Development and evaluation of Hsp90-targeting nanobodies for visualisation of extracellular Hsp90 in tumours using...

The extracellular localisation of the Heat shock protein 90 (Hsp90) is associated with the diseased state and wound healing and presents a promising opportunity for cancer targeting using Positron Emission Tomography (PET) imaging and molecularly targeted radiotherapy. The aim of this work is to develop a radiotracer with low nanomolar binding affinity to target the extracellular and particularly membrane pool of Hsp90, evaluate it in vitro, and conduct preliminary PET studies in vivo in mouse tumour models. Variable Heavy domain of Heavy chain antibodies, often referred to as Nanobodies, are suitable targeting vectors for the extracellular targets due to their favourable pharmacokinetic properties and low nanomolar target affinities. The main objective of the study is to target tumours expressing extracellular and membrane Hsp90 phenotype with minimal tracer accumulation in the non-target organs, which limited the translation of previously studied small molecule cytosolic Hsp90 tracers suffering from high non-Hsp90 specific background in the abdominal area.

Six nanobodies were obtained after llama immunization with recombinant Hsp90α and ELISA biopanning, produced in E. coli and screened for stability and affinity. We selected one nanobody, 4DAM26, with good thermal stability, no aggregation at elevated temperatures, and low nanomolar affinity towards Hsp90α and Hsp90β isoforms for translation as a PET radiotracer. The nanobody was bioconjugated to p-NCS-NODAGA and radiolabeled with gallium-68 with 75 ± 11% radiochemical yield and > 99% radiochemical purity and remained stable up to 3 h in phosphate buffered saline and mouse serum. Pilot in vivo evaluation using µPET/CT and ex vivo biodistribution demonstrated a favourable pharmacokinetic profile, but the tumour uptake was non-distinguishable from the background tissue.

Compared to the small molecule Hsp90 tracers, the studied Nb-based tracer has improved pharmacokinetics properties including renal clearance and almost no accumulation in the non-target organs. Tumour uptake, on the other hand, was minimal and could not be differentiated from the background in µPET/CT. Our experiments indicate that in the studied models, membrane and extracellular expression of Hsp90 is majorly an artifact of cellular death, as only dead/dying cells had accessible pools of Hsp90 by flow cytometry, a consequence of a leaky membrane. More fundamental research is required to reassess the role of extracellular Hsp90 in cancer, and our future efforts will be focused on improving our inventory of cytosolic Hsp90 tracers with proven Hsp90-specific tumour accumulation.