title: Phage-display selection of a human single-chain fv antibody highly specific for melanoma and breast cancer cells using a chemoenzymatically synthesized G(M3)-carbohydrate antigen.
abstract: Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-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 (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 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 G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Skaggs Institute for Chemical Biology, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Kyung Joo
authors.initials: KJ
authors.lastname: Lee
authors.forename: Shenlan
authors.initials: S
authors.lastname: Mao
authors.forename: Chengzao
authors.initials: C
authors.lastname: Sun
authors.forename: Changshou
authors.initials: C
authors.lastname: Gao
authors.forename: Ola
authors.initials: O
authors.lastname: Blixt
authors.forename: Sandra
authors.initials: S
authors.lastname: Arrues
authors.forename: Louis G
authors.initials: LG
authors.lastname: Hom
authors.forename: Gunnar F
authors.initials: GF
authors.lastname: Kaufmann
authors.forename: Timothy Z
authors.initials: TZ
authors.lastname: Hoffman
authors.forename: Avery R
authors.initials: AR
authors.lastname: Coyle
authors.forename: James
authors.initials: J
authors.lastname: Paulson
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Felding-Habermann
authors.forename: Kim D
authors.initials: KD
authors.lastname: Janda
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001943:Breast Neoplasms / Q000276:immunology*; D002240:Carbohydrate Sequence; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007129:Immunoglobulin Gm Allotypes / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D008545:Melanoma / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D013816:Thermodynamics; D014407:Tumor Cells, Cultured
chemical_list: D007128:Immunoglobulin Fragments; D007129:Immunoglobulin Gm Allotypes; D019151:Peptide Library; C055966:immunoglobulin Fv