A comparative analysis of the immunological evolution of antibody 28B4

In an effort to gain greater insight into the evolution of the redox active , catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were deter mined. Comparison with the previously determined structures of the unligand ed and hapten-bound affinity-matured Fab [Hsieh-Wilson, L.C., Schultz, P.G. , and Stevens, R.C. (1996) Proc. Nad. Acad. Sci. U.S.A. 93, 5363] shows tha t the gem-dine antibody binds the p-nitrophenyl ring of hapten 3 in an orie ntation significantly different from that seen in the affinity matured anti body, whereas the phosphonate moiety is bound in a similar mode by both ant ibodies. The affinity-matured antibody 28B4 has more electrostatic and hydr ophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR LI upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in t he germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The s tructural differences are reflected in the differential binding affinities of the germline Fab (K-d=25 muM) and 28B4 Fab (K-d=37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured ant ibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibo dies. One of the mutations, Asp95(H)Trp, leads to a change in the orientati on of the bound hapten, and its presence is a prerequisite for other somati c mutations to enhance the binding affinity of the germline antibody for ha pten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, aff inity selection, and clonal expansion model for germline antibody evolution . Two other antibodies, 20-1 and NZA6, with very different antigen specific ities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combi nations can give rise to polyspecific hapten binding sites [Romesberg, F. E ., Spiller, B.. Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929 ]. The ultimate specificity of the polyspecific germline antibody appears t o be defined by CDR H3 variability and subsequent somatic mutation. Insight s into the evolution of antibody-combining sites provided by this and other structural studies are discussed.