Isolation of peptides that inhibit binding of basic fibroblast growth factor to its receptor from a random phage-epitope library.

Abstract

Basic fibroblast growth factor (bFGF) is known to bind to its cell-surface receptors with high aitmity and in a heparin-dependent manner. In an attempt to predict the receptor recognition site on bFGF we screened phageepitope libraries with monoclonal antibodies DG2 and DE6, which inhibit bFGF binding to its receptor. On the affinityisolated phages, we identified several peptide sequences as the putative antibody-binding epitopes on bFGF. Two monoclonal antibodies (mAbs) raised against human recombinant bFGF were found to efficiently block binding of bFGF to its cell-surface receptors (17). These two mAbs, designated DG2 and DE6, inhibited bFGF-stimulated proliferation of cultured human glioma cells and retarded rat C6 glioma growth in nude mice (18). A recent study showed that the administration of neutralizing mAbs to bFGF caused significant alterations in tumor growth in vivo and that these changes were specific for tumor type and bFGF characteristics (8). The inhibitory effects of the antibodies on bFGF binding and biological activities suggest that the bFGF epitopes recognized by the antibodies may coincide with sequences recognized by the receptor. To identify these sequences we used these antibodies to screen a phageepitope library (19).The library consists of phages bearing random hexapeptides fused to the amino terminus of the coat protein PIII. Screening of the library is accomplished by the use of a monospecific binding protein, such as an antibody, to affinity-purified phages that display a high-avidity binding peptide. The amino acid sequences ofthe hexapeptides displayed on the phage are then determined by sequencing the corresponding coding region in the phage DNA (19). Here we describe the characterization of a series of hexapeptides synthesized according to the epitope sequences identified in the phage-epitope library by screening with mAbs DG2 and DE6. These peptides show a significant ability to inhibit high-affinity receptor binding and biological activity of bFGF. MATERIALS AND METHODSMaterials. Human recombinant bFGF was from Takeda (Osaka). The mAbs DG2 and DE6 have been described (17). Rabbit antibodies against human placenta alkaline phosphatase (AP) or against phage M13 were generated by immunization using a standard procedure (20). Heparin was from Hepar (Franklin, OH). The hexapeptide phage-epitope library was from G. Smith (University of Missouri, Columbia) (19).

Suppression of autocrine cell proliferation and tumorigenesis of human melanoma cells and fibroblast growth factor transformed fibroblasts by a kinase-deficient FGF receptor 1: evidence for the involvement of Src-family kinases

“…Binding, chemical cross linking and visualization of high a nity ligand-receptor complexes were as described (Li et al, 1994a;Yayon et al, 1993;Yayon and Klagsbrun, 1990b). Brie¯y, for binding activity, cells (50 000/well, 24-well plate) were washed twice with ice-cold PBS and incubated with [ 125 I]bFGF (10 ng/ml) in DMEM containing 1 mg/ml BSA and 25 mM HEPES (pH 7.4) for 2 h at 48C.…”

The epitopes for natural polyreactive antibodies are rich in proline

“…The eluate was neutralized and used to infect Escherichia coli K91 cells. After three rounds of biopanning, individual bacterial colonies were selected and amplified for DNA sequencing (14).…”

The Maltose-Binding Protein as a Scaffold for Monovalent Display of Peptides Derived from Phage Libraries

“…We have found that the "peptide-on-pins" method of producing and analyzing peptides (Chiron Mimotopes PTY Ltd., Clayton Victoria, Australia) often results in inconsistencies with the data derived from phage libraries. Similarly, others have reported that the phage itself can be important for the affinity of a selected peptide, since the corresponding synthetic peptides have reduced affinity (25)(26)(27)(28).…”