{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T20:58:17Z","timestamp":1781729897865,"version":"3.54.5"},"posted":{"date-parts":[[2026,5,20]]},"group-title":"Immunology","reference-count":64,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T00:00:00Z","timestamp":1779235200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"NCI","award":["R01CA247681"],"award-info":[{"award-number":["R01CA247681"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  T-cell engagers (TCEs) for cancer immunotherapy have traditionally relied on high affinity single chain fragment variable (scFv) domains to target CD3, specifically the \u03b5 chain, for the activation of T-cells. Despite their clinical success, there have been reports of TCEs driving systemic toxicity, non-specific T-cell activation, on-target off-tumor effects, and severe inflammation due to cytokine release. To address these limitations, we designed multivalent TCEs using Chemically Self-Assembled Nanorings (CSANs) that target the \u03b1\/\u03b2 constant region of the T-cell receptor (TCR) in the TCR\/CD3 complex using a moderate affinity \u03b1TCR nanobody (\u03b1TCR\n                  <jats:sub>VHH<\/jats:sub>\n                  ). Nanobodies offer superior physical and chemical properties over scFvs-including higher solubility, stability and lower production cost-making them increasingly popular as structural units of TCEs. We compared the efficacy and safety profile of this moderate affinity, nanobody-based TCR binder against high affinity \u03b1CD3\n                  <jats:sub>scFv<\/jats:sub>\n                  based CSANs across EGFR and PSMA expressing solid tumor models. While the \u03b1CD3\n                  <jats:sub>scFv<\/jats:sub>\n                  CSANs offered potent cytotoxicity, they also induced antigen independent T-cell activation bypassing the requirement of tumor crosslinking for cytotoxicity. In contrast the \u03b1TCR\n                  <jats:sub>VHH<\/jats:sub>\n                  CSANs required strict antigen engagement to trigger cytotoxicity, significantly reducing non-specific T-cell activation and thus enhancing the safety profile. Although the initiation of cytotoxicity was kinetically slower than the \u03b1CD3\n                  <jats:sub>scFv<\/jats:sub>\n                  counterpart, \u03b1TCR\n                  <jats:sub>VHH<\/jats:sub>\n                  CSANs achieved comparable end point cytotoxicity across multiple antigen densities, as well as in 3D tumor spheroids. Through this study we demonstrate the applicability of nanobodies as T-cell targeting domains, enhanced specificity and safety of moderate affinity T-cells binders and the diversification of T-cell targeting epitopes without compromising the efficacy of TCEs.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Abstract Graphic<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"725710v2_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.17.725710","type":"posted-content","created":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T21:55:16Z","timestamp":1779314116000},"source":"Crossref","is-referenced-by-count":0,"title":["T-cell Receptor (TCR) Targeting with Multivalent T-cell Engagers"],"prefix":"10.64898","author":[{"given":"Debasmita","family":"Paul","sequence":"first","affiliation":[{"name":"University of Minnesota, Department of Medicinal Chemistry, 2231 6th Street S.E. 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