(357n) Engineering Saccharomyces Cerevisiae to Mimic B Cell Antibody Diversification for the Rapid Enhancement and Selection of Antibody Therapeutics.

Antibody therapeutics represent a rapidly growing market within the pharmaceutical industry, with annual revenue estimated to reach $300 billion by 2025.¹ The success of the antibody market is owed to the remarkable number of diseases that can be treated by this class of drug, including autoimmune diseases, viral infections, and cancer. Antibody candidates are discovered from large libraries of distinct antibody sequences, but it is often laborious and slow to generate and screen this diversity. B cells in the human body naturally create antibody diversity through two main processes: somatic hypermutation and V(D)J recombination, but they are not easily amenable to cellular engineering. Inspired by the elegance of these two processes, we have sought to recapitulate B cell antibody diversification in a more tractable organism, Saccharomyces cerevisiae. Yeast serve as an ideal platform for this purpose. They are easily engineered to express human proteins and antibody libraries can be quickly screened using yeast display, where yeast express antibodies on their cell surface. Our efforts to create yeast that behave like B cells can be divided into three aims 1) developing a CRISPR base editor that can precisely mutate a targeted antibody sequence to mimic somatic hypermutation 2) engineering yeast to efficiently undergo V(D)J recombination using human proteins and 3) optimizing yeast display to interface with our diversifying system (Figure 1).

We will highlight advancements made in each of these three aims. In aim 1, we have designed a base editor which employs CRISPR technology to target a gene with a deaminase that intensely mutates the desired locus with minimal off-target effects. We will show how a variety of engineering approaches have increased the mutation rate of our base editor at least 14-fold. In aim 2, we have identified several human proteins necessary to carry out V(D)J recombination and integrated them into yeast. We will describe how we have used fluorescent microscopy to optimize the expression and localization of these proteins. Further, we will outline a novel assay we have developed to test for recombination events in yeast. In aim 3, we will show how our base editor can be combined with yeast display to rapidly isolate an improved antibody. We envision a future where our engineered yeast are able to sufficiently imitate human B cells such that they are routinely applied to discover novel antibody therapeutics.

1. Lu, R.-M. et al. Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science 27, 1 (2020).

Research Interests:

I am interested in applying synthetic biology for the advancement of human health and wellbeing. Given my experience in antibody engineering through yeast display, I am well equipped to work on antibody therapeutics in industry. However, I would be a strong asset to any cellular or protein engineering project using either microbial or mammalian cells. I have developed many valuable skills including molecular cloning, microscopy, flow cytometry, and next-generation sequencing. Therefore, I am confident I could make contributions in a variety of areas, from designing cellular therapies to optimizing microbial factories. Lastly, through my research I have a deep understanding of CRISPR technology and hope to implement these powerful systems in my work.