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We developed a modular plug-and-play approach for the generation of ADEPT antibody\u2013enzyme conjugates that pairs a self-labeling SNAPtag-enzyme fusion with a benzylguanine-tagged antibody for rapid generation of a series of conjugates for testing. This approach enables the fusion of pharmacy-grade antibodies that are site-specifically modified with a recombinantly expressed enzyme using bioorthogonal self-labeling chemistry. The biorthogonality of the final assembly step of the ADEPT system even provides the ability for antibody\u2013enzyme pairing in situ in the target cellular environment, improving ease of testing and potentially improving in vivo pharmokinetics. We have demonstrated conjugate formation and localization to antigen-positive cells, retained enzymatic activity upon conjugate cell surface binding, and ultimately selective killing of tumor cells. Additionally, this modular assembly allowed us to construct conditionally controlled ADEPT systems through site-specific incorporation of a light-cleavable protecting group, pairing a caged enzyme with a caged prodrug to create Boolean AND logic, potentially increasing the therapeutic window by providing conditional control over cytotoxic drug activation.<\/jats:p>","DOI":"10.1021\/acschembio.6c00539","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T09:01:19Z","timestamp":1790845279000},"page":"2725-2733","source":"Crossref","is-referenced-by-count":0,"title":["Modular Assembly of Systems for Antibody-Directed Enzyme Prodrug Therapy"],"prefix":"10.1021","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7186-4773","authenticated-orcid":true,"given":"Maura E.","family":"Charette","sequence":"first","affiliation":[{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]},{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6852-8489","authenticated-orcid":true,"given":"Madelyn H.","family":"Ratesic","sequence":"additional","affiliation":[{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]},{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gianna N.","family":"Falcone","sequence":"additional","affiliation":[{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]},{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grace V.","family":"Lewis","sequence":"additional","affiliation":[{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]},{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella R.","family":"Demyan","sequence":"additional","affiliation":[{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]},{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sagar D.","family":"Khare","sequence":"additional","affiliation":[{"name":"Rutgers University , , , ,","place":["Piscataway, New Jersey, United States, 08854"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0234-9209","authenticated-orcid":true,"given":"Alexander","family":"Deiters","sequence":"additional","affiliation":[{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]},{"name":"University of Pittsburgh , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15260"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,1]]},"reference":[{"key":"2026101523064803000_cit1","doi-asserted-by":"publisher","first-page":"1378","DOI":"10.1038\/sj.bjc.6690221","article-title":"Bystander Killing of Tumour Cells by Antibody-Targeted Enzymatic Activation of a Glucuronide Prodrug","volume":"79","author":"Cheng","year":"1999","journal-title":"Br. 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