Mild condition radiolabelling and in vivo stability of nanobody constructs with terbium-161 and terbium-155

Irwin Cassells, Michiel Van de Voorde, Ben Webster, Peter Ivanov, Andrew Burgoyne, Christopher Cawthorne, Christophe Deroose, Thomas Cardinaels, Guy Bormans, Frederik Cleeren, Maarten Ooms

    Research outputpeer-review


    Objectives: Terbium has four medically relevant radioisotopes for theragnostic applications in nuclear medicine. 152Tb (PET) and 155Tb (SPECT) are useful diagnostic radionuclides, whereas 149Tb (α decay) and 161Tb (β- decay, Auger/conversion electrons) are promising therapeutic radionuclides. Recently, we have reported a terbium radiolabelling protocol for heat-sensitive biomolecules,1 using human serum albumin as an exemplar for these conditions. Here, we developed a DOTA-GA conjugate of a nanobody (Nb), a biosynthetic single domain antibody fragment which are promising vector molecules for the development of diagnostic and therapeutic radiopharmaceuticals. The Nb-conjugate was radiolabelled using both 161Tb and 155Tb. 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 and purified by SCK CEN and NPL, respectively, as reported.1,2 Conjugates were radiolabelled with ~1.0 MBq [161Tb]TbCl3 or [155Tb]TbCl3 (0.1 M NaOAc, pH 4.7, 1 h, 40°C) and purified using size-exclusion chromatography. Biodistribution of free [161Tb]TbCl3 and [161Tb]Tb-DOTA-GA-Nb construct was evaluated in healthy NMRI mice. Mice were injected with 0.9-1.5 MBq (10 nmol) via a tail vein under anaesthesia (2.5% isoflurane in O2 at 1L/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 i.v. injected in an NMRI mice, which were subsequently scanned for 0.5 h using a Molecubes® SPECT/CT system at 4 and 24h p.i. Results: Maleimide-DOTA-GA was coupled in a site-specific way to the Nb, with a protein recovery yield of 51.9±2.0%. DOTA-GA-Nb was radiolabelled with [161Tb]TbCl3 and [155Tb]TbCl3 with a radiochemical yield of ca. 94% and ca. 89%, respectively, and radiochemical purity of 99.9% after purification. Biodistribution studies of [161Tb]Tb-DOTA-GA-Nb showed a decrease in bone radioactivity, an area known for TbCl3 accumulation (SUV = 0.5±0.1 and 0.2±0.0 at 4h and 7d, respectively), strongly indicative of a stable terbium-conjugate bond. Within the first 24 h of incubation, a high kidney retention was observed (0-24 h: SUVmean = 4.7). Plasma half-life of the radiolabelled conjugate was ca.10 hours, with >40% of the injected activity still present in blood after 4 hours. 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. Conclusion: We successfully radiolabelled Nb-DOTA-GA conjugate with two terbium isotopes using a previously developed protocol in our group. The radiolabelled conjugates were observed to be stable in vivo, with no immediate evidence of metal-conjugate bond degradation. High kidney uptake was observed, similar to other nanobodies described in literature. SPECT images of the biodistribution of the Nb was possible with as little as 0.6 MBq, and further supported the ex vivo distribution results. Further evaluation of [161Tb]Tb-DOTA-GA-Nb in tumour models is currently ongoing.
    Original languageEnglish
    Title of host publicationNuclear Medicine and Biology
    Number of pages2
    StatePublished - 22 May 2022
    Event2022 - ISRS - International Symposium on Radiopharmaceutical Sciences - Nantes
    Duration: 29 May 20222 Jun 2022

    Publication series

    NameNuclear Medicine and Biology
    ISSN (Print)0969-8051
    ISSN (Electronic)1872-9614


    Conference2022 - ISRS - International Symposium on Radiopharmaceutical Sciences
    Internet address

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