Affinity of yttrium-90 labeled anti-EGFR antibody cetuximab to head and neck squamous cell carcinoma (HNSCC) xenografts, compared to the response to combined external and internal irradiation

Objectives: Since the epidermal growth factor receptor (EGFR) is often overexpressed in HNSCCs, there is strong interest to inhibit the growth of these tumor entities via the EGFR. Thus, in this study pharmacological parameters (K_i and B_max) of the chimeric monoclonal anti-EGFR antibody cetuximab (C225), conjugated with the bifunctional chelator (BFC) CHX-A’’-DTPA (C1), labeled with the therapeutic radionuclide yttrium-90 were determined in three different HNSCC cell lines, whose EGFR expression was also estimated. Furthermore, nude mice bearing xenografts generated from these three HNSCC cells were studied in order to investigate whether internal irradiation with the radiolabeled antibody conjugate could improve the results of external irradiation alone.
Methods: C1 was conjugated to C225 via thiourea bridging. MALDI-TOF-MS revealed a BFC to C225 ratio of 4 to 1. The affinity of the yttrium-90 labeled conjugate C1-C225 was determined using membranes of the HNSCC cell lines FaDu, UT-SCC-5 and UT-SCC-8. The EGFR expression was determined by Western blotting. The tumor models were created by s.c. transplantation of pieces from the investigated tumor lines into the hind leg of nude mice (NMRI nu/nu). Different experimental groups were treated by either [⁹⁰Y]C1-C225 alone, C225 alone, external irradiation alone, external irradiation plus unlabeled C225 or external irradiation plus [⁹⁰Y]C1-C225.
Results: [⁹⁰Y]C1-C225 was routinely prepared with high specific activity (about 8 GBq/mg) and showed high affinity with K_d values in the low nanomolar range and a maximal number of binding sites (B_max) of 4.0, 4.9 and 10.4 pmol/mg in membranes of FaDu, UT-SCC-5 and UT-SCC-8 cells, respectively. In vitro the EFGR expression in these cell lines correlated well with the corresponding B_max values. In vivo the HNSCC tumors showed a different response to [⁹⁰Y]C1-C225 alone compared to unlabeled C225 receiving a specific tumor growth delay of 4.2, 0.07 and 3.7 for FaDu, UT-SCC-5 and UT-SCC-8. In FaDu the tumor control dose (TCD₅₀) was significantly decreased after external irradiation in combination with internal irradiation. In contrast to the effect in FaDu no change of the TCD₅₀ was observed in UT-SCC-5.
Conclusions: For [⁹⁰Y]C1-C225 a high affinity was determined in membranes of three different HNSCC cell lines and the B_max values correlated with the EGFR expression in vitro. Furthermore, in different HNSCC different responses to treatment were observed. However, the intertumoral heterogeneity did not correlate with the Bmax values, thus it seems that additional factors influence the response to internal irradiation.
Acknowledgements: Supported by 02NUK006A+B (Kompetenzverbund Strahlenforschung, KVSF)