DNA-based selection and screening of peptide ligands.

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.

Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage.

Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a "secondary" library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

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.

Epitope discovery using peptide libraries displayed on phage.

Peptides displayed on phage, which mimic continuous and discontinuous epitopes, can be selected using purified antibodies or preparations of polyclonal serum. This review describes recent advances in this field, discusses the application of phage-display technology to the diagnosis of human diseases, and presents new ideas for the preparation of vaccines directed against specific epitopes on a pathogen.

Recognition by human sera and immunogenicity of HBsAg mimotopes selected from an M13 phage display library.

We used two mouse monoclonal antibodies (mAb) specific for the human hepatitis B virus surface antigen (HBsAg) to screen a random peptide library of 15 amino-acid residues displayed as a fusion to protein III of filamentous phage M13. By a combination of affinity selection, immuno-screening and ELISA techniques, we selected peptides that are recognized by the anti-HBsAg mAb and show aa similarity with the natural antigen. The selected phage-displayed epitopes (phagotopes) behave as antigenic mimics of HBsAg. One phagotope is specifically recognized by human sera from HBsAg-immunized individuals, pointing to the possible use of phagotopes as markers to detect the presence of specific Ab in the serum. The same phagotope also elicits Ab directed against HBsAg in mice, indicating that mAb-selected phagotopes can also be immunogenic mimics of the natural antigen. These findings demonstrate that it is possible to identify disease-specific epitopes that can be used as diagnostic reagents and as leads for the development of acellular vaccines.

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology.

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

"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.

Phage as gene delivery vectors.

Bacteriophage can be considered as a natural system to efficiently condense and package DNA. They tolerate many different types of mutations, including those that lead to the display of polypeptides as a fusion to any of the structural proteins comprising the phage particle. In addition, they are a powerful biological system for generating and screening mutants with the desired functional properties. It has also been shown that phage vectors can be engineered for receptor-mediated gene transfer to mammalian cells. The attractive features offered by this system have paved the way for various attempts to develop phage as a vector for gene therapy applications.

Peptide and protein display on the surface of filamentous bacteriophage.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

A bipartite activation domain is responsible for the activity of transcription factor HNF1/LFB1 in cells of hepatic and nonhepatic origin.

HNF1/LFB1 is a transcription factor that controls the expression of several liver-specific genes. Previous in vitro experiments allowed us to identify two different regions in the carboxy-terminal portion of the protein responsible for most of the transcription activation potential: the first, ADI, between amino acids 546 and 628 and the second, ADII, between amino acids 281 and 318. To characterize the molecular anatomy of HNF1/LFB1 better, we have analyzed its trans-activating properties in vivo. Several HNF1/LFB1 deletion mutants were tested for their ability to induce transcription from HNF1/LFB1-dependent synthetic promoters in cells of hepatic and nonhepatic origin. These last recipient cells provide an HNF1/LFB1-deficient environment that is useful for a precise quantification of the recombinant protein. Our results confirm the importance of ADI and indicate that no activating property can be assigned to ADII in vivo. Moreover, a novel glutamine/proline-rich activation domain (ADIII) has been identified between amino acids 440 and 506. These findings are confirmed by domain-swapping experiments, carried out with the heterologous GAL4 DNA-binding domain, which also show that the activity of each individual activation domain is influenced by combining adjacent HNF1/LFB1 sequences. The data presented indicate that HNF1/LFB1 transcription activating potential relies on a complex structure and also provide important clues to understanding the different functions exerted by transcription factors of this family.

Adenovirus as vehicle for anticancer genetic immunotherapy.

Adenoviruses (Ads) are in the forefront of genetic immunization methods being developed against cancer. Their ability to elicit an effective immune response against tumor-associated antigens has been demonstrated in many model systems. Several clinical trials, which use Ad as vehicle for immunization, are already in progress. Preclinical studies have also demonstrated the efficacy of combining Ad-mediated immunization with adjuvants such as chemotherapeutic agents and cytokines. Issues related to sero-prevalence and safety of Ads, however, continue to pose a challenge and need to be addressed.

RNA polymerase III promoter elements enhance transcription of RNA polymerase II genes.

Using transient expression assays in cultured human cells we have observed that RNA Polymerase III promoter sequences exert a positive cis-acting enhancer effect on RNA Polymerase II transcription. A DNA segment containing a copy of the Alu repeated element enhances transcription of the liver specific Haptoglobin related (Hpr) promoter in Hepatoma cell lines but not in HeLa cells. A tRNA(pro) gene acts as enhancer of the SV40 promoter both in Hepatoma and in HeLa cell lines. Transcription from the SV40 promoter is also enhanced by DNA segments containing only the box A or the box B of the tRNA(pro) promoter.

Two different liver-specific factors stimulate in vitro transcription from the human alpha 1-antitrypsin promoter.

The region from -137 to -2 of the human alpha 1-antitrypsin (alpha 1AT) promoter directs liver-specific in vitro transcription. Two cis-acting elements, A and B, have been identified within this segment by site-directed mutagenesis. Competition with synthetic oligonucleotides corresponding either to the A or to the B sequence inhibits transcription from the wild-type promoter in vitro. Cis-linked A and B elements mediate liver-specific transcription from a truncated HSV-TK promoter in vitro. Five different proteins, LF-A1, LF-A2, LF-B1, LF-B2 and LF-C, bind to the alpha 1AT promoter in liver extracts. LF-A1 and LF-B1 are positive transcriptional factors which bind to the A and B elements respectively. Their absence in spleen provides an explanation for the liver specificity of transcription. A protein similar to LF-B2 is present in spleen. Binding of LF-B1 and LF-B2 to the alpha 1AT promoter is mutually exclusive, suggesting that LF-B2 might be a repressor.

Trans-dominant inhibition of transcription activator LFB1.

Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the same dominant negative mutant in human hepatoma HepG2 cells only partially inhibited endogenous LFB1 activity, due to stabilization of LFB1 dimers in these cells. Dimer stabilization in hepatoma cells is mediated by a heat-labile association with an 11kD polypeptide, analogous to the DCoH cofactor identified in rat liver by Mendel et al. (1). The property of stabilizing LFB1 dimers is also shared by HeLa cells which produce a HeLa homolog of DCoH. These results demonstrate that LFB1 dimer stabilization as well as the synthesis of 'stabilizing factors' are not restricted to cells expressing LFB1 or other members of its family.

Codon-based mutagenesis using dimer-phosphoramidites.

A new approach for the synthesis of randomized DNA sequences containing the 20 codons corresponding to all natural amino acids is described. The strategy is based on the use of dinucleotide phosphoramidite building blocks within a resin-splitting procedure. Through this protocol, a minimal number of seven dimers is sufficient to encode all 20 natural amino acids. This synthesis procedure is extremely flexible and allows codon usage from different hosts to be accommodated.