title: Cellular and molecular properties of (90)Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro.
abstract: OBJECTIVE
  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.
authors.affiliation: Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Saki
authors.forename: M
authors.initials: M
authors.lastname: Toulany
authors.forename: W
authors.initials: W
authors.lastname: Sihver
authors.forename: M
authors.initials: M
authors.lastname: Zenker
authors.forename: J-M
authors.initials: JM
authors.lastname: Heldt
authors.forename: B
authors.initials: B
authors.lastname: Mosch
authors.forename: H-J
authors.initials: HJ
authors.lastname: Pietzsch
authors.forename: M
authors.initials: M
authors.lastname: Baumann
authors.forename: J
authors.initials: J
authors.lastname: Steinbach
authors.forename: H P
authors.initials: HP
authors.lastname: Rodemann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D061067:Antibodies, Monoclonal, Humanized; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D000068818:Cetuximab; D059186:Chemoradiotherapy, Adjuvant / Q000379:methods*; D006224:Cricetinae; D006801:Humans; D009374:Neoplasms, Experimental / Q000503:physiopathology* / Q000532:radiotherapy*; D011829:Radiation Dosage; D020266:Radiotherapy, Conformal / Q000379:methods*; D015021:Yttrium Radioisotopes / Q000008:administration & dosage*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D015021:Yttrium Radioisotopes; D000068818:Cetuximab