A new paper published this week in eLife, from the University of Leeds, in conjunction with a number of institutions, illustrates the potential of Affimer technology across a range of applications. From binding to specific members of an homologous protein family and tracking tumour antigens within mouse models to accessing hidden epitopes in super-resolution microscopy, the paper offers a number of examples where Affimer reagents, due to their specificity, intracellular activity, small size and target range, can be as good as or better than antibodies.
The issues with the supply of validated and renewable antibodies have been discussed at length. One commonly accepted strategy to overcome these problems is the development of new affinity tools, engineered for purpose and offering a reliable assured supply of any specific binder. Affimer binders were developed in response to this need. Beyond this advantage is their rapid, animal-free selection that addresses the 3R agenda of reducing scientific animal use and leading to monoclonal reagents that surpass antibodies in terms of performance for many applications.
As part of this study the team, led by Dr Darren Tomlinson and Prof Mike McPherson from the University of Leeds, generated Affimer binders to twelve different targets and compared their performance with antibodies across seven different case studies. These included the modulation of ion channels, affinity-fluorescence in fixed cells, super-resolution microscopy, targeting a small organic molecule and distinguishing between homologous protein domains to decipher protein function and the inhibition of extracellular proteins.
- 120 different SH2 domains are present in over 111 proteins. The team developed Affimer binders to the SH2 domains of the Grb family members and p85? subunit of PI3K. The ability to discriminately inactivate specific SH2 domains could offer increased understanding of many signalling cascades, by providing a highly single domain-specific inhibition rather than ablating protein expression by approaches such as siRNA and CRISPR-Cas9 that removes the whole protein and sometimes related proteins thus confusing the interpretation of results. Affimer binders specific for the p85? subunit of PI3K showed no binding to either of the alternate p85? or p55? subunits, despite the sequence homology between these pairs being between 83-90%. These Affimers were subsequently expressed within NIH 3T3 cells where they were successfully able to bind the p85? target, as determined by co-IP.
- The VEGF family of proteins regulate vasculogenesis, angiogenesis, arteriogenesis and lymphaniogenesis. The VEGF-A protein has been targeted therapeutically for the treatment of a range of conditions, from metastatic cancer to macular degeneration. Affimer binders generated to the VEGF receptor, VEGFR2, were biotiylated for use in immunohistochemistry and compared to a commercially available polyclonal antibody. While both the anti-VEGFR2 antibody and Affimer binder showed identical staining patterns with VEGFR2 located in epithelial cells, revealing intense staining at the cell membrane, the stain developed more rapidly with Affimer binders, showing a higher sensitivity of labelling. Applying the anti-VEGFR2 Affimer binders to HUVEC cells showed a similar functional inhibition as documented with the use of RNAi. However, whilst RNAi requires 24-48 hours post-transfection before the effects of inhibition can be observed, with the application of Affimer proteins a decrease in VEGF-dependent tubule length and branch formation was noted 30 minutes after application.
- Ion channels are important drug targets involved in many biological processes, but a lack of sufficiently selective reagents exist to allow modulation of their function without off-target effects. An Affimer binder selected against the ion channel, transient receptor potential vanilloid 1 (TRPV1), co-localised with an anti-TRPV1 antibody in fluorescent staining of U2-OS cells. Alone the anti-TRPV1 Affimer binders showed no effect on calcium modulation of this ion channel, but in conjunction with the capsaicin ligand a significant enhancement of was observed in activity, suggestive of the ability of Affimers to modulate ion channels through the allosteric effects of their binding.
- Tenascin C shows potential for use as a tumour marker in vivo. An anti-tenascin C Affimer was tagged with rhodamine red to assess its performance in vivo as a diagnostic tool as its smaller molecular size would allow more rapid clearance from the body compared to an antibody. Ex vivo analysis showed the tumour signal for the Affimer binder was significantly higher compared to control Affimer at 24 hours following administration, thus demonstrating Affimer proteins potential in imaging as rapid detection and diagnostic reagents.
- Discriminating between two homologous viruses is important in many diagnostic situations. Marek’s disease in chickens is caused by Marek’s disease virus 1 (MDV1), but controlled by vaccination with Herpes Virus of Turkeys (HVT), thus tests to distinguish between these viral proteins are crucial in the field. HVT specific Affimer binders were selected and shown to yield distinct cytoplasmic foci in infected cells when used in fluorescent microscopy of fixed chicken embryonic fibroblasts.
- The small size of Affimer reagents makes them particularly suited to use as probes in super-resolution microscopy, where the and larger size of antibodies limits resolution and thus accurate localisation of any target proteins. Affimer proteins targeting HER4 showed accurate stochastic labelling of HER4 clusters upon cell membranes and could also be used to track HER4 molecular movement in MCF7 cells. In addition Affimers against tubulin highlighted the cytokinetic furrow, a region from which antibodies are usually excluded owing to the density of microtubule packing.
- Generating affinity reagents to low molecular mass organic compounds is challenging, particularly for toxic compounds. Affimers generated against the small organic compound 2, 4, 6-trinitrotolulene (TNT) were able to discriminate between TNT, 2,4-DNT and the other two dinitrotolulene variants, demonstrating the selectivity achievable with Affimer technology to such small molecular differences.
The range of techniques and highly specific targets included in this study showcase Affimer technology as a high-quality antibody alternative, with the potential to reach previously unmet needs across the research, diagnostics and therapeutic arenas. The authors of the study note that phage selection for each of the Affimer proteins used typically took around 12 days, highlighting another key advantage of Affimer technology – the speed of development compared to standard antibodies. Furthermore, as Affimer proteins do not require complex mammalian systems for production, moving towards next generation affinity binders like Affimer proteins, may offer a solution to the bottleneck in reagent protein production.