Potential of a Cetuximab-based radioimmunotherapy combined with external irradiation manifests in a 3-D cell assay.

Targeting epidermal growth factor receptor (EGFR)-overexpressing tumors with radiolabeled anti-EGFR antibodies is a promising strategy for combination with external radiotherapy. In this study, we evaluated the potential of external plus internal irradiation by [(90) Y]Y-CHX-A″-DTPA-C225 (Y-90-C225) in a 3-D environment using FaDu and SAS head and neck squamous cell carcinoma (HNSCC) spheroid models and clinically relevant endpoints such as spheroid control probability (SCP) and spheroid control dose 50% (SCD50 , external irradiation dose inducing 50% loss of spheroid regrowth). Spheroids were cultured using a standardized platform. Therapy response after treatment with C225, CHX-A"-DTPA-C225 (DTPA-C225), [(90) Y]Y-CHX-A"-DTPA (Y-90-DTPA) and Y-90-C225 alone or in combination with X-ray was evaluated by long-term monitoring (60 days) of spheroid integrity and volume growth. Penetration kinetics into spheroids and EGFR binding capacities on spheroid cells were identical for unconjugated C225 and Y-90-C225. Spheroid-associated radioactivity upon exposure to the antibody-free control conjugate Y-90-DTPA was negligible. Determination of the SCD50 demonstrated higher intrinsic radiosensitivity of FaDu as compared with SAS spheroids. Treatment with unconjugated C225 alone did not affect spheroid growth and cell viability. Also, C225 treatment after external irradiation showed no additive effect. However, the combination of external irradiation with Y-90-C225 (1 µg/ml, 24 hr) resulted in a considerable benefit as reflected by a pronounced reduction of the SCD50 from 16 Gy to 9 Gy for SAS spheroids and a complete loss of regrowth for FaDu spheroids due to the pronounced accumulation of internal dose caused by the continuous exposure to cell-bound radionuclide upon Y-90-C225-EGFR interaction.

Cellular and molecular properties of (90)Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro.

PURPOSE: Anti-EGFR antibody cetuximab (C225) is used in combination with radiotherapy of head and neck squamous cell carcinoma (HNSCC) patients. We investigated whether conjugation of cetuximab with trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA) and radiolabeling with (90)Yttrium affect the molecular and cellular function of cetuximab and improve its combined effect with external-beam irradiation (EBI). METHODS: The following cell lines were used: HNSCC UT5, SAS, FaDu, as well as A43, Chinese hamster ovary cells (CHO), and human skin fibroblast HSF7. Binding affinity and kinetics, specificity, retention, and the combination of (90)Y-cetuximab with EBI were evaluated. RESULTS: Control cetuximab and CHX-A″-DTPA-cetuximab blocked the proliferation activity of UT5 cells. In combination with EBI, CHX-A″-DTPA-cetuximab increased the radiosensitivity of UT5 to a similar degree as control cetuximab did. In contrast, in SAS and HSF7 cells neither proliferation nor radiosensitivity was affected by either of the antibodies. Binding [(90)Y]Y-CHX-A″-DTPA-cetuximab ((90)Y-cetuximab) to EGFR in HNSCC cells occurred time dependently with a maximum binding at 24 h. Retention of (90)Y-cetuximab was similar in both HNSCC cell lines; 24 h after treatment, approximately 90% of bound activity remained in the cell layer. Competition assays, using cell membranes in the absence of an internalized fraction of cetuximab, showed that the cetuximab affinity is not lost as a result of conjugation with CHX-A″-DTPA. Cetuximab and CHX-A″-DTPA-cetuximab blocked EGF-induced Y1068 phosphorylation of EGFR. The lack of an effect of cetuximab on EGF-induced Akt and ERK1/2 phosphorylation and the inhibition of irradiation (IR)-induced Akt and ERK1/2 phosphorylation by cetuximab were not affected by DTPA conjugation. (90)Y-cetuximab in combination with EBI resulted in a pronounced inhibition of colony formation of HNSCC cells. CONCLUSIONS: Conjugation of CHX-A″-DTPA to cetuximab does not alter the cellular and biological function of cetuximab. (90)Y-labeling of cetuximab in combination with EBI may improve radiotherapy outcome.

Flow cytometric cell-based assay to preselect antibody constructs for radionuclide conjugation.

Radiolabeled antibodies (Abs) are an attractive tool for targeting and delivering particle emitters for therapy or imaging applications. The labeling of Abs with metal radionuclides requires chelating agents and can cause loss of binding to their ligands. The aim of the present approach was to design an easy-handling flow cytometric cell-based assay to evaluate Ab-binding capacity of conjugates of the therapeutic Ab Cetuximab and to verify the most promising candidate in a competitive radioactive binding experiment. The final setup for flow cytometric assessment of cellular binding capacities of epidermal growth factor receptor (EGFR)/ErbB1-directed Ab conjugates is based on (a) the selection of a robust cell line model (b) the definition of nonsaturated staining concentrations for the unconjugated reference Ab Cetuximab plus implementation of a reasonable isotype control, and (c) the calculation of relative Ab affinities based on the flow cytometric data. Two (FaDu, SAS) out of the three cell lines with different total and cell surface expression levels of EGFR turned out to be adequate models but the application of one cell line was sufficient to estimate reduced binding capacities of conjugates relative to Cetuximab. Only 1/11 conjugate Abs exhibited a fluorescence signal comparable to unconjugated Cetuximab and was applied for radiolabeling with Yttrium-90. Unaltered binding affinity of this conjugate was proven in a competitive radioactive Ab-binding study. We conclude that the flow cytometric assay is reliable and that the relative binding capacity of Cetuximab is neither affected by covalent modification with CHX-A"-DTPA (N-[(R)-2-Amino-3-(p-isothiocyanato-phenyl) propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N",N"-pentaacetic acid) with a final chelator-to-Ab ratio of 5 nor by subsequent radiolabeling. [(90)Y]Y-CHX-A"-DTPA-Cetuximab thus qualifies for preclinical treatment testing as a prerequisite for therapeutic application.