ORIGINAL RESEARCH article
Front. Immunol.
Sec. Cancer Immunity and Immunotherapy
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1599764
This article is part of the Research TopicImmunology and Therapeutic Innovations in Hepatocellular Carcinoma: Exploring Immune Evasion and BeyondView all 3 articles
Targeting CD16A on NK Cells and GPC3 in Hepatocellular Carcinoma: Development and Functional Validation of a Therapeutic Bispecific Antibody
Provisionally accepted
- 1Huazhong Agricultural University, Wuhan, Hubei Province, China
- 2Xiang Yang No.1 People’s Hospital, Xiangyang, China
- 3Yancheng City No. 6 People's Hospital, Yancheng, China
- 4Binhai County People's Hospital, Yancheng, China
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Background: 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.Methods: 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 FcγR-mediated toxicity. Functional validation included binding kinetics (ELISA, flow cytometry, and fluorescence co-localization analysis), in vitro cytotoxicity assays (luciferase-based killing), and in vivo efficacy studies in Huh7 xenograft models. Synergy with sorafenib was assessed using CompuSyn analysis.The lead candidate, MA4-hFc-CD16AM19, exhibited nanomolar affinity (EC50 < 10 nM for human CD16A) with no murine cross-reactivity. It demonstrated potent, dose-dependent cytotoxicity against GPC3+ HCC lines (HepG2/Huh7/Hep3B, IC50=15-35 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). In vivo, BsAb treatment (5 mg/kg) significantly inhibited tumor growth in xenograft models, correlating with enhanced intratumoral NK cell infiltration without toxicity.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.
Keywords: bispecific antibody, NK cell engager, CD16A, Glypican-3, Hepatocellular Carcinoma
Received: 25 Mar 2025; Accepted: 28 May 2025.
Copyright: © 2025 Chen, Yuankui, Zuo, Feng, Guo and Ji. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Lin Guo, Yancheng City No. 6 People's Hospital, Yancheng, China
Kangkang Ji, Binhai County People's Hospital, Yancheng, China
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