Influence of Histidine-Containing Tags on Biodistribution of Radiolabelled ADAPT-Based Imaging Probes

• Aim. ADAPTs are a class of small ( ∼ 5 kDa) robust scaffold proteins suitable as probes for radionuclide molecular imaging in vivo. The attachment of a histidine-containing tags to the scaffold proteins allows efficient purification by immobilized metal ion affinity chromatography (IMAC) and permits labelling with 99mTc(CO)3. Earlier, we have demonstrated that replacement of the hexahistidine (H6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HE3)-tag reduces hepatic uptake of radiolabelled affibody molecules. The same effect has been confirmed for other scaffold proteins, DARPins, and short peptides. The aim of this study was to evaluate effect of histidine-containing tag composition on biodistribution of ADAPTs. Material and methods. A series of anti-HER2 ADAPT6 probes having DEAVDANS lead sequence and H6- or HE3-tags at N-termini has been prepared. In two variants, maleimido derivative of DOTA was conjugated to a unique cysteine was incorporated at N-terminus. DOTA-C-HE3-ADAPT6 and DOTA-C-H6-ADAPT6 were labelled with 111In. HE3-ADAPT6 and H6-ADAPT6 were labelled with 99mTc(CO)3using IsoLink kit. Binding specificity, affinity, and cellular processing of new conjugates was evaluated using HER2-expressing SKOV-3 ovarian carcinoma cells. Biodistribution at 1,4 and 24 h p.i. was evaluated in normal NMRI mice. Tumour-targeting properties of the best 99mTc(CO) 3-labelled variant, 99mTc(CO)3-H6-ADAPT6 were evaluated in BALB/C nu/nu mice bearing SKOV-3 xenografts. Results. All radiolabeled ADAPTs demonstrated specific binding to SKOV-3 cells with affinities in the range of 1.1-2.8 nM. The internalization by SKOV-3 cells was slow. In vivo, all conjugates cleared rapidly from blood via kidneys with subsequent renal re-absorption. The hepatic uptake of 111In-DOTA-C-H6-ADAPT6 was slightly but significantly (p<0.05) higher that the uptake of 111In-DOTA-C-HE3-ADAPT6 at 1 h pi, but biodistribution was very similar at later time points. Surprisingly, the uptake of 99mTc(CO)3-HE3-ADAPT6 was significantly (p<0.05) higher than uptake of 99mTc(CO)3-H6-ADAPT6 in liver, blood, and bone at 1h p.i. At 4 h p.i., hepatic uptakes were equal, but 99mTc(CO)3-H6-ADAPT6 provided lower uptake in blood and bone. 99mTc(CO)3-H6-ADAPT6 demonstrated high (19? 3 %ID/g at 4 h p.i.) and specific tumour uptake. Tumour-to-blood and tumour-to-liver ratios were 102? 29 and 12? 3, respectively. Conclusion. Influence of histidine-containing tag on biodistribution of scaffold proteins depends on composition of a scaffold protein and might differ appreciably. This should be take into account in molecular design of imaging probes based on engineered scaffold proteins

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology (hsv//eng)

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)