Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display

Abstract

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f 88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phageselected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7±26 nm range. These affinities represented, however, only 1.9±7.5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N-or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phagedisplay. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high-or low-affinity peptides will be obtained from phage display.

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

“…Phages specific for aAb T13 were selected by biopanning as described previously (46). Antibody-bound phages were eluted (i) by 3 ml of acid elution (0.1 M glycine-HCl (pH 2.2), 1 mg/ml bovine serum albumin) for 30 min at room temperature and further neutralization by adding 300 l of 2 M Tris/HCl, pH 9.2; (ii) by competition with a 180 nM (20 g/ml) TPO solution in 5 ml of NaCl/Tris (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 2 h at 37°C.…”

“…Antibody-bound phages were eluted (i) by 3 ml of acid elution (0.1 M glycine-HCl (pH 2.2), 1 mg/ml bovine serum albumin) for 30 min at room temperature and further neutralization by adding 300 l of 2 M Tris/HCl, pH 9.2; (ii) by competition with a 180 nM (20 g/ml) TPO solution in 5 ml of NaCl/Tris (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 2 h at 37°C. Phages were amplified after each round of panning using an exponentially growing culture of Escherichia coli K91 cells, and T13-specific phages were cloned and characterized by ELISA as described (46). Clones giving an absorbance more than twice the background level were selected.…”

Low Molecular Weight Peptides Restore the Procoagulant Activity of Factor VIII in the Presence of the Potent Inhibitor Antibody ESH8

“…Eluted phages were then used to infect Escherichia coli K91 cells. After three rounds of enrichment, individual phage clones were isolated and further analyzed (17).…”

Anti-Peptide Antibody Blocks Peptide Binding to MHC Class I Molecules in the Endoplasmic Reticulum

“…Surface plasmon resonance analysis was used to measure the association and dissociation affinity constants for the binding of mAb IF10.2.2 to immobilized SIINFEKL by using BIACORE X (Biacore, Uppsala, Sweden) as described previously (18).…”