Developing targeted lanthanide contrast agents with Affimer® proteins

Magnetic resonance imaging (MRI) is a non-invasive technique that provides high-resolution 3D maps detailing the anatomy, function and metabolism of tissues in vivo. A team of researchers at the University of Leeds have developed optimised procedures for the production and purification of imaging reagents that could enhance the detection of diseased cells and other tissue structures.

Labelling Affimers with Gd-DOTA reagents holds the potential for new targeted imaging reagents
Labelling Affimers with Gd-DOTA reagents holds the potential for new targeted imaging reagents

MRI scans typically make use of the two hydrogen atoms in water. Apart from differences in local water content the contrast in these images results from differences in the intrinsic relaxation times of each of the two hydrogen bonds of the water molecules, T(1) and T(2). Yet, often these factors are too limited to enable a sensitive and specific image. For this reason increasing use is being made of MRI contrast agents that increase the image contrast following intravenous injection. The degree and location of the contrast changes can then provide substantial diagnostic information and could dramatically expand the range of MRI applications.

Contrast agents based on low-molecular weight chelates of gadolinium ions (Gd3+) have been used successfully in a number of applications, including haemodynamic parameters such as blood perfusion and vascular permeability. Gd3+ are typically used to shorten the T(1) relaxation time and increase image contrast. To achieve the required level of contrast in MRI, high concentrations of Gd-contrast agent are required at the target site.

Targeting reagents can be used to direct the contrast agent to the required imaging site to increase their concentration and improve image contrast. These targeting reagents consist of the Gd-chelating agent fused with a protein-targeting molecule. The protein-targeting moiety of these compounds should recognise target cells with high affinity and specificity, yet pass through the body rapidly enough to avoid the metabolism and release of the toxic Gd3+ ions. 

As Affimer reagents are significantly smaller than traditional antibodies they could potentially penetrate the desired tissue more easily to offer improved visualisation. Affimer proteins also offer advantages in that good candidate binders can be rapidly identified from library screens, they are easier to produce in homogenous batches than antibodies and can be labelled site-specifically with suitable chemicals.

In a recent study (link is external) published in RSC Advances, a team of researchers from the University of Leeds led by Dr Robin Bon, demonstrate robust synthetic procedures that allow the generation and purification of a trivalent Gd-DOTA reagent.

Macrocyclic Gd-chelating agents, such as DOTA and DO3A, are metabolically more stable than linear structures, such as DPTA, and they bind Gd3+ much more tightly, thus decreasing potential toxicity. A previously published patent outlining the manufacturing process of [Gd-DOTA]3-X contained many potential pitfalls, which made production of high-quality batches of these reagents difficult. Bon’s team developed optimised procedures offering solutions to these possible hurdles in production.

Within this paper they also offer details of the production methods of two new trivalent Gd-DOTA reagents and first findings regarding the conjugation of these trivalent reagents to Affimers, via different chemical handles that allow easy protein labelling. Successful labelling of Affimers with the Gd-DOTA compounds was achieved through isocyanate ligation, site-specific thiol-maleimide ligation and strain-promoted azide-alkyne cycloaddition.

The scientists at Leeds University are currently investigating the use of Affimer-based Gd-contrast agents in targeted imaging. The potential for the adaptation of these compounds with Affimer proteins offers exciting prospects for the future application of Affimer reagents in this new field of research.