OPENPichia: licence-free Komagataella phaffii chassis strains and toolkit for protein expression.

The industrial yeast Komagataella phaffii (formerly named Pichia pastoris) is commonly used to synthesize recombinant proteins, many of which are used as human therapeutics or in food. However, the basic strain, named NRRL Y-11430, from which all commercial hosts are derived, is not available without restrictions on its use. Comparative genome sequencing leaves little doubt that NRRL Y-11430 is derived from a K. phaffii type strain deposited in the UC Davis Phaff Yeast Strain Collection in 1954. We analysed four equivalent type strains in several culture collections and identified the NCYC 2543 strain, from which we started to develop an open-access Pichia chassis strain that anyone can use to produce recombinant proteins to industry standards. NRRL Y-11430 is readily transformable, which we found to be due to a HOC1 open-reading-frame truncation that alters cell-wall mannan. We introduced the HOC1 open-reading-frame truncation into NCYC 2543, which increased the transformability and improved secretion of some but not all of our tested proteins. We provide our genome-sequenced type strain, the hoc1tr derivative that we named OPENPichia as well as a synthetic, modular expression vector toolkit under liberal end-user distribution licences as an unencumbered OPENPichia resource for the microbial biotechnology community.

An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2­neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti­COVID-19 biologic that is now being evaluated in the clinic.

Customized protein glycosylation to improve biopharmaceutical function and targeting.

For a long time, glycoprotein production has been limited by the inherent properties of production hosts. Glycosylation of biopharmaceuticals has been regarded as a necessary evil, often needed for protein folding or function, but also a source of heterogeneity, complicating downstream processing and product characterization. This has strongly determined the choice of production hosts. Over the last few decades, numerous glycoengineering efforts have helped solving this problem. Moreover, insights from fundamental studies have made it possible to improve therapeutic protein functionality through careful glycoengineering. Here, we will focus on how production host and in vitro glycoengineering approaches allow to design biopharmaceuticals with glycans that impart improved functionality. An important branch of research explores how glycosylation can be tuned to improve pharmacokinetics and reduce glycan heterogeneity of therapeutics. Furthermore, antibody glycoengineering to obtain homogeneous, defined glycan structures has been a major focus. An example of this is the production of Fc glycans without core fucose, exhibiting tremendously improved Antibody-Dependent Cell Cytotoxicity (ADCC). In the last part, glycoforms that allow for improved (subcellular) targeting and cellular uptake, a field that opens possibilities for enzyme replacement therapies and vaccine development, will be highlighted.