Evaluating the in vivo distribution and stability of a 161Tb-labeled nanobody for potential cancer therapy

Objectives: Terbium has four radioisotopes suitable for theranostic applications in nuclear medicine. Terbium-152 (PET) and terbium-155 (SPECT) are diagnostic radionuclides, whereas terbium-149 (a decay) and terbium-161 (b– decay) are promising therapeutic radionuclides. Recently, we reported a protocol for radiolabeling heat-sensitive biomolecules with a promising bifunctional chelator, DOTA-GA.1 Here, we developed a DOTA-GA conjugate of a bivalent half-life extended Nanobody® (Nb), for radiopharmaceutical therapy, targeting c-Met and serum albumin. The Nb-conjugate was radiolabeled using both terbium-161 and terbium-155. Methods: Maleimide-DOTA-GA (1.3 molar eq.) was conjugated with the Nb (1 eq., 20 mM HEPES, 150 mM NaCl, 25°C, 1 h) and purified by size-exclusion chromatography. [161Tb]TbCl3 and [155Tb]TbCl3 were produced by SCK CEN and NPL, respectively [1,2]. Conjugates were radiolabeled with ~1.0 MBq [161Tb]TbCl3 or [155Tb]TbCl3 (0.1 M NaOAc, pH 4.7, 1 h, 40°C). Biodistribution of free [161Tb]TbCl3 and[161Tb]Tb-DOTA-GA-Nb construct were evaluated in healthy NMRI mice. Mice were injected with 0.9-1.5 MBq (10 nmol) via a tail vein under anesthesia (2.5% isoflurane in O2 at 1 L/min), and sacrificed 10 min, 1 h, 4 h, 24 h or 7 d p.i. (n=3/time point). Organs were harvested and the radioactivity was quantified in each organ. Finally, [155Tb] Tb-DOTA-GA-Nb (0.6 MBq) was injected i.v. In a healthy NMRI mouse, which was subsequently scanned for 1 h using a Molecubes® SPECT/CT system at 4 and 24h p.i.Results: Maleimide-DOTA-GA was regio-selectively coupled to the Nb and purified (protein recovery: 51.9±2.0%). DOTA-GA-Nb was radiolabeled with [161Tb]TbCl3 or [155Tb]TbCl3 with radiochemical yields above 89% and radiochemical purity of 99.9% after purification. Biodistribution studies of [161Tb]Tb-DOTA-GA-Nb showed a decrease in bone uptake over time, an area known for TbCl3 accumulation (SUVmean = 0.5±0.1 and 0.2±0.0 at 4h and 7d, respectively), indicative of a stable terbium-conjugate bond. High kidney retention was observed (0-24 h: SUVmean = 4.7) and the plasma half-life of the radiolabeled conjugate was ca.10 hours, with >40% of the injected activity still present in blood after 4 hours, which was expected because of the half-life extending properties of this Nb. SPECT images of [155Tb] Tb-DOTA-GA-Nb after 4 h provided further insight to the distribution of the conjugate, with a large proportion of the activity observed in blood-rich organs (~75%), such as spleen, lungs and liver. Conclusions: We successfully radiolabeled a Nanobody-DOTA-GA conjugate with both terbium isotopes using a previously developed protocol. The radiolabeled conjugates were observed to be stable in vivo, with no immediate evidence of metal-conjugate bond degradation. Further evaluation of [161Tb]Tb-DOTA-GA-Nb in tumor models is currently ongoing.