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Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

Oliver Schmetzer^1,*,, Gerhard Moldenhauer, Rainer Riesenberg, José Ricardo Pires^||, Peter Schlag^¶ and Antonio Pezzutto^*,

^* Molecular Immunotherapy, Max Delbrück Centrum for Molecular Medicine, Berlin, Department of Hematology and Oncology, Charité, Humboldt University, Berlin, German Cancer Research Center, Division of Molecular Immunology (D050), Heidelberg, Ludwig Maximilians University Muenchen, University Clinic Grosshadern, Department of Urology, Tumor Immunology Group, Munich, ^¶ Department of Surgical Oncology, Robert Rössle Klinik, Charité, Humboldt University, Berlin, Germany; and ^|| Universidade Federal do Rio de Janeiro, Departamento de Bioquimica Medica, Rio de Janeiro, Brazil

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. ¹H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.

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