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Phage-Display Selection of a Human Single-Chain Fv Antibody Highly Specific for Melanoma and Breast Cancer Cells Using a Chemoenzymatically Synthesized GM3−Carbohydrate Antigen

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posted on 28.09.2002 by Kyung Joo Lee, Shenlan Mao, Chengzao Sun, Changshou Gao, Ola Blixt, Sandra Arrues, Louis G. Hom, Gunnar F. Kaufmann, Timothy Z. Hoffman, Avery R. Coyle, James Paulson, Brunhilde Felding-Habermann, Kim D. Janda
Overexpression of the cell-surface glycosphingolipid GM3 is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the GM3 epitope have potential 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 human single-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 using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (Kd) ranging from 4.2 × 10-7 to 2.1 × 10-5 M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic GM3 panning reagent can be used to isolate a fully human scFv that is highly specific for native GM3 on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.

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