experimental data will elucidate the factors that drive expression dependence and empower rational design of molecules with tailored expression dependence.