ジャーナル: Proc Natl Acad Sci U S A / 年: 2023 タイトル: Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins. 著者: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan ...著者: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan Verma / Jingjia Liu / Robyn L Stanfield / Xueyong Zhu / Hannah L Turner / Devin Sok / Po-Ssu Huang / Dennis R Burton / Andrew B Ward / Ian A Wilson / Joseph G Jardine / 要旨: 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 ...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 V3-23 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 V and V 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.