The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.
Copyright 2000 Wiley-Liss, Inc.