Synthetic Tuning of Domain Stoichiometry in Nanobody–Enzyme Megamolecules

Abstract

This paper presents a method to synthetically tune atomically precise megamolecule nanobody–enzyme conjugates for prodrug cancer therapy. Previous efforts to create heterobifunctional protein conjugates suffered from heterogeneity in domain stoichiometry, which in part led to the failure of antibody–enzyme conjugates in clinical trials. We used the megamolecule approach to synthesize anti-HER2 nanobody–cytosine deaminase conjugates with tunable numbers of nanobody and enzyme domains in a single, covalent molecule. Linking two nanobody domains to one enzyme domain improved avidity to a human cancer cell line by 4-fold but did not increase cytotoxicity significantly due to lowered enzyme activity. In contrast, a megamolecule composed of one nanobody and two enzyme domains resulted in an 8-fold improvement in the catalytic efficiency and increased the cytotoxic effect by over 5-fold in spheroid culture, indicating that the multimeric structure allowed for an increase in local drug activation. Our work demonstrates that the megamolecule strategy can be used to study structure–function relationships of protein conjugate therapeutics with synthetic control of protein domain stoichiometry.

Development of an Enzyme‐Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One‐Pot Megamolecule Assembly

“…[16] 4-Iodophenol was protected by O-acetylation (5), coupled to trimethylsilylacetylene by Sonogashira cross-coupling (6), and desilylated with potassium fluoride. The protected synthetic retinoid 8 was then conjugated through Sonogashira coupling of π-acceptor (7) and π-donor (4), followed by saponification to reveal non-covalent retinoid inhibitor 9 with a succinic acid handle for chemical ligation. We completed the synthesis by sulfonylation of the terminal phenol with [4-(acetylamino) phenyl]imidodisulfuryl difluoride (AISF) to give synthetic retinoid 10.…”

“…[4] Our previous work used the reaction of a cutinase protein with a p-nitrophenyl phosphonate (pNPP) and a SnapTag domain with a chloro-pyrimidine (CP) inhibitor to synthesize dendritic molecules, [5] therapeutic antibody mimics, [6] and to study structure-function relationships in antibody-enzyme conjugates (Figure 1A-B). [7] The development of additional enzyme-inhibitor pairs will be important for enabling the efficient synthesis of complex protein architectures and possibly for preparing structures that are immunotolerant. In this work, we describe the development of an irreversible inhibitor for cellular retinoic acid binding protein II (CRABP2) and demonstrate its use in megamolecule assembly.…”

Synthesis, Characterization, and Simulation of Four-Armed Megamolecules

“…We recently described a strategy to synthesize very large molecules by joining fusion proteins with multifunctional linkers. − The reactions occur between a covalent inhibitor on the linker and its target enzyme domain and proceed in high yield and with essentially no side products. We have demonstrated this “megamolecule” approach for the synthesis of linear and cyclic molecules, to organize fluorescent proteins for studies of energy transfer, and for the preparation of antibody mimics. , However, the characterization of these structures and understanding their conformations and dynamics are very challenging.…”

“…We recently described a strategy to synthesize very large molecules by joining fusion proteins with multifunctional linkers. − The reactions occur between a covalent inhibitor on the linker and its target enzyme domain and proceed in high yield and with essentially no side products. We have demonstrated this “megamolecule” approach for the synthesis of linear and cyclic molecules, to organize fluorescent proteins for studies of energy transfer, and for the preparation of antibody mimics. , However, the characterization of these structures and understanding their conformations and dynamics are very challenging. Here, we report the synthesis and comprehensive characterization of a four-armed megamolecule with transmission electron microscopy (TEM), , X-ray crystallography, , and simulation , and demonstrate how this comprehensive approach contributes to an understanding of the structures of this new class of molecules. − …”

To Cryo or Not to Cryo? A Consideration of Length Scales During Macromolecule Sample Preparation

“…This decision flow is exemplified through the images in Figure 2. The sample of interest, a megamolecule protein construct, is shown in the insets; the protein scaffolds undergo intramolecular reaction through enzyme domains to convert linear molecules into macrocycles [7][8]. Our imaging effort should both show examples of the stimulus-dependent conformations and yield a statistical analysis of construct size.…”