It's a match: use of the radionuclide theranostic pair (133)La/(225)Ac for the radiopharmacological characterization of EGFR-targeted single-domain antibodies.

BACKGROUND: Targeted alpha therapy represents an advanced and rapidly evolving form of precision cancer treatment with increasing importance in recent years. The alpha-emitter (225)Ac plays a key role in this clinical development due to its attractive physical and chemical properties. In this context, the macropa chelator has favorable characteristics in terms of labeling conditions and complex stability, making its derivatives exceptionally appealing for (225)Ac-labeling of heat-sensitive biomolecules. However, preclinical evaluation of such (225)Ac-containing molecules and comprehensive assessment of their pharmacokinetics, dosimetry and radiobiology necessitate a suitable diagnostic counterpart. Due to its attractive radiation properties, (133)La represents an adequate positron-emitting radionuclide to form a matched pair with (225)Ac for macropa-based radiopharmaceuticals. Herein, we describe the preparation and radiopharmacological characterization of macropa-functionalized, (133)La/(225)Ac-labeled single-domain antibodies (sdAbs) targeting the epidermal growth factor receptor (EGFR) to demonstrate the general suitability of this theranostic pair of radionuclides. RESULTS: The synthesis of a clickable, bicyclononyne-modified macropa chelator and its site-specific conjugation to azide-modified, monovalent and biparatopic sdAbs is presented. Subsequent labeling at room temperature (rt) for 15 min resulted in molar activities of 30 MBq/nmol for (133)La and 0.5 MBq/nmol for (225)Ac, respectively. In vitro studies using the (133)La-labeled sdAbs revealed comparable binding characteristics, but an enhanced cellular internalization of the biparatopic variant compared to its monovalent counterparts. This increased uptake consequently resulted in higher cytotoxicity of the (225)Ac-labeled biparatopic conjugate. In vivo PET imaging of the (133)La-labeled conjugates indicated comparable uptake and retention of the mono- and biparatopic variants in liver and kidneys, with the former showing slightly higher tumor accumulation. Ex vivo biodistribution studies conducted with (225)Ac-labeled conjugates largely confirmed the findings obtained by PET imaging, albeit with a marginally higher tumor accumulation of the biparatopic (225)Ac-radioimmunoconjugate. Final histological examinations of tumor and kidney tissues showed DNA damage in the renal cortex of the (225)Ac-radioimmunoconjugate-treated mice, but no differences in the number of γ-H2AX-positive cells in the corresponding tumor tissues could be detected. CONCLUSIONS: We present a comprehensive study on the theranostic application of (133)La and (225)Ac for antibody-based biomolecules and lay the foundation for the future application of this matched pair of radionuclides towards labeling of heat-sensitive, macropa-functionalized radiopharmaceuticals in general. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s41181-025-00354-7.