ACE2-fused nanobody targeting a cryptic RBD epitope broadly neutralizes SARS-like viruses

The SARS-CoV-2 (SARS2) and SARS-CoV (SARS1) viruses pose significant threats due to their high mutation rates and potential for cross-species transmission, which has rendered many antibodies ineffective. To counter potential future emergences of novel SARS-like viruses, it is imperative to develop nanobodies with broad-spectrum neutralizing capacity. Here, we employed an alternating immunization strategy in alpacas using SARS1 and SARS2 RBDs to generate cross-reactive nanobodies. Four crossing-binding nanobodies (aSR29, aSR196, aSR347, and aSR348) were identified, exhibiting high affinities for SARS1/SARS2 RBDs and Omicron subvariants. Structural analysis revealed that aSR29 targets a cryptic, highly conserved epitope at the inner base of the RBD, a region shielded from conventional antibodies by steric hindrance, efficiently neutralizing SARS1, SARS2, and multiple Variants of Concern (VOCs), including XBB.1.5 and BQ.1.1. Building on this unique binding mode, we engineered a bispecific fusion protein, aSR29-ACE2-Fc. This construct synergistically combines high-affinity RBD binding with the steric blockade of viral attachment, resulting in a 33-fold increase in neutralization potency against SARS2 and enhanced resilience against immune escape. Our findings validate an efficient strategy for eliciting broad-spectrum nanobodies and demonstrate the therapeutic potential of VHH-ACE2 fusion proteins against current and future SARS-like viruses.IMPORTANCEThe continued evolution of SARS-CoV-2 has rendered most existing antibodies ineffective, highlighting the urgent need for broad-spectrum countermeasures against current and future SARS-like viruses. Here, we developed an alternating immunization strategy to generate nanobodies that target a hidden, highly conserved region on the viral spike protein. One such nanobody, aSR29, potently neutralizes diverse variants, including XBB.1.5 and BQ.1.1. By fusing aSR29 with the viral receptor ACE2, we created a bispecific molecule that blocks viral entry through two independent mechanisms, achieving a 33-fold increase in neutralization potency. This work provides a practical and scalable strategy for pandemic preparedness, demonstrating that engineered nanobody-ACE2 fusions can serve as effective broad-spectrum therapeutics against emerging SARS-like viruses.