In order to provide additional information about the long-term colony-forming ability of IGROV1 cells after mono- (177Lu-DOTA-chCE7) or combined (PTX?+?177Lu-DOTA-chCE7) treatments, colony assays were assessed. a prolonged overall survival of human ovarian carcinoma-bearing nude mice compared with either monotherapy. The combination is promising for future clinical applications. Electronic supplementary material Tirapazamine The online version of this article Tirapazamine (doi:10.1186/s13550-014-0054-2) contains supplementary material, which is available to authorized users. and [15,25,26]. The antibody-antigen complex internalises into the targeted cell through endocytosis. We demonstrated that a 177Lu-labelled variant of mAb chCE7 showed high efficacy in a xenograft model of disseminated ovarian carcinoma [25]. Preclinical studies have demonstrated that combined Rabbit Polyclonal to ACTL6A treatments including RIT and radiosensitising taxanes such as paclitaxel (PTX) can be advantageous compared to monotherapies [27-29]. PTX belongs to the group of microtubule-stabilising agents and induces apoptosis and arrest of tumour cells in the radiosensitive G2/M phase of the cell cycle based on suppression of microtubule dynamics. Furthermore, it was shown that PTX influences the tumour microenvironment, resulting in reoxygenation of the tumour potentially providing radiosensitising effects [30,31]. In this study, we investigated whether the efficacy of previously developed anti-L1CAM 177Lu-RIT against ovarian carcinoma can be further increased by its combination with the radiosensitising taxane PTX. Methods Cell culture and antibody formats IGROV1 human ovarian cancer cells were kindly provided by Dr. Cristina Mller (Center for Radiopharmaceutical Sciences, Paul Scherrer Institute) and analysed by STR profiling (DSMZ, Braunschweig, Germany). IGROV1 cells were maintained in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium at 37C. The medium was supplemented with 10% fetal calf serum (FCS), 2?mM glutamine, 100 units/ml penicillin, 100?g/ml streptomycin and 0.25?g/ml fungizone (BioConcept, Allschwil, Switzerland). mAb chCE7 is a IgG1-subtype chimeric monoclonal antibody (human light chain and human 1 Tirapazamine heavy chain). It was produced in HEK293 cells and purified from cell culture supernatant using a protein G-Sepharose column (GE Healthcare, Glattbrugg, Switzerland) as described by Grnberg et al. [32]. An unspecific isotype-matched IgG was used as a control for experiments. Ligand substitution and antibody radiolabelling Ligand substitution was performed as previously described by Fischer et al. [25]. For ligand conjugation, the molar excess of p-SCN-Bn-DOTA (Macrocyclics, Dallas, TX, USA) was adapted individually for each antibody to achieve similar DOTA ligands to mAb ratios. The reaction mixture was adjusted to pH?9 to 10 using a saturated Na3PO4 solution and was incubated for 16?h at 4C. Excess ligands were removed and buffer was exchanged into 0.25?M CH3COONH4 (pH?5.5) using a NAP-5 column (GE Healthcare, Glattbrugg, Switzerland). Immunoconjugates were stored at Tirapazamine ?80C. The average number of coupled chelators per mAb was determined by mass spectrometry as previously described [25]. 177Lu (ITG, Garching, Germany) was utilised for radiolabelling 1 to 3?days post calibration date. Briefly, a reaction mixture containing 250 to 900?g of the immunoconjugates and 200 to 600?MBq 177Lu was incubated in 0.25?M CH3COONH4 buffer (pH?5.5) for 1?h at 37C. After incubation, EDTA was added to a final concentration of 5?mM for 5?min in order to complex free lutetium. Radioimmunoconjugates (RICs) were purified via FPLC size exclusion chromatography on a Superose 12 column (GE Healthcare, Glattbrugg, Switzerland) in phosphate-buffered saline (PBS) with a flow rate of 0.5?ml/min. Both radiolabelled chCE7 and unspecific control IgG eluted at a retention time of 21?min. In order to test the stability of 177Lu-labelled antibodies, RICs were incubated in human plasma at 37C and analysed by FPLC size exclusion chromatography on a TSKgel G3000Wxl column (Tosoh Bioscience, Stuttgart, Germany). The flow rate of the mobile phase (0.3?M NaCl, 0.05?M Na2HPO4, pH?6.2) was set to 1 1?ml/min (Additional file 1: Figure S1). FACS cell cycle analysis upon PTX treatment For cell cycle analysis, IGROV1 cells.