Combinatorial infection and in vivo recombination: a strategy for making large phage antibody repertoires.

Antibody fragments, comprising paired heavy (VH) and light (VL) chain variable domains, can be displayed on the surface of filamentous bacteriophage, and rare phage (encoding antigen binding activities) selected by binding to antigen (1). The process mimics immune selection and has been used to make human antibody fragments in bacteria, without immunisation, by random combinatorial linkage (2) of diverse repertoires of VH and VL genes from lymphocytes (3, 4). Fragments with a range of binding specificities have been isolated with binding affinities in the range 10 M~'-10 M" (for reviews see (5, 6)). However larger 'primary' repertoires of phage antibodies should allow higher affinity fragments to be isolated (7, 8). The size of phage antibody repertoires (10) is limited by the efficiency of transformation of E.coli. In principle, larger repertoires could be made by combinatorial infection, for example by transforming E. coli with a repertoire of heavy chains (encoded on plasmids) then infecting with a repertoire of light chains (encoded on phage) (9). Since infection is extremely efficient, and most E.coli cells in an exponential culture can be infected, the combinatorial diversity of Fab fragments displayed on phage could be as large as the number of E.coli in culture (10 per litre). However the heavy and light chain genes would not be packaged together within the same phage particle, and so could not be simultaneously co-selected. Here we describe a model system, involving the lox-Cre site-specific recombination system of bacteriophage PI, to lock together the heavy and light chain genes from two different replicons within an infected bacterium.