The Role of MegaMolecule Antibody Structure in Internalization and Signaling.

This manuscript describes the synthesis of 26 megamolecule-based antibody scaffolds that target the receptor tyrosine kinase HER2 (ERBB2). The scaffolds include mono-, bi-, and trivalent structures that present high- or low-affinity Fab or nanobody domains. Cell binding, internalization, and cytotoxicity were compared with those of the parent monoclonal antibody trastuzumab.

Megamolecule Self-Assembly Networks: A Combined Computational and Experimental Design Strategy.

This work describes the use of computational strategies to design megamolecule building blocks for the self-assembly of lattice networks. The megamolecules are prepared by attaching four Cutinase-SnapTag fusion proteins (CS fusions) to a four-armed linker, followed by functionalizing each fusion with a terpyridine linker. This functionality is designed to participate in a metal-mediated self-assembly process to give networks.

Synthesis and Activity of T-Cell Tumor-Directing MegaMolecules.

This paper describes the synthesis, characterization, and functional activity of 26 MegaMolecule-based bispecific antibody mimics for T-cell redirection toward HER2+ cancer cells. The work reports functional bispecific MegaMolecules that bind both receptor targets, and recruit and activate T-cells resulting in lysis of the target tumor cells. Changing the orientation of linkage between Fabs against either HER2 or CD3ε results in an approximately 150-fold range in potency.

Synthesis, Characterization, and Simulation of Four-Armed Megamolecules.

This paper describes the synthesis, characterization, and modeling of a series of molecules having four protein domains attached to a central core. The molecules were assembled with the "megamolecule" strategy, wherein enzymes react with their covalent inhibitors that are substituted on a linker. Three linkers were synthesized, where each had four oligo(ethylene glycol)-based arms terminated in a -nitrophenyl phosphonate group that is a covalent inhibitor for cutinase.

Synthetic Tuning of Domain Stoichiometry in Nanobody-Enzyme Megamolecules.

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.

Design and Synthesis of Megamolecule Mimics of a Therapeutic Antibody.

This communication describes the design, synthesis, and biological activity of a megamolecule mimic of an anti-HER2 antibody. The antibody mimic was prepared by linking two Fabs from the therapeutic antibody trastuzumab, which are fused through the heavy chain variable domain to either cutinase or SnapTag, with a linker terminated in an irreversible inhibitor for each enzyme. This mimic binds HER2 with comparable avidity to trastuzumab, has similar activity in a cell-based assay, and can arrest tumor growth in a mouse xenograft BT474 tumor model.

Solid-Phase Synthesis of Megamolecules.

This paper presents a solid-phase strategy to efficiently assemble multiprotein scaffolds-known as megamolecules-without the need for protecting groups and with precisely defined nanoscale architectures. The megamolecules are assembled through sequential reactions of linkers that present irreversible inhibitors for enzymes and fusion proteins containing the enzyme domains. Here, a fusion protein containing an N-terminal cutinase and a C-terminal SnapTag domain react with an ethyl -nitrophenyl phosphonate (pNPP) or a chloro-pyrimidine (CP) group, respectively, to give covalent products.

Long-Range Energy Transfer in Protein Megamolecules.

In this investigation, we report evidence for energy transfer in new protein-based megamolecules with tunable distances between donor and acceptor fluorescent proteins. The megamolecules used in this work are monodisperse oligomers, with molecular weights of ∼100-300 kDa and lengths of ∼5-20 nm, and are precisely defined structures of fusion protein building blocks and covalent cross-linkers. Such structures are promising because the study of energy transfer in protein complexes is usually difficult in this long length regime due to synthetic limitations.

An Immobilized Enzyme Reactor for Spatiotemporal Control over Reaction Products.

This paper describes a microfluidic chip wherein the position and order of two immobilized enzymes affects the type and quantity of reaction products in the flowing fluid. Assembly of the chip is based on a self-assembled monolayer presenting two orthogonal covalent capture ligands that immobilize their respective fusion enzyme. A thiol-tagged substrate is flowed over a region presenting the first enzyme-which generates a product that is efficiently transferred to the second enzyme-and the second enzyme's product binds to an adjacent thiol capture site on the chip.

Synthesis of Cyclic Megamolecules.

This paper describes the synthesis of giant cyclic molecules having diameters of 10-20 nm. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (pNPP) and benzylguanine (BG) groups, respectively.

Acid-Resistant Mesoporous Metal-Organic Framework toward Oral Insulin Delivery: Protein Encapsulation, Protection, and Release.

Diabetes affects millions of people worldwide and the number of diagnoses continues to climb annually. Though several effective medications and therapeutic methods have been developed to treat type 1 (T1DM) and type 2 (T2DM) diabetes mellitus, direct insulin injection remains the only effective treatment for insulin resistant (IR) diabetes patients. Here, we immobilize insulin in a crystalline mesoporous metal-organic framework (MOF), NU-1000, and obtain a high loading of ∼40 wt % in only 30 min.

A self-adjuvanting supramolecular vaccine carrying a folded protein antigen.

This work illustrates a strategy for the design of molecularly defined immunotherapies, using a blend of supramolecular peptide self-assembly and active site-directed protein capture.

Modular assembly of protein building blocks to create precisely defined megamolecules.

Enzyme-promoted assembly: The construction of a hetero-bifunctional protein building block, HaloTag-cutinase, that reacts rapidly and selectively with a small-molecule linker is described. The step-wise combination of these building blocks generates a 300 kDa "megamolecule" with precisely defined domain orientation, connectivity, and composition.