Affinity for the cognate monoclonal antibody of synthetic peptides derivedfrom selection by phage display - Role of sequences flanking the binding motif

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are gene rally further used in the form of synthetic peptides; however, as such, the ir affinity for the selecting antibody is extremely variable and factors in fluencing this affinity have not been fully deciphered. We have used an f88 .4 phage-displayed peptide library to identify ligands of mAb 11E12, an ant ibody reactive to human cardiac troponin I. A majority of the sequences thu s selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH mot if identified by multiple peptide synthesis as the critical motif recognize d by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides d erived from the phage-selected sequences was used in BIACORE to characteriz e their interaction with mAb 11E12. Most peptides exhibited affinities in t he 7-26 nm range. These affinities represented, however, only 1.9-7.5% of t he affinity of the 15-mer peptide epitope. In circular dichroism experiment s, the peptide epitope showed a propensity to have some stabilized conforma tion, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new pep tides were prepared by grafting the N- or the C-terminal sequence of the pe ptide epitope to the Y-TEPK motif of a low-affinity peptide selected by pha ge-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in compe tition ELISA), compared with the parent sequence. Thus, the sequences flank ing the motif, although not containing critical residues, convey some deter minants necessary for high affinity. The affinity of a given peptide strong ly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether hig h- or low-affinity peptides will be obtained from phage display.