Phage Display Breast Carcinoma cDNA Libraries: Isolation of Clones Which Specifically Bind to Membrane Glycoproteins, Mucins, and Endothelial Cell Surface

We proposed to identify, by using a novel phage display technique, carbohydrate-binding proteins lectins, which may play an important role in progressionon and metastasis of breast cancer. Using blood-group H-expressing glycoprotein fraction as a bait, we observed selective amplification from a HeLa cell transformant phage display cDNA library of phage clones expressing sequences from galectin-3, a lectin with an affinity with the blood-group substance. We then constructed additional libraries including MCF-7 human breast carcinoma and EJG bovine capillary endothelial cell libraries. We have performed in vitro biopanning experiments using purified fractions of glycoproteins and glycolipids as baits and obtained several dozens of candidate phage clones. However, except for galectin-3, none of these candidates contained cDNA sequences encoding the same proteins. In vivo biopanning was performed using cultured EJG cells as bait. PCR amplification of the inserts from the phage lysates produced a smear of fragments rather than discrete bands even after the fifthsixth round of selections, indicating that the numbers of clones in the selected populations were still large. We will identify the clones of interest to pursue and characterize those clones in the second year.