Picking a winner - developing effective AND manufacturable antibody therapeutics by directed evolution

Background

Antibody-based therapeutics are revolutionising medicine but their production and formulation can be problematic, jeopardising successful development. For example, protein self-association or aggregation results in major hurdles which must be overcome for the translation of a promising candidate to a blockbuster bio-therapeutic and can result in the failure of promising candidate biologics during pre-clinical development. This lack of understanding of the relationship between sequence and aggregation will also present a significant hurdle to the manufacture of more complex “next generation” biopharmaceuticals such as bispecifics that are showing great promise in the field of oncology.

To address this unmet need, the team at Leeds, together with long term collaborators at AstraZeneca, have developed an in vivo bacterial aggregation screen (Saunders et al. Nat Chem Biol 2016, Ebo et al. Nature Commun 2020) as both a tool for fundamental understanding and to efficiently re-design problematic candidate sequences. Our assay called the tripartite beta-lactamase assay (TPBLA) is able to identify candidate sequences which are resistant to aggregation and therefore more likely to pass the development process. In addition to this screening function the method can improve aggregation prone sequences using directed evolution and identify the precise residues that lead to poor bio-physical properties by generation of mutation hotspot profiles.

In this studentship we plan to build on this success by increasing the applicability of the BPBLA to the biopharmaceutical development pipeline and to increase its utility for fundamental research.

Aim 1: we will adapt the TPBLA to allow selection of aggregation resistant sequences while maintaining target binding affinity. This will be achieved by introducing one half of a split fluorescence reporter protein to the beta-lactamase_candidate fusion and the remaining portion of the fluorescence reporter to the target antigen. Re-designed candidates with optimal aggregation resistance AND target binding will be identified by sequential screening by ampicillin resistance and fluorescence sorting.

Aim 2: the ability to probe the effects of sequence on target affinity and protein aggregation/stability opens the door to exploration of the relationship between specificity and avidity and stability/affinity trade-offs, furthering our ability to identify sub-optimal sequences early in development. As these relationships are very complex, key to success is the ability to generate very large datasets which can be subjected to modern data analysis methods.  A large dataset will be generated by a deep mutational sequencing approach to the TPBLA whereby the “fitness” of each of thousands of individual variants is quantified by next generation sequencing using antibiotic resistance and fluorescence as the fitness parameters.

The project builds on strong on-going collaborations between the academic supervisors, who have a track record of successful student supervision of industry-facing projects (two PhD students graduated and one submitted with AstraZeneca as industrial partner).

Training

The supervisors’ different skillsets, and the excellent facilities and opportunities at AstraZeneca provide a superb training environment for the student.

You will be trained in a wide range of techniques covering modern protein science including molecular biology, phenotype screening, protein expression and biophysical characterisation. The Astbury Centre for Structural Molecular Biology at the University of Leeds brings together researchers from across the University – largely from the biological sciences, chemistry and physics – to allow interdisciplinary approaches to be harnessed to understand the molecular basis of life. The Centre has outstanding expertise and research infrastructure in chemical biology, biophysics and all of the major techniques in structural molecular biology. Together, these approaches are combined with analyses of biological function with the ultimate aim of understanding the molecular basis of biological mechanisms in living cells. This PhD project is part of a Collaborative Training Partnership (CTP) between AstraZeneca (Cambridge) and the University of Leeds.