Characterization of bispecific antibodies using structural mass spectrometry and imaging techniques

The majority of drugs currently in development are biotherapeutics, i.e. based on proteins, antibodies or other biomolecules. They promise unsurpassed specificity and selectivity in how they target molecules or structures in the cell, but can also carry cargo to their destination. Bispecific antibodies constitute a promising biotherapeutic modality with increasing presence in clinical development and several approved drugs. Bispecific antibodies combine the specificities of two or more antibodies and engage different epitopes. This multi-target functionality makes them especially appealing as biotherapeutics.
Data from a library of bispecific antibodies generated at the industrial partner (MedImmune, Cambridge) suggests that the relative potency and pharmacokinetics of bispecific antibodies are dependent on the chosen scaffold, but remain difficult to predict. Moreover, these molecules are challenging to develop as therapeutic drugs due to the high propensity to aggregation and lower thermal stability. This PhD project is designed to use emerging structural biology techniques to better understand the impact of bispecific engineering strategies on their structure and dynamics whilst evaluate the utility of these techniques for future use.
Structural Mass spectrometry based techniques such as native MS combined with ion mobility, hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP, a novel technique unique to Leeds) followed by high resolution LC-MS/MS analysis provide a unique opportunity to investigate the characteristics of these molecular formats. In this study we propose to compare solvent accessibility differences with FPOP, with particular focus in the scFv addition. This understanding can not only help identify unstable or prone aggregation regions but potentially also shed light on target binding affinity differences.
Understanding relative domain stability can be derived from native MS in combination with ion mobility. The relative collisional cross sections, population distribution and stability after low energy collisional energy of the different bispecific formats can be used to gain molecular understanding of their differences in solution. Secondary and tertiary structural information gained from the above studies will be complemented by quaternary structural analyses using imaging techniques such as optimized negative-staining electron microscopy and individual-particle electron tomography, taking advantage of the world-class Titan Krios equipment available at Leeds. Together these studies will seek to describe changes in domain structure and dynamics across formats.
The availability of a well characterized library of bispecific formats in combination with structural MS and imaging techniques provide a unique opportunity to help elucidate differences in the relative stability and surface properties of bispecific antibodies.