Development of ¹⁷⁷Lu‐nanobodies for radioimmunotherapy of HER2‐positive breast cancer: evaluation of different bifunctional chelators

Abstract

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor-to-background-ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2-overexpressing minimal residual disease. The therapeutic radionuclide Lutetium-177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators - p-SCN-Bn-DOTA, DOTA-NHS-ester, CHX-A"-DTPA or 1B4M-DTPA - in order to select the optimal chemical link between Lutetium-177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator-conjugation. High stability in time was observed, together with nanomolar affinities on HER2-expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro-CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M-DTPA-coupled conjugate was further evaluated in a high HER2-expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor-to-background ratios. No specific binding was observed in a HER2-negative model. In conclusion, the in-house developed anti-HER2 nanobody 2Rs15dHIS can be successfully labeled with (177) Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M-DTPA-based conjugate.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

“…With regard to the effect of radionuclide and labeling method on these parameters, we note that in a previous study with 5F7 (with a cys-tag) labeled using [ 125 I]SGMIB, intracellular retention in this cell line was maintained at about 60 % for up to 24 h as was the case with another residualizing radioiodination agent [36]. The 2Rs15d sdAb has been radiolabeled with several radionuclides including F-18 [18], Ga-68 [24], Tc-99m [23], and Lu-177 [38, 39]; however, these publications did not include internalization experiments for comparison to the current study.…”

Synthesis, Preclinical Validation, Dosimetry, and Toxicity of 68Ga-NOTA-Anti-HER2 Nanobodies for iPET Imaging of HER2 Receptor Expression in Cancer

“…The observation that the removal of the His 6 results in much lower kidney retention could be explained by differences in the overall charge of the Nanobodies because the pH in the kidney is below the pK a (negative logarithm of the acid ionization constant) of the imidazol groups. Presumably, other factors besides charge effects also are involved in the mechanism of kidney retention, and it is important to address this issue especially with regard to therapeutic applications of Nanobodies radiolabeled with b 2 -emitting radionuclides (28).…”

Targeted Radionuclide Therapy with A 177Lu-labeled Anti-HER2 Nanobody

“…Subsequently, the characterisation of 68 Ga-labeled 2Rs15d was also described in terms of clinical immuno-PET imaging 16. Finally, the His-tagged anti-HER2 2Rs15d was also radiolabeled with 177Lu and biodistribution was assessed in xenografted mice 20. In the latter study we demonstrated that the bifunctional chelator 1B4M-DTPA was the preferred chelator for 177 Lu-labeling as it exhibited the most favorable biodistribution.…”