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Selective interaction of a conformationally-constrained Arg-Gly-Asp (RGD) motif with the integrin receptor alphavbeta3 expressed on human tumor cells.

Abstract
Two antigenized antibodies (AgAbs) were engineered to express peptidic Arg-Gly-Asp (RGD) motifs present in extracellular matrix molecules. The RGD tripeptide sequence was inserted in the third hypervariable loop of an immunoglobulin human/mouse chimeric heavy chain gene as a single or three repeat yielding two antibodies termed gamma1RGD and gamma1(RGD)3, respectively. The antibodies were used to target specific cell-surface receptors of the integrin type expressed by three human tumor cell lines, a melanoma (M21), and osteosarcoma (KRIB) and a fibroblastoma (WI-38). Based on in vitro adhesion assays and flow cytometric analysis, we found that all three cell lines interacted with gamma1(RGD)3 but not with gamma1RGD. Binding of tumor cells to surface-immobilized gamma1(RGD)3 was inhibited in a dose-dependent manner by the RGD-containing synthetic peptides GdRGDSP and RGDS. These synthetic peptides, but no a GDR-containing control peptide, interfered with the binding of tumor cells to surface-immobilized human fibronectin. In their soluble form, neither fibronectin nor gamma1(RGD)3 inhibited tumor cell adhesion to surface-immobilized fibronectin. Gamma1(RGD)3 specifically recognized integrin alphavbeta3 based on two criteria: reactivity with purified integrin receptors and binding to variants of M21 melanoma cells expressing alphavbeta3, alphaIIbbeta3 or no beta3 integrins, respectively. Collectively, our results indicate that the (RGD)3 loop in the antigenized antibody mimics the ligand function of natural extracellular matrix proteins and has a restricted receptor specificity for the alphavbeta3 integrin which is not inherent to short RGD containing peptides.
AuthorsP Lanza, B Felding-Habermann, Z M Ruggeri, M Zanetti, R Billetta
JournalBlood cells, molecules & diseases (Blood Cells Mol Dis) Vol. 23 Issue 2 Pg. 230-41 (Aug 1997) ISSN: 1079-9796 [Print] UNITED STATES
PMID9268674 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • Fibronectins
  • Immunoglobulin Heavy Chains
  • Oligopeptides
  • Peptide Fragments
  • Receptor-CD3 Complex, Antigen, T-Cell
  • Receptors, Vitronectin
  • Recombinant Fusion Proteins
  • arginyl-glycyl-aspartic acid
Topics
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Adhesion (drug effects)
  • Fibronectins (chemistry)
  • Genes, ras
  • Genetic Variation
  • Humans
  • Immunoglobulin Heavy Chains (biosynthesis, chemistry, genetics)
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Oligopeptides
  • Peptide Fragments (chemistry, pharmacology)
  • Protein Conformation
  • Receptor-CD3 Complex, Antigen, T-Cell (biosynthesis, chemistry)
  • Receptors, Vitronectin (biosynthesis, chemistry, genetics)
  • Recombinant Fusion Proteins (biosynthesis, chemistry)
  • Transfection
  • Tumor Cells, Cultured

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