Optimising the radiolabelling properties of technetium tricarbonyl and His-tagged proteins

Abstract

BackgroundTo date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging.MethodsFour different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, α-CD33 scFv, α-VCAM-1 scFv and α-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins.ResultsWe used 260 and 350 μL [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink® kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+].ConclusionWe have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification.

A 99mTc-labelled scFv antibody fragment that binds to prostate-specific membrane antigen

“…The formation of the 99m Tc tricarbonyl was monitored by TLC on glass-backed silica gel 60 plates developed with 1% HCl in methanol 20. The R f values in this system are as follows: 99m Tc tricarbonyl, 0.2–0.8; 99m Tc colloids, 0.0 and 99m Tc-pertechnetate, 0.9.…”

High-throughput high-volume nuclear imaging for preclinical in vivo compound screening§

“…Small aliquots of all 25 proteins were radiolabelled first for testing, following the recommendations on 99m Tc-tricarbonyl labelling given in [19]. Subsequently, larger aliquots were radiolabelled for imaging.…”

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

“…A number of laboratories have labeled antibody fragments bearing C-terminal (His) 6 tags with the [ 99m Tc(CO) 3 ] + synthon for SPECT imaging [43–46]. Kogelberg, et al ., for example, labeled the α v β 6 -targeting diabody B6.3 via the coordination of [ 99m Tc(CO) 3 ] + to a C-terminal (His) 6 tag, and it was found that the resultant conjugate retained nanomolar affinity for its target and demonstrated tumor-specific targeting in vivo .…”