Ma Yunping, Liu Qianying, Rehati Palizhati, Zhang Zhaoyong, Wang Yingdan, Liu Zhimin, Chen Jiali, Bai Xuezhou, Hao Aihua, Wang Yanqun, Wen Yumei, Chen Zhenguo, Lu Lu, Zhao Jincun, Wu Fan, Sun Lei, Huang Jinghe
Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Fifth People's Hospital, Institutes of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Shanghai Immune Therapy Institute, Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital, Shanghai, China.
iScience. 2025 Jul 22;28(8):113175. doi: 10.1016/j.isci.2025.113175. eCollection 2025 Aug 15.
The emergence of SARS-CoV-2 variants like KP.2 and KP.3.1.1 challenges COVID-19 countermeasures. Bispecific antibodies (bsAbs) targeting two antigens show promise, but the impact of scFv sequence arrangement on functionality remains unclear. Using neutralizing mAbs 7F3 (Omicron XBB.1-specific) and GW01 (cross-neutralizing), we engineered four bsAbs with diverse scFv configurations. Despite similar RBD binding affinities, only G7-Fc neutralized all 22 tested pseudoviruses, including KP.2, with 18-fold enhanced potency over S309. Cryo-EM revealed that G7-Fc uniquely stabilizes a BA.2.86 spike trimer-dimer apex-to-apex configuration, blocking RBD-ACE2 interaction via two conserved epitopes distinct from S309. Structure-guided A227S/T and Y229N mutations restored G7-Fc activity against KP.3.1.1. In hACE2-transgenic mice, G7-Fc demonstrated robust prophylactic and therapeutic efficacy against EG.5. This study systematically evaluates how scFv positioning affects bsAb performance, demonstrating rapid antibody optimization through structural insights. G7-Fc's unique trimer-dimer engagement and conserved epitope targeting provide a framework for designing potent, variant-resistant bsAbs against emerging SARS-CoV-2 strains.
像KP.2和KP.3.1.1这样的SARS-CoV-2变体的出现对COVID-19应对措施构成了挑战。靶向两种抗原的双特异性抗体（bsAbs）显示出前景，但单链抗体片段（scFv）序列排列对功能的影响仍不清楚。利用中和单克隆抗体7F3（奥密克戎XBB.1特异性）和GW01（交叉中和），我们构建了四种具有不同scFv构型的双特异性抗体。尽管受体结合域（RBD）结合亲和力相似，但只有G7-Fc能中和所有22种测试的假病毒，包括KP.2，其效力比S309增强了18倍。冷冻电镜显示，G7-Fc独特地稳定了一种BA.2.86刺突三聚体-二聚体顶点对顶点的构型，通过两个不同于S309的保守表位阻断RBD与血管紧张素转换酶2（ACE2）的相互作用。结构引导的A227S/T和Y229N突变恢复了G7-Fc对KP.3.1.1的活性。在人ACE2转基因小鼠中，G7-Fc对EG.5表现出强大的预防和治疗效果。本研究系统评估了scFv定位如何影响双特异性抗体的性能，通过结构见解证明了抗体的快速优化。G7-Fc独特的三聚体-二聚体结合和保守表位靶向为设计针对新兴SARS-CoV-2毒株的强效、抗变体双特异性抗体提供了框架。
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