Introduction
Since 2021, Phenotypeca has been collaborating with Isogenica in an IUK-funded project worth up to £400,000 to design, produce, and test therapeutic antibody fragments. Isogenica are experts in developing small antibody fragments (VHHs). During their >20-year history, Isogenica have established successful discovery partnerships with biopharma companies across the globe, as well as research collaborations with other SMEs and academic groups. VHHs have been on the radar of life scientists and medical practitioners for their use in clinical therapeutics and immunodiagnostics since their discovery in llamas and other camelids in the early 1990s, with the first VHH-based therapeutic — caplacizumab — approved in 2019.
Figure 1. Traditional antibody and VHH
Biotherapeutics are most commonly manufactured in mammalian systems due to their complexity. These molecules, such as conventional antibodies or cytokines, contain multiple domains and often require distinct glycosylation patterns. VHHs are approximately 1/10th the size of conventional antibodies and generally do not require glycosylation, making them more straightforward to manufacture in microbial systems.
While these molecules can be produced in Escherichia coli for early-stage research, the presence of endotoxins makes this material unsuitable for later-stage assays, while mammalian cell production systems are often prohibitively expensive, and it is challenging to increase throughput. Caplacizumab is manufactured in the yeast Pichia pastoris. However, this yeast strain is fed by methanol, which both has toxicity risks and poses fire hazards during large-scale manufacture. This presents a market opportunity for alternative bioproduction hosts with low cost of goods.
Phenotypeca has the world’s largest unique collection of Saccharomyces cerevisiae strains bred and engineered for stable recombinant protein production. By transitioning early R&D to a final manufacturing host, drug discovery can select for high-producing clones sooner and eliminate future issues during manufacturing scale-up, such as those encountered with other microbes or mammalian cells. With this in mind, Isogenica tasked Phenotypeca with investigating whether high-quality, functional VHH proteins could be produced, secreted, and displayed on the surface of a S. cerevisiae yeast cell.
Approach
The first task involved verifying that Phenotypeca’s unique collection of strains could be modified to successfully produce a range of VHH proteins. Expression of these proteins was confirmed in several different yeast strains (Figure 2), showing a very clean homogeneous product even in un-purified culture supernatant (Figure 3). The absence of significant host-derived protein in these samples means that the product can be purified without the need for immunogenic tags or expensive affinity resins, further reducing cost of goods. In addition, it showed that Phenotypeca’s strains could also produce multi-target (bi-specific) VHH antibodies, a particular challenge for E. coli systems.
Figure 2. Phenotypeca’s strain collection: Expression levels of VHHs in yeast strains from Phenotypeca’s diverse library were assessed using fluorescence, allowing the best strains to be picked for further development.
Figure 3: VHHs were secreted by yeast into culture media, which has been visualised here using SDS-PAGE, showing high levels of purity even before purification.
Phenotypeca then sought to improve the production and secretion of these VHHs in various yeast strains, as well as developing a method to successfully display these molecules on the surface of the yeast cell (Figure 4).
Figure 4: VHHs were expressed with fluorescent tags, and surface display was determined by assessing whether the fluorescent signal was retained on solid culture media after the cells were washed away. The fluorescent patches in the middle of the second plate show where cells grew that were secreting product, whereas no fluorescence is retained where the VHHs were displayed on the surface of the cells (top and bottom). This was also confirmed by flow cytometry and ELISA-based studies showing antibody binding to the surface displayed VHHs.
Results
Phenotypeca successfully produced several different VHHs, both secreted and displayed, in a range of engineered yeast strains, improving secretion levels from 10- to 20-fold for a variety of antibody fragments. In addition, Isogenica has been able to confirm that VHH molecules produced by Saccharomyces at Phenotypeca are fully active and able to bind to target antigens, with affinities comparable to antibody fragments produced in E. coli (Figure 5).
Figure 5: ELISA showing equivalent target binding of anti-PD-L1 VHHs, produced using E. coli and Phenotypeca’s Saccharomyces system, including a bi-specific variant.
Conclusion
Phenotypeca’s platform has paved the way for the creation of a multi-functional toolbox for VHH discovery and production. This will lead to a reduction in time and cost of producing high-quality, functional VHHs when compared to production in E. coli and mammalian systems, and improved safety both for manufacturing and for patients compared with production in Pichia.
