Abstract Tambo is developing a platform to activate drugs at specific sites in the body, thus enhancing their efficacy while minimizing systemic toxicity and adverse drug events (ADEs). Most drugs distribute throughout the body; due to lack of specificity for the pathological site, high doses are required to achieve effective therapeutic concentrations, causing toxicity and ADEs in healthy tissues. Each year, there are ~1.2 million reports of ADEs in the U.S., with overall costs of ADE-related morbidity and mortality thought to exceed $177 billion. ADEs also contribute to the 90% failure rate of drug candidates. Common prodrug approaches to overcome these critically limiting ADEs rely on biological or physiological factors, such as selected biomarkers, enzymatic activity, altered pH, or reactive oxygen species, which have found limited success due to inherent variability among patients. Tambo’s Click Activated Protodrugs (CAP) platform was designed to achieve higher concentrations of active drugs at specific pathological sites while minimizing systemic toxicity using an activation method independent of native biological processes; thus the approach is not affected by variations across patients. The platform also enables the reinvestigation and repurposing of drug candidates that may have previously been shelved due to off-target effects. A CAP therapeutic consists of 2 components: 1) a tetrazine (Tz)-modified activating agent that is not therapeutically active but is targeted to a disease site; and 2) a systemically administered trans-cyclooctene (TCO)-modified payload (a protodrug) with attenuated activity/toxicity. At the target site, the activating agent selectively and rapidly captures the protodrug via a bioorthogonal click chemistry reaction, followed by local release of the payload. One challenge the CAP platform has encountered is that the low solubility of certain protodrugs limits the feasible dose to subtherapeutic levels. To address this, the Phase I project successfully synthesized a selection of TCO-modified protodrugs carrying different solubilizing groups, including branched amino acids, linear PEG chains, and both positively and negatively charged groups. Solubility of a paclitaxel- based protodrug reached 16 mg/mL in an aqueous formulation buffer; free paclitaxel requires a toxic cremophor/ethanol mixture to be formulated. Solubility of an exatecan protodrug was similarly high, enabling high doses of each protodrug when tested in vivo. The protodrugs were tolerable in mice, and when the exatecan protodrug was tested in a murine tumor model with a biopolymer targeting agent carrying the activator, the tumors were completely eliminated. Phase II aims to further advance the CAP platform and to demonstrate its modularity by undertaking three specific aims: 1) synthesizing and characterizing protodrugs from novel therapeutic classes, 2) confirming in vitro and in vivo efficacy of developed protodrugs with a targeting agent, and 3) un...