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  • Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins. Proceedings of the National Academy of Sciences of the United States of America Misson Mindrebo, L., Liu, H., Ozorowski, G., Tran, Q., Woehl, J., Khalek, I., Smith, J. M., Barman, S., Zhao, F., Keating, C., Limbo, O., Verma, M., Liu, J., Stanfield, R. L., Zhu, X., Turner, H. L., Sok, D., Huang, P. S., Burton, D. R., Ward, A. B., Wilson, I. A., Jardine, J. G. 2023; 120 (24): e2216612120


    Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

    View details for DOI 10.1073/pnas.2216612120

    View details for PubMedID 37276407