Whole IgG surface display on mammalian cells: Application to isolation of neutralizing chicken monoclonal anti-IL-12 antibodies.

We have developed a mammalian cell surface display vector, suitable for directly isolating IgG molecules based on their antigen-binding affinity and biological activity. Using an Epstein-Barr virus-derived episomal vector, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and fluorescence-activated cell sorting. Plasmids encoding antibodies with desired binding characteristics are recovered from sorted cells and are converted to the form for production of soluble IgG. Transiently expressed soluble IgG antibodies are individually tested for binding to target antigens, as well as for biological activities, such as neutralization. This vector system was used to generate antibody display libraries derived from spleen cDNA of chickens immunized with human and mouse IL-12. Chicken-human chimeric IgG1 antibodies that neutralize human and mouse IL-12 were successfully isolated from the library. The mammalian surface display vector developed in this work facilitates the isolation of monoclonal antibodies from essentially any species.

Humanization of a chicken anti-IL-12 monoclonal antibody.

Chicken anti-IL-12 monoclonal antibodies were isolated by phage display using spleen cells from a chicken immunized with human and mouse IL-12 as a source for library construction. One of the chicken monoclonal antibodies, DD2, exhibited binding to both human and mouse IL-12 in the single-chain Fv form and also after conversion to chicken-human chimeric IgG1/lambda antibody. The chicken DD2 variable regions were humanized by transferring their CDRs and several framework amino acids onto human acceptor variable regions. In the Vlambda, six chicken framework amino acids were identified to be important for the conformation of the CDR structure by computer modeling and therefore were retained in the humanized form; likewise, five chicken amino acids in the VH framework regions were retained in the humanized VH. The affinities of humanized DD2 IgG1/lambda to human and mouse IL-12 measured by competitive binding were nearly identical to those of chicken-human chimeric DD2 IgG1/lambda. This work demonstrates that humanization of chicken monoclonal antibodies assisted by computer modeling is possible, leading to a new way to generate therapeutic humanized antibodies against antigens to which the rodent immune system may fail to efficiently raise high affinity antibodies.