By-passing immunization: building high affinity human antibodies by chain shuffling.

Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.