ISOLATION AND CHARACTERIZATION OF NUCLEIC ACID-BINDING ANTIBODY FRAGMENTS FROM AUTOIMMUNE MICE-DERIVED BACTERIOPHAGE DISPLAY LIBRARIES

The display of antibody fragments (Fab) on the surface of filamentous bacteriophage and selection of phage that bind to a particular antigen has enabled the isolation of Fab with numerous specificities, includi ng haptens, proteins and viral particles. We have examined the possibi lity of isolating nucleic acid-binding Fab by constructing a combinato rial library of phage displaying Fab derived from autoimmune (MRL/lpr) mice. Autoimmune mice were chosen because they contain antibodies (Ab ) reactive against nuclear components, including DNA, RNA and protein complexes. The library was panned against single-stranded (ss) calf th ymus (CT) DNA and the selected Fabs were analyzed further. Characteriz ation of the nucleic acid-binding phage led to the identification of t wo kinds of Fab with quite different properties. One Fab bound with hi gh affinity a variety of ssDNA molecules, as well as several model RNA substrates. This Fab has been affinity purified to greater than 95% a nd competition studies revealed a marked preference for binding to pol y(dT). The second Fab showed a reduced binding to RNA ligands and a re stricted number of ssDNA molecules. Analysis of the deduced amino acid (aa) sequences of the Fab variable (V) regions revealed that the heav y (H) chain V region from the strong nucleic acid-binding Fab was deri ved from a V-H gene that is used recurrently in autoantibodies. This V -H domain was most similar to an anti-ssDNA autoimmune monoclonal anti body (mAb) suggesting that antigen-binding specificities present in an autoimmune repertoire may be directly accessed by this approach. A li ght (L) chain shuffling experiment was performed to investigate how in discriminate a H chain fragment from a nucleic acid-binding Fab could be and still retain antigen binding. In contrast to previous reports e mploying haptens or proteins, only a few L chains were isolated that e nabled DNA and RNA binding. Furthermore, the deduced aa sequences of t hese L chains were almost identical to that of the original Fab. The f ew aa changes present resulted in a shift in antigen specificity, sugg esting that fine tuning of substrate recognition may be possible. This report demonstrates that phage Fab display libraries will be a powerf ul method for the isolation of nucleic acid-binding Ab in vitro and ma y be applicable to the study of immune recognition, especially as it o ccurs in autoimmune disease.