A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529).

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have risen to dominance, which contains far more mutations in the spike protein in comparison to previously reported variants, compromising the efficacy of most existing vaccines or therapeutic monoclonal antibodies. Nanobody screened from high-throughput naïve libraries is a potential candidate for developing preventive and therapeutic antibodies. Methods: Four nanobodies specific to the SARS-CoV-2 wild-type receptor-binding domain (RBD) were screened from a naïve phage display library. Their affinity and neutralizing activity were evaluated by surface plasmon resonance assays, surrogate virus neutralization tests, and pseudovirus neutralization assays. Preliminary identification of the binding epitopes of nanobodies by peptide-based ELISA and competition assay. Then four multivalent nanobodies were engineered by attaching the monovalent nanobodies to an antibody-binding nanoplatform constructed based on the lumazine synthase protein cage nanoparticles isolated from the Aquifex aeolicus (AaLS). Finally, the differences in potency between the monovalent and multivalent nanobodies were compared using the same methods. Results: Three of the four specific nanobodies could maintain substantial inhibitory activity against the Omicron (B.1.1.529), of them, B-B2 had the best neutralizing activity against the Omicron (B.1.1.529) pseudovirus (IC50 = 1.658 µg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC50 = 0.653 µg/mL). The results of peptide-based ELISA indicated that LS-B-B2 might react with the linear epitopes in the SARS-CoV-2 RBD conserved regions, which would clarify the mechanisms for the maintenance of potent neutralization of Omicron (B.1.1.529) preliminary. Conclusion: Our study indicated that the AaLS could be used as an antibody-binding nanoplatform to present nanobodies on its surface and improve the potency of nanobodies. The multivalent nanobody LS-B-B2 may serve as a potential agent for the neutralization of SARS-CoV-2 variants.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment.

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future. Systematic Review Registration.