Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins

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.

Automated summary

Researchers have developed a fully synthetic nanobody library for the treatment of SARS-CoV-2 infection. They generated nanobodies by immunizing camelids and screened and analyzed their structures to find that they can bind to and neutralize SARS-CoV-2. This study provides a blueprint for the development of multispecific molecules.