Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody.
Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteine-constrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.
De Ciechi
P A
PA
G. D. Searle & Company, Chesterfield, MO 63198, USA.
Devine
C S
CS
Lee
S C
SC
Howard
S C
SC
Olins
P O
PO
Caparon
M H
MH
D000595:Amino Acid Sequence; D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D018122:B7-1 Antigen / Q000235:genetics; D001483:Base Sequence; D002614:Chelating Agents; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D006801:Humans; D017912:Inovirus / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D018122:B7-1 Antigen; D002614:Chelating Agents; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D004247:DNA