靶向自然杀伤细胞上的CD16A和肝细胞癌中的GPC3：一种治疗性双特异性抗体的研发与功能验证

Targeting CD16A on NK cells and GPC3 in hepatocellular carcinoma: development and functional validation of a therapeutic bispecific antibody.

作者信息

Chen Liu, Zhu Yuankui, Feng Mingqian, Zuo Dianbao, Chen Guoping, Ji Kangkang

机构信息

College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China.

College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, Hubei, China.

出版信息

Front Immunol. 2025 Jun 12;16:1599764. doi: 10.3389/fimmu.2025.1599764. eCollection 2025.

DOI:10.3389/fimmu.2025.1599764

PMID:40574860

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12198245/

Abstract

INTRODUCTION

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), cytotoxicity assays (luciferase-based killing), and efficacy studies in Huh7 xenograft models. Synergy with sorafenib was assessed using CompuSyn analysis.

RESULTS

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). , BsAb treatment (5 mg/kg) significantly inhibited tumor growth in xenograft models, correlating with enhanced intratumoral NK cell infiltration without toxicity.

CONCLUSION

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.

摘要

引言

晚期肝细胞癌（HCC）由于化疗耐药性和当前靶向治疗的疗效有限，带来了重大的治疗挑战。为了满足这一未被满足的需求，我们开发了一种双特异性抗体（BsAb）平台，该平台靶向自然杀伤（NK）细胞上的CD16A和磷脂酰肌醇蛋白聚糖-3（GPC3），GPC3是一种在70%的HCC病例中过表达的肿瘤特异性抗原。

方法

通过噬菌体展示选择高亲和力抗CD16A单链可变片段（scFv），随后对Fc稳定化的BsAb（MA4-hFc(N297A)-CD16A系列）进行工程改造，以最小化FcγR介导的毒性。功能验证包括结合动力学（酶联免疫吸附测定、流式细胞术和荧光共定位分析）、细胞毒性测定（基于荧光素酶的杀伤）以及在Huh7异种移植模型中的疗效研究。使用CompuSyn分析评估与索拉非尼的协同作用。

结果

主要候选药物MA4-hFc-CD16AM19表现出纳摩尔亲和力（对人CD16A的EC50 < 10 nM），且无小鼠交叉反应性。它通过激活NK细胞对GPC3+ HCC细胞系（HepG2/Huh7/Hep3B，IC50 = 15 - 35 ng/mL）表现出强效的、剂量依赖性细胞毒性，超过了传统的抗体依赖性细胞毒性（ADCC）。与索拉非尼联合使用时，MA4-hFc-CD16AM19实现了协同肿瘤抑制（CI = 0.41）。此外，BsAb治疗（5 mg/kg）在异种移植模型中显著抑制肿瘤生长，这与肿瘤内NK细胞浸润增强相关且无毒性。