Page 53 - 89Zr-Immuno-PET:Towards a Clinical Tool to Guide Antibody-based Therapy in Cancer
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                                Immuno-PET with 89Zr-cMab U36 in head and neck cancer
data on CT and MRI were combined, and if patients had undergone both diagnostic modalities only the best performing modality was used for analysis.
Human anti-cMAb U36 and anti-N-sucDf-cMAb U36 responses
To evaluate the immunogenicity of cMAb U36 and 89Zr-N-sucDf-cMAb U36, a human-anti-cMAb U36 (anti-isotypic) and a human anti-89Zr-N-sucDf-cMAb
U36 assay were done. Human antibody response was tested in patient sera before 3 administration of 89Zr-N-sucDf-cMAb U36 and at 1 and 6 weeks after administration. The concentrations of human anti-cMAb U36 and anti-89Zr-N- sucDf-cMAb U36 antibodies were measured, essentially as described previously
(4). In short, microtiter plates (Costar Europe LtD, Badhoevedorp, the Netherlands)
were coated with cMAb U36 IgG or with 89Zr-N-sucDf-cMAb U36 (after decay), 2
μg/well, in PBS (pH 7.2), and incubated overnight at room temperature. After
blocking with assay buffer [PBS with 1% FCS (BioWhittaker, Verviers, Belgium)
and 0.02% Tween 20 (Sigma, Zwijndrecht, the Netherlands)] and extensive
washing with wash buffer (PBS with 0.05% Tween 20), 100 μL of standard dilutions
of rabbit anti-human IgG (DAKO, Glostrup, Denmark) and diluted patient serum
(1:10 in assay buffer) were pipetted into the wells and incubated for 1 hour at room temperature. Human antichimeric antibody-positive sera from previous clinical
trials with cMAb U36 served as reference samples. The rabbit anti-human IgG was
used to construct a calibration curve. After extentive washing with wash buffer
and PBS, 100 μL biotinylated cMAb U36 or biotinylated 89Zr-N-sucDf-cMAb U36
were added (± 1 μg/well), and the plate was incubated for 1 hour at room temperature. All subsequent steps, including incubation with horseradish peroxidase-conjugated streptavidin (CLB, Amsterdam, the Netherlands) and tetramethylbenzidine substrate, as well as absorption measurement at 450 nm,
were exactly the same as previously described (4). Also, criteria for considering a
sample positive were the same (4).
STATISTICS
In this feasibility study, descriptive statistics were used with assessment of sensitivity, specificity and accuracy for 89Zr-immuno-PET as well as for the routine diagnostic modalities palpation, CT/MRI, and FDG-PET. This was considered most appropriate because (a) in routine diagnostic work up CT/MRI might have
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