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Antibody-drug conjugates (ADCs) are a method of targeted drug delivery that transport a payload to a desired cell type. By directly targeting the cell type of choice, off-target effects experienced by non-diseased cells can be mitigated. Currently, the majority of clinical-stage ADCs are directed to tumor cells and contain a cytotoxic payload. In contrast, this work focuses on the design and evaluation of ADCs that deliver Toll-Like Receptor (TLR)-7/8 agonists to B-cells. TLR-7/8 agonists activate an intracellular cytosolic receptor that leads to the activation of NFκB, which results in the production of proinflammatory cytokines. This forms a link between the innate and adaptive immune system by promoting T cell activity. We will present work that focuses on 3 aspects of these TLR-activating ADCs: 1) Evaluation of payload and linker-payload efficacy in B-cells; 2) Understanding ADC stability and catabolism; and 3) Evaluating the specificity and activity of the TLR-activating ADCs. Nine payloads were synthesized and evaluated and found to exhibit sub-µM potency: Resiquimod, E66, and E104. Of these three, E104 had the highest potency followed by Resiquimod. Each payload was evaluated using three different linkers: mc, mc_ValCit, and mc_ValCitPABC. Mc has been shown to be a non-cleavable linker via catabolism studies, while mc_ValCitPABC has been shown to be cleavable. Quanti-blue assays in Ramos Blue cells demonstrated that mc_E104 is the most potent linker-payload when attached to a B-cell targeting antibody. This non-cleavable ADC was significantly more potent than the corresponding cleavable (ValCitPABC) ADC. We will present catabolism and permeability data that may explain this unexpected finding.



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Targeted Delivery of TLR-Agonists Using ADC Technology