{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T02:23:18Z","timestamp":1791685398542,"version":"4.3.4"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,6,12]],"date-time":"2025-06-12T00:00:00Z","timestamp":1749686400000},"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><jats:title>Introduction<\/jats:title><jats:p>Advanced hepatocellular carcinoma (HCC) poses significant therapeutic challenges due to chemotherapy resistance and limited efficacy of current targeted therapies. To address this unmet need, we developed a bispecific antibody (BsAb) platform targeting CD16A on natural killer (NK) cells and glypican-3 (GPC3), a tumor-specific antigen overexpressed in 70% of HCC cases.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>High-affinity anti-CD16A single-chain variable fragments (scFvs) were selected via phage display, followed by engineering of Fc-stabilized BsAbs (MA4-hFc(N297A)-CD16A series) to minimize Fc\u03b3R-mediated toxicity. Functional validation included binding kinetics (ELISA, flow cytometry, and fluorescence co-localization analysis), <jats:italic>in vitro<\/jats:italic> cytotoxicity assays (luciferase-based killing), and <jats:italic>in vivo<\/jats:italic> efficacy studies in Huh7 xenograft models. Synergy with sorafenib was assessed using CompuSyn analysis.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The lead candidate, MA4-hFc-CD16AM19, exhibited nanomolar affinity (EC50 &amp;lt; 10 nM for human CD16A) with no murine cross-reactivity. It demonstrated potent, dose-dependent cytotoxicity against GPC3+ HCC lines (HepG2\/Huh7\/Hep3B, IC50 = 15\u201335 ng\/mL) via NK cell activation, surpassing conventional antibody-dependent cellular cytotoxicity (ADCC). Combined with sorafenib, MA4-hFc-CD16AM19 achieved synergistic tumor suppression (CI=0.41). <jats:italic>In vivo<\/jats:italic>, BsAb treatment (5 mg\/kg) significantly inhibited tumor growth in xenograft models, correlating with enhanced intratumoral NK cell infiltration without toxicity.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study introduces three innovations: (1) a species-specific CD16A binder overcoming polymorphism limitations, (2) Fc domain engineering (N297A) to optimize stability and safety, and (3) a synergistic combination strategy with sorafenib. The results provide a translatable framework for GPC3+ solid tumor immunotherapy.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1599764","type":"journal-article","created":{"date-parts":[[2025,6,12]],"date-time":"2025-06-12T05:21:24Z","timestamp":1749705684000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":9,"title":["Targeting CD16A on NK cells and GPC3 in hepatocellular carcinoma: development and functional validation of a therapeutic bispecific 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