A new approach in protein phosphorylation for Nanofitin radiolabelling

Recently, new strategies emerged in the field of monoclonal antibodies radiolabeling for PET imaging with the use of positron emitter such as 89Zr or 68Ga. Despite their important role in the therapeutic world, antibodies have many disadvantages related to their structure making their production difficult and expensive: limited stability, disulfide bridges and post-translational modifications. Moreover, conjugation of chelating agent often occurs on lysines, which is non-regioselective and leads to a heterogeneous mixture of products. In addition, the long clearance of antibodies can be a problem to obtain a good contrast when they are used in imaging. To address these limitations, we developed a chemistry-free system consisting of a highly phosphorylatable peptide tag to chelate stereoselectively radionuclides for PET imaging. To ensure an efficient targeting of the radionuclide, the tag was genetically fused to a Nanofitin®, a protein scaffold developed as an alternative to antibodies. Nanofitins share the advantages of being small, cystein-free, easy to engineer, highly stable both to temperature and pH and expressed with high yield in E. coli.

We developed an in vivo phosphorylation system consisting in the co-expression of a Nanofitin® fused to the phosphorylatable tag and the α subunit of casein kinase II, in order to save purification steps and decrease production costs. Effectiveness was demonstrated by electrophoretic mobility shift assay and staining with a specific phosphoprotein staining gel. To further optimize and validate the system, the rate of phosphorylation is evaluated by an isoelectric focusing method (based on pHi variability). In addition, we are developing a phosphoprotein purification protocol based on gallium(III) or iron(III) functionalized chromatography beads. Eventually, the use of a single tag for both the purification and the labeling together with Nanofitins as a highly stable targeting moiety will provide a much simpler and straightforward system.