Identification of a human anti-CD55 single-chain Fv by subtractive panningof a phage library using tumor and nontumor cell lines

A large naive human single-chain (sc) Fv phage library was used to search f or tumor-associated antigens by panning with a lung adenocarcinoma cell lin e, 1264, and counter-selecting with a nontumor bronchial epithelial cell li ne, BEAS-2B, After three rounds of subtractive panning, 239 of 673 clones a nalyzed bound selectively to 1264 tumor cells in a phage ELISA. Diversity a nalysis of these tumor-selective clones by BstNI fingerprinting and nucleot ide sequencing revealed 14 distinct scFv fragments. Four clones bound selec tively to 1264 over BEAS-2B cells when analyzed by a more discriminating fl ow cytometric assay using scFv, Moreover, these clones showed only Limited cross-reactivity to several primary human fell lines. One clone, LU30, also cross-reacted strongly with the lung adenocarcinoma line, A549, The LU30 a ntigen was identified as decay-accelerating factor (CD55) by expression clo ning 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 count er-selection was estimated as 75,000 +/- 5,000 and 13,000 +/- 10,000, respe ctively. Thus; phage library panning combined with-expression cloning permi ts identification of antibodies and their cognate antigens for proteins tha t are differentially expressed on the surface of distinct cell populations.