In vitro and in vivo evaluation of yttrium-90 labelled cetuximab in combination with external X-ray irradiation – radiooncology and nuclear medicine shake hands

Introduction
Results from preclinical and clinical trials indicate that combination of the Epidermal Growth Factor Receptor (EGFR) specific antibody Cetuximab (C225) with classical radiotherapy improves local tumor control and overall survival of patients with head and neck cancers^1-4. First results of Meller et al. showed a synergistic effect of ¹³¹I-labelled C225 in combination with external irradiation in vitro⁵. These data indicate that the combination of internal radiation dose delivered by radiolabelled C225 plus the therapeutic effect of C225 in combination with radiotherapy might significantly lower the overall external dose applied to patients, reducing undesired side effects on healthy tissue. Thus, bimodal cancer therapy seems a promising approach. This encouraged us to investigate the combined effect of ⁹⁰Y[Y]-CHX-A"-DTPA-Cetuximab (⁹⁰Y-C225) and radiation dose delivered by external beam irradiation in vitro and in vivo in our joint project.

Materials and Methods
CHX-A´´-DTPA was conjugated to C225 via thiourea bridging. Radiolabelling was performed under mild conditions using [⁹⁰Y]YCl₃. Binding affinity of ⁹⁰Y-C225 was studied by flow cytometric analysis as well as by competitive binding assays using cells in 2D and 3D culture or cell membrane preparations. Distribution in spheroids (FaDu) was studied in a concentration- and time-dependent manner. Cellular uptake, EGFR binding specificity and kinetics, retention of ⁹⁰Y-C225 as well as clonogenic activity of ⁹⁰Y-C225 treated cells in combination with external irradiation were studied. The capacity of ⁹⁰Y-C225 to induce DNA double-strand breaks (DSBs) was assessed through the Gamma-H2AX/53BP1 foci technique and cell survival by colony formation. Ex vivo autoradiography was performed with ⁹⁰Y-C225. Biodistribution and in vivo kinetics were measured with PET using ⁸⁶Y as radiolabel. FaDu tumour bearing nude mice were treated with ⁹⁰Y-C225 (2.8 MBq 13 μg C225 / mouse, i.v.) and external beam irradiation (20 Gy single dose, 1.1 Gy/min). Experimental endpoints are the tumour growth delay and the local tumour control after 180 d.

Results
Radiolabelling of the conjugate resulted in specific activities up to 9 GBq/mg. For the present study, a labelling protocol to achieve a reproducible specific activity of 1.2 GBq/mg was established.

in vitro: Flow cytometric analysis showed that affinity of ⁹⁰Y-C225 is not significantly reduced compared to native C225. A K_i of 0.4 nM vs 0.22 nM for native C225 was determined by a competitive binding assay. Saturation of FaDu spheroids with ⁹⁰Y-C225 was achieved after 24 h of incubation at 5 μg/ml. A saturation test showed that the optimal concentration to block all EGFR (SAS and UT5 cells) by C225 in monolayer culture is about 3-5 nM. No unspecific binding on an EGFR-negative CHO cell line was observed. Binding ⁹⁰Y-C225 to EGFR in HNSCC cells occurred time dependently with a maximum after 24 h. 24 h after treatment approximately 10% of bound activity was found in the supernatant. Conjugation of CHX-A"-DTPA to C225 does not alter the cellular and biological function of C225. Combination of single doses of 2 or 4 Gy with ⁹⁰Y-C225 reduced clonogenic survival in the UT5 and SAS cell lines. For UT5 cells the D37 value for non-treated control cells was 3.7 Gy with X-rays. Treatment with C225 reduced D37 of UT5 cells to 2.7 Gy. When ⁹⁰Y-C225 was combined with X-rays, the D37 of UT5 cells dropped to 1.8 Gy. Thus, in comparison to C225, a decrease of D37 by about 33 % was observed in cells treated with ⁹⁰Y-C225. D37 of untreated cells was only reduced from 3.9 Gy to 3.7 Gy after incubation with native C225. A D37 of 3.3 Gy resulted upon treatment with ⁹⁰Y-C225 which reflects only a reduction of about 11%, compared to C225.
Induction of DNA DSBs by ⁹⁰Y-C225 in cell lines with varying EGFR expression showed that the amount of ⁹⁰Y-C225 binding and the number of induced DSBs is proportional to the EGFR on the cell membrane and that the cytotoxicity is dependent on the number of residual DSB (clonogenic survival).

in vivo: Autoradiography revealed high tumour accumulation 48 h p.i., also PET showed an increasing accumulation of activity in the tumour, which was abundant after 24 h p.i.
The combined treatment was well tolerated by all mice and no histological alterations in organs were found. A clear dose-dependent effect of the external irradiation was observed. A significant improvement of the local tumour control after X-ray irradiation with 20 Gy was achieved after application of 13 μg of ⁹⁰Y-C225 compared to native C225 or to the treatment by external irradiation alone.

Conclusion
The results from in depth investigations of the effects of ⁹⁰Y-C225 on various EGFR expressing cell lines validate it as a powerful tool for in vivo studies. The combined treatment of tumours in our experimental mouse model permits a reduction of the external radiation dose of 12 Gy. Our in-vivo data support the concept that bimodal cancer treatment results in a potentially relevant improvement of local tumour control. This encourages us to follow this promising scientific concept.

Research Support: The Bundesministerium für Bildung und Forschung (grant 02NUK006, framework “Kompetenzverbund Strahlenforschung” (KVSF)) is gratefully acknowledged for the financial support.

References
1 Bonner JA, Harari PM, Giralt J et al. [2010] Lancet Oncol.; 11: 21-8.
2 Bonner JA, Harari PM, Giralt J et al. [2006] N Engl J Med.; 354: 567-78.
3 Curran D, Giralt J, Harari PM et al. [2007] J Clin Oncol.; 25: 2191-7.
4 Zhang N, Erjala K, Kulmala J et al. [2009] Radiother Oncol.; 92: 388-92.
5 Meller B, Rades D, Wolff C et al. [2009] IJROBP 75: 1226-1231.