The ongoing SARS-CoV-2 pandemic continues to pose a significant global health threat, exacerbated by the emergence of variants of concern (VOCs) that evade vaccine-induced immunity and cause breakthrough infections. The Omicron variant, with its numerous spike protein mutations, exemplifies this challenge. While vaccines remain crucial, the need for effective prophylactic and therapeutic drugs to combat VOCs, particularly Omicron, is paramount. Neutralizing antibodies, particularly those targeting the receptor-binding domain (RBD) of the spike protein, offer a promising approach. However, many approved antibodies exhibit diminished or absent activity against emerging VOCs. Nanobodies (Nbs), derived from camelids, present attractive alternatives due to their smaller size, ease of engineering, and potential for lower immunogenicity. While several Nbs targeting SARS-CoV-2 RBD have been identified, they often face challenges in neutralizing VOCs. This study focuses on two hetero-bivalent Nbs, aRBD-2-5 and aRBD-2-7, previously shown to effectively neutralize the wild-type SARS-CoV-2 strain. The goal is to investigate their efficacy against VOCs, including Omicron, through structural analysis and *in vitro* and *in vivo* testing.

The literature extensively covers the challenges posed by SARS-CoV-2 VOCs and the search for effective neutralizing antibodies. Studies highlight the reduced efficacy of many existing antibodies against Omicron. The advantages of nanobodies over conventional antibodies, including their smaller size, ease of production, and potential for reduced immunogenicity, have been well-documented. Numerous research papers describe the identification and characterization of individual nanobodies against SARS-CoV-2 RBD, but many struggle to maintain efficacy against rapidly evolving variants. The development of multivalent nanobodies as a potential solution to overcome this challenge has also garnered significant attention. This study builds upon previous research on hetero-bivalent nanobodies and aims to expand their demonstrated effectiveness against a broader range of SARS-CoV-2 variants.

This study employed a multi-pronged approach combining structural biology, *in vitro* binding and neutralization assays, and *in vivo* animal models. **Structural Analysis:** Crystal structures of aRBD-2-7 in complex with wild-type SARS-CoV-2 RBD and aRBD-5 in complex with monomeric wild-type SARS-CoV-2 RBD were determined using X-ray crystallography. These structures were analyzed to identify the binding epitopes of the individual nanobodies and assess potential steric clashes or competition with ACE2 binding. Superposition analysis with cryo-EM structures of the spike protein in various conformations was performed to understand the binding mode of the hetero-bivalent nanobodies to the intact spike. **Binding Affinity Assays:** Surface plasmon resonance (SPR) was used to determine the binding affinities (K_D) of aRBD-2-Fc, aRBD-5-Fc, and aRBD-2-5-Fc for the RBDs of various SARS-CoV-2 variants, including wild-type, Alpha, Beta, Gamma, Delta, Delta plus, Kappa, Lambda, and Omicron BA.1 and BA.2. Enzyme-linked immunosorbent assay (ELISA) was used to assess the binding of aRBD-7-Fc and aRBD-2-7-Fc. **Neutralization Assays:** A micro-neutralization assay was used to determine the neutralizing activity of the hetero-bivalent nanobodies against authentic Alpha, Gamma, and Kappa variants. Plaque reduction neutralization test (PRNT) evaluated the neutralization of wild-type, Beta, Delta, and Omicron BA.1 variants. HIV-1-based pseudoviruses were used to assess neutralization against Omicron BA.1, BA.1.1, and BA.2. **In Vivo Studies:** Prophylactic protection was evaluated in K18-hACE2 mice challenged with wild-type SARS-CoV-2 and in BALB/c mice challenged with mouse-adapted SARS-CoV-2 (MA10). Prophylactic and therapeutic efficacy of aRBD-2-5-Fc against Omicron BA.1 was assessed in Syrian golden hamsters. Viral RNA copies and infectious virus titers were quantified. Pharmacokinetic profiles of aRBD-2-5-Fc were determined in mice and hamsters. **Data Analysis:** Statistical analysis, including unpaired t-tests with Welch's correction, was used to compare groups. Binding and neutralization data were analyzed using GraphPad Prism.

The crystal structures revealed that aRBD-2 targets a highly conserved epitope on the RBD, explaining its ability to bind to various SARS-CoV-2 variants. In contrast, aRBD-5 and aRBD-7 bind to less-conserved epitopes and show reduced binding to some variants. Importantly, the fusion of aRBD-2 with aRBD-5 or aRBD-7 resulted in hetero-bivalent nanobodies with significantly enhanced binding affinities and neutralization potencies compared to their individual components. Specifically, aRBD-2-5-Fc and aRBD-2-7-Fc potently neutralized all tested authentic and pseudotyped viruses, including wild-type, Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.1.1, and BA.2 variants. *In vivo* studies demonstrated that aRBD-2-5-Fc provided prophylactic protection against wild-type and mouse-adapted SARS-CoV-2 in mice, and prophylactic and therapeutic protection against Omicron BA.1 in hamsters. The pharmacokinetic analysis revealed that aRBD-2-5-Fc exhibited a long half-life in both mice and hamsters. The results suggest that aRBD-2-5-Fc may be a promising therapeutic agent for the prevention and treatment of COVID-19 caused by emerging SARS-CoV-2 VOCs, including Omicron. Comparative analysis highlighted the superior neutralizing potency of aRBD-2-5-Fc against Omicron variants compared to Sotrovimab, a clinically used monoclonal antibody. Notably, aRBD-2-5-Fc exhibited a significantly higher binding affinity for the RBD of several variants than aRBD-2 alone, suggesting a synergistic effect from the fusion with aRBD-5, even when aRBD-5 exhibited limited binding individually.

The findings address the critical need for broad-spectrum antivirals against SARS-CoV-2 variants. The potent and broad-spectrum neutralization activity of the hetero-bivalent nanobodies, particularly aRBD-2-5-Fc, demonstrated in both *in vitro* and *in vivo* studies, underscores their potential as effective therapeutics. The long half-life observed in the pharmacokinetic studies further supports their suitability for clinical applications. The superior neutralization potency of aRBD-2-5-Fc compared to Sotrovimab highlights its potential advantage in addressing the challenge posed by Omicron. The mechanism of enhanced binding affinity through the fusion of aRBD-2 and aRBD-5 warrants further investigation. The development of hetero-bivalent nanobodies using this strategy presents a promising avenue for the development of effective treatments against emerging SARS-CoV-2 variants.

This study demonstrates that hetero-bivalent nanobodies, particularly aRBD-2-5-Fc, exhibit potent and broad-spectrum neutralization of SARS-CoV-2 variants, including Omicron. The *in vivo* data show strong prophylactic and therapeutic efficacy. The long half-life suggests clinical viability. Future research should focus on elucidating the precise mechanism of the synergistic effect between aRBD-2 and aRBD-5 and optimizing the design for further enhanced potency and broader neutralization coverage against future variants. Clinical trials are needed to evaluate the safety and efficacy of aRBD-2-5-Fc in humans.

The study has several limitations. The precise mechanism by which aRBD-5 contributes to the enhanced binding affinity of aRBD-2-5-Fc when fused to aRBD-2 requires further investigation, potentially through structural analysis of complexes with variant RBDs. The assessment of aRBD-2-5-Fc's antiviral effect on Omicron BA.1 in hamsters was conducted at a relatively late time point (4 dpi), when viral loads were already reduced. Earlier time points should be considered in future studies. Further dose optimization studies for aRBD-2-5-Fc in hamsters are also needed.