Roberto Burioni

Human monoclonal recombinant Fabs specific for HCV antigens obtained by repertoire cloning in phage display combinatorial vectors

Molecular cloning of the antibody repertoire in phage display combinatorial vectors is a powerful method enabling the dissection of the immunoresponse against a given pathogen. In this paper we describe the construction of a combinatorial library displayed on phage surface, containing the antibody repertoire of a patient with high serological response against hepatitis C virus (HCV) antigens. Following selection of the library against solid-phase-bound antigen, sixteen human antibody Fab fragments able to bind to HCV-specific antigens were generated and studied for binding characteristics. The majority of them appeared to have specificity for the HCV c33 peptide. All the clones reacting with the c33 peptide shared the same heavy-chain CDR3 sequence. This is the first report of molecular cloning in a combinatorial phage display vector of the antibody repertoire of an anti-HCV-positive patient.

A vector for the expression of recombinant monoclonal Fab fragments in bacteria

The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.

A new subtraction technique for molecular cloning of rare antiviral antibody specificities from phage display libraries

The preparation of random combinatorial libraries exposed on the surface of phage provides a route for the selection of diverse high affinity human monoclonal antibodies. However, in particular settings, the isolation of genes coding for a rare antibody can be elusive because some epitopes are predominant and because, in the case of impure antigens, the protein or any compound of interest can be present in relatively minimal amount. In this paper, we describe the successful utilization of a new strategy of preadsorption panning that allowed us to clone a rare human monoclonal antibody fragment and to access a different antibody repertoire. The procedure is easy, fast, inexpensive, can be used together with other panning techniques and can be particularly useful in cloning antibodies against rare or unknown determinants.

An improved phage display vector for antibody repertoire cloning by construction of combinatorial libraries.

Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small stuffer fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.

Probing the natural antibody repertoire by combinatorial cloning of IgM and IgD isotypes in phage display vectors

It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD phenotype and can help elucidate the role played by natural antibodies in defence against infectious agents.