Radiosynthesis and evaluation of 68Ga-Cys2-AnxA5 and 68Ga-Cys165-AnxA5 as apoptosis imaging agents

1536 Objectives Radiolabelled AnnexinV (AnxA5) measures cell death both in vitro and in vivo. We radiolabelled AnxA5 with 68Ga using two variants of AnxA5 with a single cysteine residue at position 2 or 165, respectively Cys2-AnxA5 and Cys165-AnxA5, allowing site-specific coupling to 68Ga-Dota-maleimide. Methods In vitro cell binding was studied in control- and anti-Fas treated Jurkat cells. Biodistribution and pharmacokinetics were studied with µPET in healthy mice and in a hepatic apoptosis model (anti-Fas Ab treated) up to 60 min p.i. Daudi (Burkitt lymphoma) tumour bearing mice were scanned before and after treatment with combined chemotherapy (125 mg/g Endoxan) and radiotherapy (10 Gy/tumour) using µPET and µMRI. Tracer uptake was measured and imaged ex vivo using autoradiography and correlated to histological evidence of apoptosis (TUNEL). Results 68Ga-Dota-maleimide labelling yield was ≥ 98% and coupling yield of 68Ga-Dota-maleimide to Cys2-AnxA5 and Cys165-AnxA5 was ~70%. Labelling and purification took about 60 min, with a final radiochemical purity of ≥ 98%. In vitro binding of 68Ga-Cys2-AnxA5 and 68Ga-Cys165-AnxA5 to anti-Fas treated cells was 5 times higher compared to normal cells. Biodistribution data in normal mice indicated fast urinary clearance with only minor hepatobiliary clearance but high kidney retention. Anti-Fas treated animals showed a 3 to 8 times higher liver uptake (for respectively 68Ga-Cys2-AnxA5 and 68Ga-Cys165-AnxA5) as compared to healthy animals. Tumour uptake of 68Ga-Cys2-AnxA5 and 68Ga-Cys165-AnxA5 was respectively only 0.5 ± 0.1 % ID/g and 1.0 ± 0.3 % ID/g, but significantly increased to 1.5 ± 0.2 % ID/g and 1.6 ± 0.1 % ID/g after therapy. Conclusions Both 68Ga-Cys2-AnxA5 and 68Ga-Cys165-AnxA5 show a clear binding to apoptotic cells and are promising tracers for follow-up of cancer therapy. Research Support This study was financially supported by the European Union (IVA-VLANED-1.20)