Design of ModProtease through High-Throughput Screening Technologies for Therapeutic Applications

The integration of ModProtease with antibody-based systems represents a promising strategy for targeted cancer therapy, particularly in addressing the challenges posed by KRAS mutations. These mutations are notoriously difficult to target directly with antibodies due to their involvement in intracellular signalling pathways and the development of resistance mechanisms. Strategies to overcome these limitations could include the development of protease-antibody conjugates and these conjugates might be directed to cleave specific substrates within tumour microenvironment, thereby enhancing the therapeutic effect of the antibody while minimizing off-target effects. However, engineered proteases remain underrepresented amongst protein-based biopharmaceuticals. This situation is due to fundamental challenges that exist in redirecting the substrate specificity of proteases. We propose a high-throughput screening platform that employs monoclonal hydrogel microbeads containing target substrates with FRET-based detection systems allowing for the precise identification of protease activity. This enables the rapid evaluation of various ModProtease variants and their effectiveness when conjugated to antibodies. The interplay of the SpyCatcher-SpyTag system enables the efficient and specific conjugation of ModProteases to antibodies, facilitating the creation of potent antibody-protease. Following high-throughput screening using fluorescence-activated bead sorting (FABS), the platform facilitates the recovery and analysis of protease genotypes from the microbeads. The group of Prof. Hollfelder (University of Cambridge) has substantial expertise in high-throughput methods and regarding the biology of proteases and protein binders. These conditions make the Host laboratory ideal for this project and will provide critical insights and technical support. This project will also collaborate with AstraZeneca, and targeted proteolysis against cancer cells will be assessed.