(700a) Solid-Phase Synthesis of Megamolecules: A New Class of Nanoscale Therapeutics

Proteins are versatile building blocks in the field of synthetic biology. Novel proteins can be engineered to perform unique functions and chemistry for both biological and industrial applications. However, synthesis of non-natural molecules greater than 100 kDa presents a large metabolic burden on bacterial hosts, leading to issues in aggregation, low yield, and purity. Most approaches for the in vitro construction of solution-phase multi-protein assemblies require high concentrations of protein building blocks, costly purification steps, and result in heterogeneous products. Taking inspiration from approaches such as solid-phase peptide synthesis, we present a synthetic strategy to produce homogeneous protein-based nanoscale structures, referred to herein as megamolecules, by immobilization of protein building blocks on SnapTag-functionalized resin beads. Building from the SnapTag capture chemistry, protein building blocks are constructed by step-wise assembly into large molecules using multifunctionalized bi-, tri-, and tetra-functional crosslinker molecules. Expanding upon the library of traditional capture chemistries, the crosslinks capitalize upon the specificity and irreversibility of the active site-ligand complex. Through the construction of a toolbox of oligomers and the paired inhibitor molecules, the creation of a library of megamolecules is possible. Upon proteolytic cleavage from the resin by an engineered TEV recognition site between the bead and the megamolecule assembly, this method produces a monodisperse product that requires no formal purification process - suggesting ease and feasibility in scale up for industrial applications. Through rational design, we generated cost-effective biomaterial therapeutics that exhibit non-linear connectivity in high yield with masses up to 500 kDa. Using this synthetic-strategy, we constructed a panel of antibody-enzyme conjugates for prodrug therapy in a breast cancer model. Leveraging the ability to control the stoichiometry of both the enzyme and antibody domains assembled on the nanoscale structure, we assembled biorthogonal megamolecules to target overexpressed HER2+ breast cancer cells in 3D cell culture. Once the megamolecule is bound to the cancer cell membrane, the turnover of the non-cytotoxic prodrug into a cytotoxic drug is localized to the cell, minimizing off target toxicity to healthy cells. We believe that the construction of antibody-enzyme conjugate megamolecules will enhance the established techniques of nanotherapeutic delivery of prodrug cargo through improvements in spatial ligand binding, which is dependent on specificity, uptake, binding, and recognition of the nanostructure. Based on the function-structure relationship of multi-protein complexes, we expect that tuning the topology of the nanostructure will enable the generation of a new class of cell-specific drug delivery therapeutics.