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Identification of a Human Anti-CD55 Single-Chain Fv by Subtractive Panning of a Phage Library Using Tumor and Nontumor Cell Lines

Departments of Molecular Oncology [J. B. B. R., E. N., P. C.], Molecular Biology [J. L., J. B., A. G.], and Protein Chemistry [J. A. K.], Genentech Inc., South San Francisco, California 94080

A large naïve human single-chain (sc) Fv phage library was used to search for tumor-associated antigens by panning with a lung adenocarcinoma cell line, 1264, and counter-selecting with a nontumor bronchial epithelial cell line, BEAS-2B. After three rounds of subtractive panning, 239 of 673 clones analyzed bound selectively to 1264 tumor cells in a phage ELISA. Diversity analysis of these tumor-selective clones by BstNI fingerprinting and nucleotide sequencing revealed 14 distinct scFv fragments. Four clones bound selectively to 1264 over BEAS-2B cells when analyzed by a more discriminating flow cytometric assay using scFv. Moreover, these clones showed only limited cross-reactivity to several primary human cell lines. One clone, LU30, also cross-reacted strongly with the lung adenocarcinoma line, A549. The LU30 antigen was identified as decay-accelerating factor (CD55) by expression cloning from a 1264 cDNA library. The mean number of decay-accelerating factor molecules on the surface of 1264 and BEAS cells used for panning and counter-selection was estimated as 75,000 ± 5,000 and 13,000 ± 10,000, respectively. Thus, phage library panning combined with expression cloning permits identification of antibodies and their cognate antigens for proteins that are differentially expressed on the surface of distinct cell populations.

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