LambdaZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage.

We describe a vector, lambdaZLG6, combining the high efficiency of cDNA library cloning in bacteriophage lambda with filamentous phage display of cDNA-encoded products. The cDNAs are expressed as fusions to the 3' end of M13 gene VI. The lambdaZLG library is converted to a pZLG6-cDNA phagemid library by in vivo mass excision. Helper phage infection generates a library of phagemid particles displaying the cDNA-encoded products and containing the corresponding nucleotide sequences within.

Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI.

We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.

Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen.

We have developed a strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template. Thus the heavy chain of a rodent antibody (MAb32) directed against human tumor necrosis factor alpha (TNF alpha) was cloned and paired as a template chain with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage were selected by binding to the antigen. The selected human light chains were in turn paired with a repertoire of human heavy chains displayed on phage, and the phage selected again. The isolated phage displaying human antibody fragments binding to TNF alpha also bound to a peptide comprising the N-terminal region of TNF alpha as with MAb32. One of the human Fab fragments was recloned for expression as a glycosylated human antibody in mammalian cells: Binding to TNF alpha could be competed with MAb32 or with anti-serum to the peptide, indicating the same epitope. The human antibody was found to have a binding affinity (Kd = 15 nM) similar to MAb32 (Kd = 26 nM). The process contrasts with existing means of "humanizing" rodent monoclonal antibodies in that the antibodies derived are completely human.