Paola Fortugno

Selection of biologically active peptides by phage display of random peptide libraries.

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein-protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.

Identification of tumor-associated antigens by screening phage-displayed human cDNA libraries with sera from tumor patients

Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage‐display technology. We designed a new lambda vector to express protein fragments as N‐terminal fusions to the D capsid protein and generated high‐complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage‐displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients.

Affinity maturation of ligands for HCV-specific serum antibodies.

We have previously screened a phage-displayed random peptide library using sera from patients and identified ligands binding to antibodies specifically associated with the hepatitis C virus infection. The ability of these peptides to detect HCV-specific antibodies was improved through an in vitro procedure which mimics the natural process of antibody affinity maturation operating in secondary immune response. Libraries were generated by mutating the sequence of the original peptide through a protocol that efficiently introduced substitution, insertion and deletion mutations on a single or population of clones. Screening these libraries isolated mutants that displayed increased specific reactivity with a broader range of sera from HCV-infected patients. Several variants of the original peptide were identified which discriminate between the various components of the specific polyclonal response. This methodology to select artificial ligands from RPL using sera and to enhance their diagnostic properties by affinity maturation makes the development of a diagnostic assay to detect disease-associated antibodies feasible, without requiring the natural antigen.

Antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of Entamoeba histolytica.

Entamoeba histolytica is the protozoan parasite responsible for intestinal amoebiasis and amoebic liver abscess, which cause significant morbidity and mortality in many countries of the world. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) represent dominant surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against these components protected SCID mice from amoebic liver abscess, so PPGs might be regarded as vaccine candidates; however, their structure is very complex and only known in part. They are glycosylphosphatidylinositol‐linked polypeptides of unknown sequence carrying glycan side‐chains linked to serine residues via phosphodiester bonds. In order to identify peptide mimics of the E. histolytica PPG antigens, we screened six different phage‐displayed random peptide libraries with the antibody EH5. Various peptide mimics of different length were identified and, in all the peptides, a distinct consensus sequence Gly‐Thr‐His‐Pro‐X‐Leu could be identified. The phages strongly bound to the antibody, and the natural antigen inhibited binding of the phages to antibody EH5. In addition, several of the phages induced a significant immunoglobulin G response against amoebic antigens in immunized mice.