Development of anti-MUC1 DNA aptamers for the imaging and radiotherapy of breast cancer

Background

Aptamers have shown great potential as novel targeted radiopharmaceutical entities for the diagnosis and imaging of disease. They offer reduced immunogenicity, good tumour penetration, rapid uptake and clearance compared with their monoclonal antibody counterparts. In previous work we have reported the labelling of such aptamers against breast-cancer-related biomarkers with radionuclide ligands.

Methods

We have now conjugated previously selected aptamers against the protein core of the MUC1 glycoprotein tumour marker with chelating agents and labelled them with ^99mTc, for the diagnostic imaging of breast cancer. The conjugation is achieved using standard peptide coupling reactions between an amino modification on the aptamer and the carboxylic group on the ligands. Labelling with ^99mTc used tin chloride as the reducing agent, and analysis was by HPLC where both the UV and the gamma emission was monitored. Radiolabelled aptamer conjugates were separated from free, unconjugated ^99mTc using microcon filters. For the analysis of the pharmacokinetic properties of the aptamer–radionucleotide conjugate we used gamma-camera imaging in MCF-7 breast cancer tumour model systems.

Results

We coupled the aptamer with the highest affinity for the MUC1 glycoprotein to different ligands (MAG2 or meso-2,3-dimercaptosuccinic acid) and labelled it with active ^99mTc to obtain stable complexes that were used in pharmacokinetic studies. This allows us to compare the properties of a single conjugate with a biaptamer conjugate, as two of the DMSA–aptamer conjugates can coordinate the metal core. An efficient and convenient labelling of the aptamer with short half-life radioisotopes was achieved as the last step of the synthesis (postconjugation labelling). The labelled aptamers were separated from free ^99mTc using microcon filter separation and were monitored by HPLC at all stages, to ensure that only radiolabelled aptamers were injected and imaged for their pharmacokinetic properties.

Conclusion

The aptamer–chelator conjugates have strong ^99mTc binding properties and the resulting complexes are highly stable in vivo both in terms of nuclease degradation and leaching of the metal. The presence of more than one molecule of aptamer per complex alters the binding and pharmacokinetic properties of the radiolabelled products, allowing the complex to remain longer in circulation and thus offering improved tumour imaging properties, without affecting the tumour penetration of the aptamer. Furthermore, different ligands affect accumulation of the aptamer in different organs, as they alter the lipophilic properties of the conjugate. These results aim to open new possibilities for the diagnostic imaging of, and potentially the targeted radiotherapy of, breast cancer.