Direct labelling of proteins using Affimer-conjugate warheads for imaging

The ability to magnify objects that are invisible to the naked eye using a technique called microscopy revolutionised science. Microscopy has been widely used to study organisms and has increased the ability to understand processes in health and disease. The ability to detect individual objects in organisms using antibodies has further pushed the bounderies and enabled the study of even smaller objects, such as proteins. Proteins regulate every process in the body, and there are over 20,000 different proteins. Antibodies are routinely used in every molecular and cellular biology laboratory to indirectly visualise proteins but as microscopy technologies have improved and become 'super-resolution' the size of the antibody has become one of the limiting factors. To overcome this, super-resolution microscopy has adopted using smaller probes, often referred to an non-antibody binding proteins, that places the detection reagent closer to the protein. However, this is still an indirect method of visualising proteins and as super-resolution techniques improve these will become the next limiting factor to increasing resolution. This project aims to develop a method of directly labelling the target protein for visualisation. This will utilise a recently developed non-antibody binding proteins, called Affimers, to deliver the detection reagent and chemically conjugate it to the target protein. This will provide the first example of a reagent to directly conjugate imaging reagents to endogenous proteins for visuallisation and super-resolution microscopy and represents and exciting method that could be adapted for many other techniques.

Recently there has been an increase in the use of alternatives to antibodies in super-resolution microscopy, including nanobodies, Affibodies, and Affimers. These reagents are approximately 10 times smaller than antibodies, and bind with similar levels of specificity and affinity. Anti-GFP nanobody, used to target GFP-fusion proteins, reduced the linkage error to ~2nm. The anti-HER-2 Affibody has also shown promise in super-resolution microscopy, enabling visualisation of the intra- and intercellular distribution patterns of HER-2 in over-expressing cancer cells. Affimers, developed by DT, have been used in a range of assays from diagnostics to blocking protein-protein interactions. In super-resolution microscopy Affimers have been used to image tubulin and HER4, as well as other unpublished targets including actin and actinin.
Although binding reagents like Affimers provide improve resolution by placing the fluorophore in close proximity to the protein, as super-resolution microscopy techniques and technologies improve the resolution will increase, making even small binding reagents a limiting factor. Potential methods of overcoming this issue are to use over-expressed tagged target proteins fused or genetically modified for direct incorporation of fluorophores in non-natural amino acids. Although useful these methods still require over-expression or genetic modification of target and cell to enable visualisation. A more appealing approach would be to generate a technique capable of directly labelling endogenous proteins therefore reducing the chances of altering protein function by overexpression or tagging. Hence, the aim is to develop methods to directly label endogenous proteins with fluorophores by targeting them with Affimers functionalised with a reactive fluorophore warhead.

The ease with which Affimer technology can be developed greatly enhances their utility compared to other technologies. The implications for health and disease and the ability to image proteins will be important for industrial beneficiaries through the potential for development of molecules that modulate protein function and for other healthcare products including diagnostics.
We already have an established network of academic partners in numerous Universities and have had funding with industry eg Tecrea (Innovate feasability grant), Pfizer (CASE studentship), Fera (CASE studentship) and Avacta Life Sciences (CASE studentship, license agreement). Some of the Affimers generated in our laboratory for Avacta Life Science are available to purchase and we regularly screen targets for Avacta's industrial collaborators and clients. Some of our unpublished results have generated commercial interest and investment in the Affimer brand which recently helped Avacta fund raise for a therapeutic programme, some of which will be fed back into the academic projects at Leeds. This demonstrates our drive to develop impact through the Affimer technology. This porfolio of collaborators is only likely to grow after publication of the ability to directly label proteins providing further impact for our research.

The project thus has the potential to make a significant impact on the wider public, and also on the UK economy. Leeds have services focused upon building appropriate relationships with businesses and other external organisations. There are mechanisms in place for protecting and managing intellectual property that may be produced and for assisting in the exploitation of outputs from the research. We have existing links with Avacta Life Sciences in relation to the Affimer Technology through the University of Leeds. The strong interactions with both industrial partners will provide opportunities for exploring technology and reagent deployment, and create further opportunities such as CASE studentships and project grants.