{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,17]],"date-time":"2026-02-17T07:20:27Z","timestamp":1771312827050,"version":"3.50.1"},"reference-count":48,"publisher":"BMJ","issue":"1","license":[{"start":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T00:00:00Z","timestamp":1768867200000},"content-version":"unspecified","delay-in-days":19,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Jiangsu Province Key Research and Develpoment Program","award":["BE2023654"],"award-info":[{"award-number":["BE2023654"]}]},{"DOI":"10.13039\/501100001809","name":"The National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82272811"],"award-info":[{"award-number":["82272811"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Jiangsu Provincial Medical Key Discipline","award":["ZDXK202233"],"award-info":[{"award-number":["ZDXK202233"]}]},{"name":"Nangjing Jiangbei New Area key research and development program","award":["NA"],"award-info":[{"award-number":["NA"]}]},{"name":"Nanjing Medical Key Laboratory of Oncology","award":["NA"],"award-info":[{"award-number":["NA"]}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2025,12,31]]},"published-print":{"date-parts":[[2026,1]]},"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Bispecific T cell engagers have demonstrated promising clinical progress in both hematologic malignancies and solid tumors; however, their therapeutic efficacy is still limited by multiple challenges including T cell anergy resulting from single-signal activation exclusively through CD3 engagement. As a critical co-stimulatory molecule, CD28 enhances T-cell functionality through synergistic interaction with the TCR-mediated primary activation signal, thus potentiating antitumor efficacy. Non-superagonistic CD28 bispecific antibodies, while enhancing antitumor efficacy, can minimize systemic toxicity. Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1), a cancer-testis antigen overexpressed in diverse malignancies, emerges as a promising target for tumor-specific immunotherapy. Herein, the development of a dual-signal T-cell engager strategy targeting KK-LC-1, using a non-superagonist CD28-based co-stimulatory mechanism, is of critical importance.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Based on the successful acquisition of the designed ankyrin repeat proteins targeting KK-LC-1 and CD28 through phage display technology (KD=8.985\u2009nM and 7.43\u2009nM), we designed two T cell engagers (KK-LC-1\u00d7CD3\u2009and KK-LC-1\u00d7CD28). The tumor-specific binding activity of KK-LC-1 designed ankyrin repeat protein was verified by surface plasmon resonance, flow cytometry, confocal microscopy, and in vivo imaging. We then fused it with CD3 single-chain variable fragment and CD28-designed ankyrin repeat protein, respectively, to construct two T cell engagers. Their biological activities and antitumor efficacy were systematically evaluated both in vitro and in vivo (n=5) using flow cytometric analysis, confocal microscopy imaging, and bioluminescence quantification.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      T-cell engagers KK-LC-1\u00d7CD3\u2009and KK-LC-1\u00d7CD28 were successfully engineered and demonstrated high binding affinity for both KK-LC-1-positive tumor cells and T cells. Co-administration of these engagers significantly augmented T-cell activation and antitumor efficacy (88% vs 66%, p&lt;0.001) compared with KK-LC-1\u00d7CD3 monotherapy. In vivo, the combination suppressed tumor growth by 59.6% vs monotherapy (p&lt;0.05) with enhanced intratumoral CD8\n                      <jats:sup>+<\/jats:sup>\n                      infiltration (5.4-fold, p&lt;0.001) and CD4\n                      <jats:sup>+<\/jats:sup>\n                      infiltration (2.7-fold, p&lt;0.001), while triple therapy incorporating PD-1\u00d7CTLA-4 bispecific antibodies extended median survival from 44 to 48 days (p&lt;0.05).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>We validated the feasibility of the KK-LC-1-targeted dual-signal T-cell engager strategy for the treatment of solid tumors and demonstrated that its combination with PD-1\u00d7CTLA-4 bispecific antibodies synergistically enhanced antitumor efficacy in preclinical studies.<\/jats:p>\n                  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