The ongoing emergence of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the urgent need for developing antivirals targeting both SARS-CoV-2 variants and related sarbecoviruses. To this end, we designed novel trispecific antibodies, Tri-1 and Tri-2, engineered by fusing the single-chain variable fragments (scFvs) of a potent antibody (PW5-570) to the Fc region of "Knob-into-Hole" bispecific antibodies (bsAbs) composed of two distinct broad antibodies (PW5-5 and PW5-535). Compared with the parental antibodies, Tri-1 and Tri-2 displayed enhanced binding affinities to the receptor-binding domains of SARS-CoV, SARS-CoV-2 wild type, and Omicron XBB.1.16, with each arm contributed to the overall enhancement. Furthermore, pseudovirus neutralization assays revealed that Tri-1 and Tri-2 effectively neutralized all tested SARS-CoV, SARS-CoV-2 variants, and related sarbecoviruses (Pangolin-GD, RaTG13, WIV1, and SHC014), demonstrating potency and breadth superior to any single parental antibody. Consistently, Tri-1 and Tri-2 were found to effectively neutralize authentic SARS-CoV and SARS-CoV-2 variants with IC₅₀ values comparable to or better than those of parental antibodies. Taken together, this study highlights the potential effectiveness of Tri-1 and Tri-2 as novel formats for harnessing cross-neutralizing antibodies in the development of multivalent agents to combat both current and future SARS-like coronaviruses.

Keywords: SARS‐CoV‐2; bispecific antibody; broadly neutralizing antibody; coronavirus; sarbecovirus; trispecific antibody.

© 2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.

严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）的新变种不断出现，强调了开发针对 SARS-CoV-2 变异株及相关 sarbecovirus 的抗病毒药物的紧迫性。为此，我们设计了新型三特异性抗体 Tri-1 和 Tri-2，通过将强效抗体 (PW5-570) 的单链可变片段 (scFvs) 融合到由两种不同广谱抗体（PW5-5 和 PW5-535）组成的“Knob-into-Hole”双特异性抗体 (bsAbs) 的 Fc 区域来构建。与亲本抗体相比，Tri-1 和 Tri-2 对 SARS-CoV、SARS-CoV-2 基因型和 Omicron XBB.1.16 的受体结合域表现出增强的结合亲和力，每臂都对整体提升有贡献。此外，假病毒中和试验显示，Tri-1 和 Tri-2 有效中和了所有测试过的 SARS-CoV、SARS-CoV-2 变异株及相关 sarbecovirus（穿山甲-GD、RaTG13、WIV1 和 SHC014），显示出比任何单个亲本抗体更优的效力和广谱性。一致地，Tri-1 和 Tri-2 被发现能够有效中和真正的 SARS-CoV 和 SARS-CoV-2 变异株，其 IC₅₀ 值与亲本抗体相当或更好。总而言之，这项研究突显了 Tri-1 和 Tri-2 作为新型格式的潜在有效性，在开发用于对抗当前和未来 SARS 相关冠状病毒的多价药物方面具有跨中和抗体的应用潜力。

关键词：SARS-CoV-2；双特异性抗体；广谱中和抗体；冠状病毒；sarbecovirus；三特异性抗体。