{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T06:04:35Z","timestamp":1790229875659,"version":"4.1.0"},"reference-count":41,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>TCR-mimetic CAR-T combines antibody-based targeting with physiological TCR signaling, yet suboptimal co-stimulation and safety risks remain key barriers for solid tumor therapy. The prevailing assumption that stronger co-stimulation yields superior efficacy has not been systematically tested in TCR-mimetic CAR-T architectures.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We developed a Mesothelin-targeted scFv-CD3\u03f5 TCR-mimetic CAR-T platform and systematically compared co-stimulatory modules\u20144-1BB, BRM-truncated 4-1BB\n                      <jats:sup>\u0394BRM<\/jats:sup>\n                      , and the 80BB chimera\u2014alongside CD3\u03b6 overexpression. To define the requirements for toxicity, we generated a series of control constructs and compared SS1 versus MOR-based scFvs targeting the same antigen, which differ in their binding properties including affinity.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The optimized SS1-\u03f5\u03b6-80BB TCR-mimetic CAR-T exhibited potent anti-tumor activity but unexpectedly induced dose-dependent lethal toxicity. Systematic controls identified the intact 80BB chimera as the construct associated with lethality. Deleting the 4-1BB intracellular domain, rerouting the 4-1BB intracellular domain to an antigen-dependent format, or replacing SS1 with MOR each abrogated toxicity while preserving anti-tumor efficacy. The SS1-\u03f5\u03b6-80BB architecture was associated with excessive T-cell expansion and multi-organ infiltration while the MOR-\u03f5\u03b6-80BB architecture was moderate.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>These observations suggest that the mode of co-stimulatory configuration, rather than its mere presence, influences safety outcomes in TCR-mimetic CAR-T cells. Modifying either the affinity-related properties or the 80BB-based architecture preserved efficacy while reducing toxicity, providing potential design considerations for affinity selection and co-stimulator design in the mesothelin-targeted TCR-mimetic CAR-T platform.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1947351","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T05:44:02Z","timestamp":1790228642000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Affinity and costimulatory architecture-associated toxicity in mesothelin-targeted TCR-mimetic CAR-T cells"],"prefix":"10.3389","volume":"17","author":[{"given":"Wei","family":"Du","sequence":"first","affiliation":[{"name":"Nanjing CART Medical Technology Co. Ltd.","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siqi","family":"Li","sequence":"additional","affiliation":[{"name":"Clinical Pathological Diagnosis & Research Center, Youjiang Medical University for Nationalities","place":["Baise, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaomei","family":"Gan","sequence":"additional","affiliation":[{"name":"Clinical Pathological Diagnosis & Research Center, Youjiang Medical University for Nationalities","place":["Baise, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing CART Medical Technology Co. Ltd.","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Lu","sequence":"additional","affiliation":[{"name":"Nanjing CART Medical Technology Co. 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