Effect of charges on the kidney retention of site-specific labelled Nanobodies

Introduction: Homogeneous Nb-based products are important when used as radiopharmaceuticals (1,2). It has been shown that sortase A catalyses the site-specific conjugation between a sortagged protein and an oligoglycine functionalized probe (3). The kidneys are the main route of elimination and previous studies have shown that the C-terminal amino acid composition of Nbs have an effect on the kidney retention. In this regard, probes with different charges are coupled to a α-HER2 Nb. Nb properties also have an influence on kidney retention, therefore in vivo studies with three different Nbs are evaluated.
Methods: In a first experiment, the α-HER2 Nb is site-specifically coupled, via the sortase reaction, to different probes (H-GGGXnYK(DTPA-CHX-A’’)-NH2, with n=0 or 4 and X= A, E or D) and a no-tagged α-HER2 Nb is used as a control. In a second experiment Nbs against different targets (further referred as NbT1, NbT2 and NbT3) are coupled via the sortase reaction, to the DTPA-CHX-A’’ chelator (H-GGGYK(DTPA-CHX-A’’)-NH2). In both experiments, Nbs are labelled with 177Lu and in vivo studies are performed in C57BL/6 mice (n=3).
Results: Sortase reaction yields between 20% and 60% were obtained. All functionalized Nbs, were obtained with high radiochemical purity (99%) and radiochemical yields between 65% and 87%. α-HER2 Nb coupled to the probe H-GGGXnYK(DTPA-CHX-A’’)-NH2, with n = 0 resulted in 141.6±6.7% injected activity/g (IA/g) kidney accumulation, when n = 4 and X = A, E or D, accumulations of 128.9±12.0%, 221.6±26.5% and 299.9%±58.9% IA/g were observed, respectively. In comparison, the notagged Nb showed a retention of 58.9±4.7% IA/g.
When comparing the ex vivo results of the three different targeting Nbs, differences in kidney retention were observed. The highest accumulation was shown for the NbT2 (96.0±12.6% IA/g), followed by the NbT1 (71.8±7.0% IA/g) and NbT3 (14.5±1.9% IA/g).
Conclusion: Introducing extra negatively charged amino acids at the C-terminal of the sortagged α-HER2 Nb resulted in higher kidney retention in comparison with the sortagged α-HER2 Nb coupled to the H-GGGYK(DTPA-CHX-A’’)-NH2 probe. The notagged Nb showed the lowest kidney retention. Comparison of the ex vivo results of different targeting Nbs reveals a difference in kidney retention for all three Nbs. This implicates the importance of the characteristics of the Nb on the accumulation in the kidneys.
References
1. Xavier, C. et al. (2013). Synthesis, preclinical validation, dosimetry and toxicity of 68Ga-NOTA-Anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer. J. Nucl. Med. 54, 1-9.
2. D’Huyvetter, M. et al. (2014). Targeted Radionuclide Therapy with A 177Lu-labeled Anti-HER2 Nanobody. Theranostics. 4(7), 708-720.
3. Theile, C.S. et al. (2013). Site-specific N-terminal labeling of proteins using sortase mediated reactions. Nat. Protoc. 8(9), 1800-1807.