Yeast-based production platform for potent and stable heavy chain-only antibodies

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast. ### Competing Interest Statement C.L., H.E., K.R., B.S., S.D.C., D.F., W.N., J.N., X.S., L.v.S., and N.C. are named as inventors on patent application Coronavirus Binders (WO 2021/156490 A2), published on 12 August 2021. C.L., B.S., W.W., L.v.S. and N.C. are named as inventors on patent application Engineered stabilizing aglycosylated fc-regions (WO 2023/148397 A1), published on 10 August 2023. X.S. and N.C. are scientific founders of and consultants for ExeVir Bio and are in receipt of ExeVir Bio share options. All other authors declare that they have no competing interests.