文摘
Overexpression of the cell-surface glycosphingolipid GM3 is associated with a number of differentcancers, including those of the skin, colon, breast, and lung. Antibodies against the GM3 epitope havepotential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two GM3-derived reagents and their use in the panning of a phage-displayed humansingle-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones,GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized usingBIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociationconstants (Kd) ranging from 4.2 × 10-7 to 2.1 × 10-5 M. Flow cytometry was used to evaluate the abilityof each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanomacells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayedcross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activitytoward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showedno measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic GM3 panningreagent can be used to isolate a fully human scFv that is highly specific for native GM3 on the surface oftumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.