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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 G(M3)-carbohydrate antigen

Academic Article
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Overview

related to degree

  • Kaufmann, Gunnar, Ph.D. in Chemistry, Scripps Research 2001 - 2006
  • Gao, Changshou, Ph.D. in Biology, Scripps Research 1994 - 2002

authors

  • Lee, K. J.
  • Mao, S. L.
  • Sun, C. Z.
  • Gao, Changshou
  • Blixt, O.
  • Arrues, S.
  • Hom, L. G.
  • Kaufmann, Gunnar
  • Hoffman, T. Z.
  • Coyle, A. R.
  • Paulson, James
  • Felding, Brunhilde
  • Janda, Kim

publication date

  • October 2002

journal

  • Journal of the American Chemical Society  Journal

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.

subject areas

  • Amino Acid Sequence
  • Antibody Specificity
  • Breast Neoplasms
  • Carbohydrate Sequence
  • Flow Cytometry
  • Humans
  • Immunoglobulin Fragments
  • Immunoglobulin Gm Allotypes
  • Kinetics
  • Melanoma
  • Molecular Sequence Data
  • Peptide Library
  • Thermodynamics
  • Tumor Cells, Cultured
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Identity

International Standard Serial Number (ISSN)

  • 0002-7863

Digital Object Identifier (DOI)

  • 10.1021/ja020737j

PubMed ID

  • 12381184
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Additional Document Info

start page

  • 12439

end page

  • 12446

volume

  • 124

issue

  • 42

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