Cambridge Healthtech Institute’s Kent Simmons recently spoke with Dr. Darren Tomlinson, an Associate Professor at the University of Leeds, about his upcoming presentation “Generation of Positive Allosteric Modulators of TRPV1 Using Affimer Reagents,” to be delivered in the Antibodies Against Membrane Protein Targets (Part 2) meeting at the 2018 Discovery on Target. DOT is scheduled for September 25-28, 2018 in Boston, with Part 2 of the membrane proteins program set for September 27-28.
What is the Affimer platform?
We recently described the Affimer platform in an article published in eLife. Affimers proteins are antibody-mimetic which have been designed to outperform antibodies in many aspects. The platform is based on cystatins – one is human in origin and the other is a consensus of plant proteins. Both scaffolds have similar structures and have been engineered to present one or two randomized nine amino acid loops for molecular recognition. The loops mimic the CDRs of antibodies and are involved in binding to the target molecule. We have generated multiple large libraries containing billions of Affimer molecules and we isolate target specific binders from these libraries using phage display. To date we have screened over 500 target (proteins and other molecules) which can be used in a broad range of molecular and biotechnology techniques. These include, amongst others, staining for targets in cells and tissues, blocking protein function, capture and detection reagents and therapeutics per se.
What are the benefits of Affimers vs. standard antibody technologies?
Affimers offer many benefits over standard antibody-based molecules. Unlike antibodies, Affimers can be isolated against target proteins in a few weeks and because the Affimer scaffold is very simple – monomeric 100aa protein, no cysteine and no post-translational modifications – they can be produced at high-yield using standard microbial E.coli processes. Both identification and production are highly reproducible animal-free processes and our Affimer eLife paper was highly recommended by the NC3Rs for helping reduce animal usage. Additionally, Affimers offers multiple easy formatting options. They can be multimerized to provide multivalency or multispecificity and can also be fused to various protein domains such as Fc, fluorescent reporter proteins or receptor traps. They can easily be conjugated to small molecules such as toxins or fluorescent tags and efficiently immobilized and oriented on a variety of solid surfaces. These characteristics make the Affimer platform a very versatile option for many research, diagnostics and therapeutics applications.
Why have you been using Affimers to target ion channels?
We were awarded a grant from BBSRC (a UK research funding agency) to develop phage screening protocols to isolate Affimer reagents against membrane proteins which historically have been difficult targets to address using standard antibody approaches. The grant was awarded with Dr Jon Lippiat, an ion channel expert at the University of Leeds. We isolated Affimers against TRPV1 and demonstrated their ability to bind to the ion channel on cells. Interestingly the Affimers did not directly stimulate or inhibit the function of the ion channel but acted as positive allosteric modulators. This means they only regulate already activated ion channels.
Why are you excited about these Affimers?
What is really unusual about these Affimers is their ability to act as positive allosteric modulators. TRPV1 is a therapeutic target in chronic pain syndromes. There have been a few small molecules developed against TRPV1 but because they either inhibit or activate ion channel function throughout the body they have fairly severe side effects such as burning pain and numbness. By using positive allosteric modulator Affimer binders, we could specifically target the receptors involved in the region of chronic pain and selectively deactivate them, hence reducing the systemic side effects. We have generated X-Ray crystallography images of the complex of the Affimer and TRPV1 peptide and we have been able to pinpoint the exact amino acids involved in binding. Using this information, we’ve done two things: the first one was to improve the affinity of the Affimers against TRPV1 by affinity maturation. The second one, was to demonstrate that we can use the information about the small interaction sites between the Affimer and the receptor to design small molecules that modulate function with a similar allosteric mode of action.
Darren Tomlinson, PhD, Associate Professor, University of Leeds, United Kingdom
I studied for a PhD in developmental biology at the University of Edinburgh in the MRC Reproductive Biology Unit. After completing my PhD I moved to Leeds and worked as a PDRA studying the function of growth factor receptors in bladder cancer. In 2010 I set up two facilities in Leeds - a siRNA screening facility and a facility to produce non-antibody binding proteins, which led to the development of Affimers. In 2015 I became a University Academic Fellow and in 2018 became an Associate Professor at the University of Leeds to continue the exploitation of isolating Affimers for studying protein function in disease.