Engineering Antibody-Drug Conjugates for Enhanced Cancer Immunotherapy

Over the past decades, the field of antibody-drug conjugates (ADCs) has grown rapidly with 15 FDA-approved drugs and over 100 candidates in clinical trials. These complex therapeutics, comprised of a monoclonal antibody and a cytotoxic payload conjugated through a linker, were initially developed to selectively deliver cytotoxins to tumor cells. Despite recent clinical successes, ADC efficacy in solid tumors remains limited by heterogeneous distribution and dose-limiting toxicities. ADCs rapidly bind to their target antigens after extravasation, restricting tissue penetration and reducing efficacy. Although higher dosing may improve distribution, systemic toxicity narrows the therapeutic window. This dissertation addresses these challenges through rational design strategies aimed at improving payload delivery and immune engagement, thereby enhancing therapeutic efficacy and durability. The first focus centers on optimizing ADC and payload delivery through strategic antibody combinations. We examined competitive and non-competitive co-administration approaches to modulate intratumoral distribution and internalization using co-culture assays, 3D tumor spheroids, and HER2-positive xenograft models. Competitive combinations involved co-administering unconjugated carrier antibodies targeting the same epitope as the ADC, thereby improving ADC distribution. These antibodies were further engineered with reduced binding affinity and enhanced Fc-effector function – termed enhanced Fc, high-avidity, low-affinity (eFc-HALA) antibody – to “auto-tune” distribution based on antigen density while enhancing Fc-mediated immune responses. In contrast, non-competitive antibodies, binding different epitopes on the same antigen, promoted faster internalization and intracellular payload delivery. We further investigated epitope selection in ADCs and immune-stimulating antibody conjugates (ISACs) combinations. Our results demonstrated that optimal targeting depends on payload function (cytotoxic vs. immune-activating) and is critical for therapeutic outcomes. Immunofluorescence imaging and co-culture assays revealed that competitive ISACs and non-competitive antibodies could effectively deliver both ADC and ISAC payloads to tumors. In vivo, the combination of ADC, competitive ISAC, and non-competitive antibody produced the strongest antitumor responses: competitive binding improved tissue-level distribution, while non-competitive binding enhanced cellular delivery. These findings highlight importance of rational epitope selection in combination therapy to coordinate distribution, internalization, and immune stimulation for maximal therapeutic benefit. The second focus of this work investigates how Fc-Fcγ receptor (FcγR) interactions influence ADC efficacy by modulating immune cell engagement and payload distribution in the tumor microenvironment. Using syngeneic mouse models, we evaluated Fc-engineered ADCs, including Fc-silent and FcγRI-muted variants, in combination with the eFc-HALA antibody. Multiscale pharmacokinetic analyses including plasma clearance, biodistribution, and tumor histology, were used to assess the impact of Fc modifications on therapeutic outcomes. Our findings demonstrated that Fc-effector functions enhanced ADC efficacy in immunostimulatory (“hot”) tumors, while FcγRI-mediated uptake of highly potent payloads such as PBD reduced efficacy, likely by depleting beneficial immune populations. This outcome contrasts with observations in immunosuppressive (“cold”) tumors, where Fc-effector functions impaired responses and FcγRI-mediated uptake of PBD instead improved efficacy. With less potent agents and different mechanisms of action, such as the tubulysin derivative MMETA, FcγRI-mediated uptake into immune cells resulted in better responses. These results underscore the context-dependent role of FcγRs, with their contribution determined by both immune landscape and payload potency. Overall, this dissertation illustrates how epitope selection, Fc engineering, and payload potency can be strategically integrated to design next-generation ADCs that optimize delivery, immune engagement, and antitumor activity. By tailoring ADCs to tumor biology and immune context, this work provides a framework for advancing ADC therapies beyond targeted cytotoxins toward multifunctional immunotherapeutics to achieve more effective and durable responses.