Effect of charges of radiolabeled Nanobodies on kidney retention

Introduction
Nanobodies (Nbs) can be radiolabeled and used for diagnostics, therapy or as a theranostic tool (1,2). The kidneys are the main route of elimination and previous studies have shown the impact of C-terminal charges of radiolabeled Nbs on the kidney retention (2). Hereby was shown that removing polar positively charged amino acids was decreasing the kidney retention. In this regard, the effect of extra negatively charged amino acids was determined. Herefore the sortase A technique, wich catalyses the site-specific conjugation between a sortagged protein and an oligolycine functionalized probe (3), was used to insert extra C-terminal amino acids. Further the effect of overall charges was determined.

Method
In a first experiment, a α-HER2 Nb was site-specifically coupled, via the sortase reaction, to different probes (H-GGGXnYK(DTPA-CHX-A’’)-NH2, with n=0 or 4 and X= A, R, E or D). In a second experiment different Nbs (NbT1, NbT2, NbT3 and NbT4), were site-specific coupled via the sortase reaction, to the DTPA-CHX-A’’ chelator. Hereby NbT1 contained the highest pI followed by NbT2, NbT3 and NbT4. All Nbs were labeled with 177Lu.

Results
Sortase reaction yields between 20% and 60% were obtained. After radiolabelling, Nbs with high radiochemical purity (99%) and radiochemical yields between 65% and 87% were obtained. At 1 hour post injection (p.i.) the α-HER2 Nb coupled to the H-GGGXnYK(DTPA-CHX-A’’)-NH2 probe, with n = 0 resulted in 26.6±3.1% injected activity (%IA) in the kidneys, when n = 4 and X = A, R, E or D, accumulations of 30.2±2.5%, 48.8±2.2%, 44.5±7.7% and 42.5±2.0% IA were observed, respectively. The same trend was seen at 3 and 24 hours p.i. In the second experiment, an accumulation of 50.2±8.3% IA was observed for NbT1 at 1 h p.i.. For NbT2, NbT3 and NbT4 accumulations of 39.8±5.7%, 33.3±3.6% and 14.2±1.3% IA were observed. The same trend was seen at 3 and 24 hours p.i..

Conclusion
Introducing extra charges, e.g. negative or positive, at the C-terminal of a α-HER2 Nb resulted in higher kidney retention. In the second experiment a trend can be seen between the overall charge of the Nb and the kidney retention.

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.