Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161comparedtolutetium-177 in an ovarian cancer therapy model

Jürgen Grünberg1, Dennis Lindenblatt1, Holger Dorrer2, Susan Cohrs1, Konstantin Zhernosekov3, Ulli Köster4, Andreas Türler 2,5, Eliane Fischer1, and Roger Schibli1,6

1Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, Villigen-PSI, Switzerland; 2Laboratory of Radiochemistry and Environmental Chemistry, Paul Scherrer Institute, Villigen-PSI, Switzerland; 3ITM Isotopen Technologien München AG, Garching, Germany; 4Institut Laue-Langevin, Grenoble, France; 5Department of Chemistry and Biochemistry, University of Bern, Berne, Switzerland; 6Department of Chemistry and Applied Biosciences, ETH, Zurich, Switzerland

For correspondence or reprints contact: Roger Schibli, Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland; Phone +41-(0)56-3102837. Fax +41-(0)56-3102849. Email:

This work was supported by the Cancer Research Foundation (Project No. KFS-2546-02-2010) to Jürgen Grünberg

Cell culture conditions and antibodies

IGROV1 cells were cultured in RPMI 1640 medium at 37 0C in a humidified atmosphere containing 5% CO2. The medium was supplemented with 10% fetal calf serum (FCS), 2 mM of glutamine, 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 0.25 mg/mL of fungizone. All media and additives were obtained from BioConcept (Allschwil, Switzerland).

The anti-L1CAM mAb chCE7 is a chimeric monoclonal antibody of the IgG1 subtype and was produced in HEK-293 cells and purified from cell culture supernatant as previously described (Grünberg et al. 2003). As controlsnon specific isotope matched IgGs were used.

L1-CAM expression on IGROV1 cells

L1-CAM expression on IGROV1 cells was analyzed via an indirect immunofluorescence staining. Cells were detached with PBS/EDTA and resuspended in ice cold PBS (1.25 × 106 cells/0.2 ml). After incubation with the primary mAb chCE7 (1 μg/ml, 3% BSA/PBS) for 2 h, cells were washed with PBS and resuspended in 3% BSA/PBS containing a fluorochrome-conjugated secondary antibody (goat anti-human-FITC, 1:200, Sigma-Aldrich, Buchs, Switzerland). After incubation for 30 min at room temperature (RT) in the dark, cells were washed twice with PBS and resuspended in 0.2ml PBS for flow cytometry analysis (Guava easyCyte HT flow cytometer, Millipore, Zug, Switzerland). Resutls were evaluated with FlowJo software (Tree Star, Ashland, USA, version 9.7.1).

Fig. 1 Expression of the tumour-associated L1-CAM antigen on the surface of IGROV1 cells. L1-CAM expression (98.6%) is demonstrated by increased fluorescence intensity. Untreated control samples were used for background fluorescence.

Stability of the labelled antibodies after incubation in human plasma at 37 0C was analysed by FPLC size exclusion chromatography on a TSKgel G3000Wxl column (Tosoh Bioscience, Stuttgart, Germany) with sodium phosphate buffer (0.3 M NaCl, 0.05 M Na2HPO4, pH 6.2) as mobile phase at a flow rate of 1 mL/min.

Statistical analysis

For biodistribution experiments analysis of data wasperformed using the Student’s t test (unpaired, two-tailed). To evaluate the anti-tumour effects the percentage of tumour growth inhibition (%TGI) was calculated. TGD is calculated as the time in days required for the relative tumour volume (RTV) to increase threefold over the initial RTV at therapy start. The equation 100 – (T/C x 100), T = mean RTV treatment group, C = mean RTV control group, both at the time when the first mouse in the control group reached the humane endpoint gave the percentagetumour growth inhibition (TGI). A TGI > 42% was considered as effective, inhibition of more than 90% was considered as highly effective according to the National Cancer Institute standard (Bissery et al. 1991). The mean RTVs in treated and control groups were compared using the Student’s t test (unpaired, two-tailed). Statistical significance was defined as p < 0.05, highly significant as p < 0.001.

Representative picture of a mouse 13 days after receiving adose one level above the MTD

Fig.2 Representative picture of a mouse treated with adose one level above the MTD 13 days after injection of 14 MBq 177Lu-DOTA-chCE7. Low number of white and red blood cells characterizes the mouse. Bleeding under the skin is seen (red dots).We dissected all mice in the treatment groups one dose level over the MTD and found in some of them blood in the gastrointestinal tract and an altered spleen.

Biodistribution of 177Lu-DOTA-chCE7 144 h after injection in the censored mouse

Organ / %IA/g
Tumour / 14.5
Blood / 1.6
Liver / 6.5
Spleen / 7.2
Kidney / 1.6
Heart / 1.1
Stomach / 0.4
Intestine / 0.7
Muscle / 0.8
Bone / 1.6

Grünberg et al. (2003) Clin. Cancer Res. 11: 5112-5120

Bissery et al. (1991) Cancer Res. 51: 4845-4852

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