Page 50 - 89Zr-Immuno-PET:Towards a Clinical Tool to Guide Antibody-based Therapy in Cancer

Chapter 3
Monoclonal antibody U36
Selection and production of MAb U36 and its chimeric (mouse/human) IgG1 derivative (cMAb U36) have been described previously (4). MAb U36 binds to the v6 region of CD44 (CD44v6). Homogeneous expression of CD44v6 has been observed in squamous cell carcinoma of the head and neck, lung, skin, esophagus, and cervix, whereas heterogeneous expression was found in adenocarcinomas of the breast, lung, colon, pancreas, and stomach. In normal tissues, expression has been found in epithelial tissues such as skin, breast, and prostate myoepithelium, and bronchial epithelium (13).
Production of 89Zr-cMAb U36
The production and purification of 89Zr and its coupling to cMAb U36 have been described previously (9). 89Zr was produced by a (p,n) reaction on natural yttrium (89Y).
Labeling of cMAb U36 was achieved starting from the chelate desferrioxamine B (Df; desferal, Novartis, Basel, Switzerland). All procedures were done under aseptic conditions in a shielded laminar flow hood. In short, Df was succinylated (N-sucDf), temporarily filled with stable iron [Fe(III)], and coupled to the lysine residues of cMAb U36 by means of a tetrafluorophenol-N-sucDf ester. After removal of Fe(III) by transchelation to EDTA, the premodified cMAb U36 was purified on a PD10 column. Subsequently, N-sucDf-cMAb U36 (5 mg) was labeled with 89Zr (185 MBq). Finally, 89Zr-N-sucDf-cMAb U36 was purified on a PD10 column [eluent: 0.9 % NaCl/gentisic 5 mg/mL (pH 5.0)]. 89Zr-N-sucDf- cMAb U36 will be abbreviated to 89Zr-cMAb U36 in the rest of this article. The mean labeling efficiency was 87.4 ± 11.0%. Finally, cold cMAb U36 and for 14 of the 20 patients 186Re-MAG3-cMAb U36 were added and the conjugates were filter sterilized (total amount of cMAb U36 to be administered was 10 mg). These procedures resulted in a sterile final product with endotoxin levels < 5 EU/mL. The molar ratio N-sucDf to cMAb U36 was always <2. The radiochemical purity was always >94.9 % (mean, 96.0 ± 1.2%). After each preparation of 89Zr-cMAb U36, the immunoreactivity was determined by measuring binding to a serial dilution of UM-SCC-11B cells as described previously (10). The immunoreactive fraction of the 89Zr-cMAb U36 preparations ranged from 74.8% to 91.0% (mean, 85.1 ± 4.5%) at the highest cell concentration. In addition, the data were graphically analyzed in a modified Lineweaver-Burk plot and the immunoreactivity was determined by extrapolating to conditions representing infinite antigen excess. By doing so, the immunoreactive fraction ranged from 89.4% to 100 % (mean, 98.7 ± 3.1%).
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