Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines.
A large naive 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.
Ridgway
J B
JB
Department of Molecular Oncology, Genentech Inc., South San Francisco, California 94080, USA.
Ng
E
E
Kern
J A
JA
Lee
J
J
Brush
J
J
Goddard
A
A
Carter
P
P
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis*; D018958:CD55 Antigens / Q000032:analysis*; D002460:Cell Line; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000032:analysis*; D014407:Tumor Cells, Cultured
D015295:Antigens, Tumor-Associated, Carbohydrate; D018958:CD55 Antigens; D007135:Immunoglobulin Variable Region