Development of [⁹⁰Y]Y-CHX-A´´-DTPA-Cetuximab for combination of radioimmunotherapy and external radiotherapy: Radiochemistry and first in vivo results

Objectives:

Radiochemical development, characterisation and preparation of [⁹⁰Y]Y-CHX-A´´-DTPA-Cetuximab ([⁹⁰Y]C225). Preclinical evaluation of [⁹⁰Y]C225 by combination of radioimmunotherapy and external radiotherapy.
Methods:
CHX-A´´-DTPA was conjugated to C225 via thiourea bond formation in HEPES-buffer at pH 7.2 by reacting 20 equivalents of chelator with C225 for 24 h. Purification of the conjugate was done by ultrafiltration. Chelator to C225 ratio was determined by MALDI-TOF MS and the conjugate’s affinity determined by flow cytometric analysis. Radiolabeling was performed in MES buffer at pH 6.1 at 30°C for 30 min using [⁹⁰Y]YCl₃. Free DTPA chelator (0.1 μg/μL) was added to the reaction mixture for quenching the labeling reaction and fixing unreacted radionuclide. Ex vivo autoradiography ([⁹⁰Y]C225) was performed, in vivo kinetics were measured with PET using ⁸⁶Y as radiolabel and biodistribution studies were performed. FaDu tumour bearing nude mice were treated with [⁹⁰Y]C225 (2.8 MBq 13 g C225 / mouse, i.v.) and external beam irradiation (X-rays, single dose, 1.1 Gy/min). Experimental endpoints are the tumour growth delay and the local tumour control. Some animals are still in follow-up and not all cohorts are completed. The data are preliminary (figure 1B).
Results:
MALDI-TOF MS analysis of the purified conjugate showed that approximately 5 CHX-A´´-DTPA moieties have been conjugated to C225. The conjugate has not lost affinity to EGFR on cell surfaces when compared to the unconjugated C225 at 0.33 nM (flow cytometry). CHX-A´´-DTPA-C225 was labelled with [⁹⁰Y]YCl₃ with radiolabelling yields >95% and a reproducible specific activity of 1.2 GBq/mg was achieved. Autoradiography studies revealed high tumour accumulation 48 h p.i., also PET showed an increasing antibody accumulation in the tumour, which was abundant after 24 h p.i. These data validate the conjugate to be suitable for therapeutical studies: The combined treatment (internal and external irradiation) was well tolerated by all mice and no histological alterations in organs could be found. The preliminary data in Figure 1B clearly show a dose-effect of the external irradiation. A significant improvement of the local tumour control after external irradiation with 26 Gy was achieved after application of 13 g of radiolabelled C225 compared to unlabelled C225 or to the treatment by external irradiation solely.
Conclusions:
[⁹⁰Y]C225 has been successfully prepared, characterized and it can be produced in routine with high avidity and specific activity as prerequisite for long-term preclinical therapeutic studies. Our in-vivo preliminary data support the concept that multimodal cancer treatment results in a potentially relevant improvement of local tumour control in an experimental tumour model.
Research Support:
The Bundesministerium für Bildung und Forschung (grant 02NUK006, framework “Kompetenzverbund Strahlenforschung” (KVSF)) is gratefully acknowledged for the financial support.