Antibody alternatives coming of age in imaging applications

Antibody alternatives were developed with the aim of overcoming some of the inherent problems found with the use of antibodies, such as their large size, irreproducibility, and the difficulties and high costs of engineering and manufacturing them. Designing new affinity proteins has typically involved the modification of naturally occurring proteins or protein domains to carry variable insert sequences that form a binding region analogous to that of a standard antibody. This strategy has led to the development of over 50 different affinity proteins, including DARPins, Monobodies, Affibodies and Affimer proteins. Each of these different scaffolds offer a range of target suitability, function across different applications and improvement over standard antibody performance.

A new review published this week in Biophysical Reviews highlights the progress that has been made with these reagents in imaging, across research and diagnostic applications. These advances demonstrate that the intended benefits of alternative affinity proteins over antibodies have in many cases come to fruition and emphasise how these next generation technologies are offering new and improved options in in vivo imaging for research and diagnostics.


The use of antibody alternatives in histochemistry has shown very positive results for a number of scaffolds.

Detection of the human epidermal growth factor receptor 2 (HER2) in samples can provide both prediction of prognosis in cases of breast and ovarian cancer and guide therapeutic treatment options. Use of an anti-HER2 DARPin in histochemical analysis of human tissue samples showed that this reagent was just as reliable as the FDA approved antibody 4B5, but was in fact more specific than the antibody.

Histochemistry of a polyclonal anti-VEGFR2 antibody and of representative Affimers B8 and A9. Staining is shown as a light brown color, haemotoxylin counter staining (blue). Arrows show similar staining patterns.

A recent report showed that Affimer binders directed to VEGFR2, which is important in blood vessel formation in tumours, gave rise to the same staining pattern as a VEGFR2 polyclonal antibody, but the Affimer binders showed increased sensitivity. The increased sensitivity of Affimer binders is thought to be due to their smaller size allowing increased tissue penetration. Within the same report anti-tenascin C Affimer binders also were shown to give comparable performance to an anti-tenascin C antibody.

The ability to easily format many of these alternative affinity proteins as Fc-fusions allows them to directly replace primary antibodies in such applications with the use of Fc-based detection strategies. 

In vivo imaging of tumours

The ability to image tumours in vivo offers a non-invasive method that is proving to be an important tool in cancer diagnosis. Benefits offered by antibody alternatives include the lack of an Fc portion and their overall smaller size. This gives these scaffold proteins increased tissue penetration and more rapid clearance from the body, which can result in more sensitive analysis, and quicker imaging meaning shorter hospital stays for patients undergoing diagnostic tests.

Anti-HER2 DARPins and anti-EGFR nanobodies have both shown improved sensitivity and lower background in in vivo imaging compared to standard monoclonal antibodies. Additionally, reduced off-target effects were noted with the anti-EGFR nanobody due to its rapid clearance from the body. Compared to both the approved antibodies, trastuzumab and cetuximab, that have clearance times of over 24 hours the anti-EGFR antibody cleared the system within 45 minutes from administration.

Fluorescent imaging probes

While the fluorescent labelling of fixed cells with antibodies is common practice in many imaging applications, the ability to modify alternative affinity proteins at specific sites in the scaffold with various tags, means that they are also capable of carrying fluorophores and labelling epitopes in an analogous manner. This has been directly demonstrated with the labelling of live cells with Affimer proteins directed to the calcium channel protein, TRPV1.

Due to the highly stable nature of many affinity proteins they are able to function intracellularly, whereas standard monoclonal antibodies are sensitive to the reducing intracellular environment. Examples of this include the intracellular labelling of cytokeratin-B, lamin Dm0 and an HIV1 precursor protein by nanobodies, which allowed the visualisation of HIV viral particle assembly in vivo.

The removal of the resolution limit of microscopy with the advent of super resolution light microscopy, means that we can now accurately visualise beyond the previous 200nm limit, down to distances of approximately 20nm. The large size of antibodies limits the potential resolving power of these new techniques, placing any fluorescent signals at a greater distance from the labelled epitope of interest. In comparison, the small size of alternative binding proteins reduces this error in super resolution imaging, from as large as 30nm using primary and secondary antibodies for labelling or 10nm with the use of a single antibody to as little as 2nm with the use of an anti-GFP nanobody to capture GFP-tagged proteins.

Similar effective use of these smaller antibody alternatives has been seen with anti-tubulin Affimer binders. A similar staining pattern was seen of interphase microtubules for both the anti-tubulin Affimer protein and a widely used anti-tubulin antibody. However, the smaller Affimer proteins were also able to label the central region of the cytokinetic furrow in the microtubules, from which antibodies are excluded owing to the density of tubulin in this region. 

These results show that the initial proposed benefits of engineered antibody alternatives are indeed coming to fruition. Besides the simple production in E.coli and the increased stability of many of the non-antibody scaffolds, within the field of imaging the smaller size of these molecules is of huge benefit, offering increased tissue penetration for increased assay sensitivity, and bringing detection agents such as fluorophores into closer contact with targeted epitopes for increased accuracy in imaging.