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J. the megamolecule approach to synthesize anti-HER2 nanobodyCcytosine 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 structureCfunction relationships of protein conjugate therapeutics with synthetic control of protein domain stoichiometry. Graphical Abstract INTRODUCTION Many chemotherapy drugs target cancer cells due to their proliferative growth. However, proliferative noncancer cells are also affected, resulting in the death of healthy cell populations and severe side-effects for patients. Local delivery of chemotherapeutics to tumors could improve patient care and treatment outcomes. One strategy to achieve local delivery of chemotherapeutics is antibody-directed enzyme prodrug therapy (ADEPT).1 This strategy employs a heterobifunctional antibodyCenzyme conjugate that specifically binds to a tumor-specific antigen and enzymatically activates a nontoxic prodrug into a toxic chemotherapeutic within the tumor microenvironment (Figure 1A).2,3 Synthesis of the antibodyCenzyme NSC 23766 conjugate requires the antibody and enzyme domains to be covalently joined.4,5 However, previous approaches to synthesize antibodyCenzyme conjugates yielded heterogeneous conjugates, making it difficult to investigate structureCfunction relationships and leading to stalled early-stage translation.6 Open in a separate window Figure 1. Design and synthesis of nanobodyCenzyme megamolecules. A. Scheme of a nanobodyCenzyme megamolecule binding to a HER2+ cancer cell followed by activation of the nontoxic prodrug 5-FC into the chemotherapeutic drug 5-FU. B. The anti-HER2 NSC 23766 nanobody N (orange) is fused to the cutinase (blue) protein, and the yeast cytosine deaminase enzyme E (pink) is fused to the SnapTag (yellow) protein. The two fusion proteins are conjugated by use of a heterobifunctional linker, which is terminated by a NSC 23766 and having improved tumor penetration when compared to a monoclonal antibody.18 We hypothesized Hdac8 that the efficacy of nanobody-directed enzyme prodrug therapy depends on the structure of the nanobodyCenzyme conjugate. We tested this hypothesis by synthesizing nanobodyCenzyme megamolecules with an increasing number of nanobody or enzyme domains. Homogeneous megamolecules containing tunable numbers of nanobody and enzyme domains for prodrug cancer therapy allows us to address the role of stoichiometryparticularly as it relates to bivalent binding to the cell surface and dimerization of the enzymeand enables the synthesis of homogeneous antibody conjugates for structureCfunction studies of therapeutic efficacy of next-generation biologics for cancer therapy.19C23 RESULTS Design and Synthesis of NanobodyCEnzyme Megamolecules. We synthesized nanobodyCenzyme megamolecules with the megamolecule approach, where fusion proteinscontaining either the nanobody or enzyme domainsreact covalently with small molecule cross-linkers (Figure 1B). The fusion proteins possess two domains: a functional domain (either the nanobody for binding or the enzyme for prodrug activation) and a linking domain (either cutinase or SnapTag).24,25 Fusion proteins were produced in and purified by affinity chromatography (Figure 1E,?,F).F). The cross-linkers are terminated with substrates that covalently bind to the nucleophilic residues in the active site of either linking domain and are orthogonally reactive in NSC 23766 solution.11,12 We used a and em k /em cat for the E fusion protein agree well with previously reported values for yCD of 0.16 0.01 mM and 17 0.4 s?1, respectively.39 We found that the 1N:2E megamolecule had the highest catalytic efficiency, suggesting that the intramolecular dimerization increased enzyme activity. Open in a separate window Figure 3. Characterization of yCD activity and nanobodyCenzyme megamolecule structure. A. Size-exclusion chromatograms of megamolecule purification and.