Overview of Research
Our research is directed towards taking an interdisciplinary approach to generate effective therapeutics for cancer and autoimmune disease. This involves a combination of antibody/protein engineering, fluorescence microscopy and in vivo studies in mice. These studies are funded by the Wellcome Trust and CRUK. Broadly, the work can be described as follows:
Antibody Engineering for Tumor Targeting
We have ongoing projects in the area of tumour targeting as follows:
- The generation of engineered antibodies to generate antibody-drug conjugates that more efficiently deliver their cytotoxic payload to tumour cells.
- The use of state-of-the-art microscopy methods to understand how tumour targets traffic within cells, and how their trafficking is affected by (therapeutic) antibodies that recognise them.
- The development of engineered antigen-delivery vehicles to elicit anti-tumour immunity.
Our overall goal in this area is to use a combination of mechanistic studies and protein engineering to produce a new generation of biologics for the treatment of cancer.
Engineering Antibodies to Treat Autoimmune Disease
A major interest of our laboratory is to investigate the mechanisms that regulate the transport and concentrations of IgG at different sites in the body. This has significance to understanding how an effective humoral response develops, which in turn relates to multiple aspects of human health (autoimmunity, immunodeficiency, pathogen resistance etc.). The successful use of diagnostic and therapeutic antibodies also depends, in part at least, on understanding the factors that regulate their distribution and persistence.
An area of interest in the laboratory is to develop engineered antibodies that are altered in their binding properties for FcRn with the goal of altering antibody dynamics in vivo. Earlier work in our laboratory involved the generation of antibodies that have longer in vivo persistence and transport better across cellular barriers. Such "half-life extended" antibodies are being developed for use as therapeutics in the biopharma industry. A second class of engineered antibodies that we have developed is called Abdegs, for "antibodies that enhance IgG degradation". Abdeg delivery results in the lowering of IgG levels; the licensing of Abdeg technology to the biopharma company, argenx, has resulted in the translation of an Abdeg called Efgartigimod, to the clinic, with the recent successful completion of phase 3 trials. As a follow up to Abdeg technology, we have also developed engineered Fc fusions, called Seldegs, that selectively degrade antibodies of defined specificities.
The development of approaches to engineer antigen delivery vehicles to induce immunological tolerance in a mouse model of multiple sclerosis is currently an area of active investigation. These studies involve the engineering of antibody Fc fragment-antigen fusion proteins with different intracellular trafficking properties and dynamic behaviour, with the overall aim of correlating these properties with immunological outcome.
Image Analysis and Single Molecule Microscopy
An important component of our work relates to the development of methodology for image analysis for cellular microscopy. Special emphasis is being placed on the development of image analysis approaches for single molecule microscopy, which allows the properties of individual (protein) molecules to be studied. Due to the low signal-to-noise ratio that is characteristic of fluorescence microscopy and the quantum limited nature of the detection process, this area presents novel problems of both a theoretical and experimental nature.
A central component of this work has involved an investigation of the accuracy with which a single molecule can be localised using a fluorescence microscope. We have also derived a new resolution criterion for two point sources. This new resolution measure overcomes several deficiencies of classical criteria such as Rayleigh’s criterion and has been validated in experimental single molecule studies. Ongoing efforts include the development of parameter estimation algorithms that are of importance for the tracking of fluorescently or quantum dot labelled single molecules in, for example, tubules and vesicles within cells. A fundamental aspect of our work has been to incorporate parameter estimation problems in fluorescence microscopy
into a well-founded analytical framework.
To be able to carry out advanced fluorescence microscopy experiments we have invested a substantial amount of effort into the building of high performance microscopy imaging stations. Each of the workstations is equipped with several laser lines and multiple cameras that allow the rapid, simultaneous imaging of different fluorophores in individually selectable combinations of widefield and total internal reflection excitation.
We are also actively continuing the development of our imaging approach in which different focal planes can be imaged simultaneously to build up three dimensional, dynamic images of cells. Combining this imaging approach with a multi-color labelling strategy of the cellular proteins allows us, for example, to simultaneously investigate processes on the cell surface using the high sensitivity of total internal reflection microscopy together with the intracellular events that correlate with the membrane events. This has, for example, allowed us for the first time to visualise the trafficking pathways from intracellular sorting endosomes to the plasma membrane (exocytosis) and from the plasma membrane to sorting endosomes (endocytosis).
Software development is another active component in our work. Novel microscopy technologies, such as fast and high sensitivity imaging detectors generate new challenges for software design, not least of which is the large amount of data that is being produced. For example, due to the lack of appropriate software, biologists often spend an extraordinary amount of time analysing acquired imaging data. We are therefore developing software that allows for the efficient acquisition, processing and analysis of the acquired data. (See MIATool for our software package for the analysis of microscopy images).