Structure-function correlation and biostability of antibody CDR-derived peptides as tumour imaging agents.

Abstract

Based on the CDR3 V(H) sequence of a monoclonal antibody (ASM2) raised against epithelial cancer cells, the synthetic peptide YCAREPPTRTFAYWG (EPPT1) has been found to have an appreciable affinity (Kd = 20 microM) for the deglycosylated mucin-derived peptide antigen YVTSAPDTRPAPGST (PDTRP). The technetium-radiolabelled form of this peptide has been found to be a good tumour-imaging candidate for diagnosis of breast carcinoma. Several EPPT1 peptide analogues were synthesised. A differential biostability was obtained blocking the end groups of EPPT1. The susceptibility to proteolytic degradation was significantly decreased for the C-amidated form of EPPT1 than the N-acetylated form. Using resonant mirror biosensor technique, the EPPT1 analogues were classified as active and non-active peptides according to their PDTRP-binding properties. The binding of EPPT1 to PDTRP in free solution was also determined unambiguously by CD spectroscopy. CD spectra of both active and non-active peptides showed the presence of irregular conformations in H2) and SDS above cmc. In TFE, significant degree of ordered conformations of alpha-helix or beta-turn type were induced but did not correlate well with their binding properties. In SDS below cmc a conformational difference was observed between the active and non-active peptides. The active peptides exhibited CD spectra of aggregation of beta-strand type whilst the non-active showed CD spectra similar to those in H2O and SDS above cmc, critical micelle concentration. A good correlation between the extended conformation of beta-strand type and the binding affinity of the active peptides suggests this conformation as the binding feature of the EPPT tumour-imaging peptides. These information are vital for the design of novel EPPT analogues. Any modification to improve binding affinity must retain the ability of the peptides to adopt the extended conformation of beta-strand type.