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. Increasing scaffold valency from Fab dimers up to tetramers improves the potency of the antibody mimics up to 5-fold, but with diminishing returns in effective dose beyond trimeric formats. Antibody mimics that present either one or two Fabs against either receptor target allows for initial engagement of one cell type over the other. Finally, the antibody mimics significantly reduce HER2+ tumor volumes in a humanized xenograft model of breast cancer.

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. 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.

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. A panel of 16 bivalent anti-HER2 antibodies were prepared wherein each varied in the orientation of the fusion domain on the Fabs. The analogs displayed a range of cytotoxic activity, and surprisingly, the most active mimic binds to cells with a 10-fold lower avidity than the least active variant suggesting that structure plays a large role in their efficacy. This work suggests that the megamolecule approach can be used to prepare antibody mimics having a broad structural diversity.

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. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. Because these are coupled first- and second-order processes, an analysis of product yields from reactions performed at a range of linker concentrations gives rate constants for cyclization. We determined these to be 9.7 × 10-3 s-1, 2.3 × 10-3 s-1, and 8.1 × 10-4 s-1 for the dimer, tetramer, and hexamer, respectively. This work demonstrates an efficient route to cyclic macromolecules having nanoscale dimensions and provides new scaffolds that can be generated using the megamolecule approach.

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.