Evolution of catalytic antibody repertoire in autoimmune mice.

Abstract We have attempted to efficiently obtain catalytic antibodies (catAbs) with amidase/esterase activity in the expanded sequence space of the antibody repertoire. In doing so, we used an autoimmune mouse strain, MRL/lpr, that is known to produce enhanced levels of autoantibodies. We applied different types of haptens, such as alkylphosphonate (1) , alkylphosphonamidate (2) , alkylphosphonamidate (OMe) (3) , and difluoroketone (4) , that are supposed to mimic the transition state of the substrate in the ester/amide hydrolysis. Among them, hapten ( 2 ) could not be used, as it was readily broken down after synthesis. Upon immunization with hapten ( 1 ), catAbs preferentially evolved in MRL/lpr mice, but this did not happen upon immunization with haptens ( 3 ) and ( 4 ). Independently, immunization to MRL/lpr mice with vitamin B6 phosphonate hapten (5) successfully elicited the catAbs with the ability to activate vitamin B6 prodrugs. The common observation seen in these two cases is that most of the catAbs derived from MRL/lpr mice by hapten ( 1 ) and half of them by hapten ( 5 ) had a Lys at H95, which is at the junctional N region between the VH and JH gene segments. Despite the conservation of Lys (H95), analyses of the N-region and utilization of the D gene segment in the heavy chain gene showed that these catAbs were from several independent clones of the same family. Studies of site-directed mutagenesis suggest that, in the catAbs elicited from hapten ( 1 ), a Lys (H95) and a His (L91) are involved in the catalytic function. Both residues are known to interact with the phosphonate moiety of hapten ( 1 ). Such studies also suggest that, in the catAbs elicited from hapten ( 5 ), a Lys (H95) and a His (H35) are involved in the catalytic function. These basic amino acids seem to be important for binding to the phosphonate hapten, as they were not changed even after extensive evolution following multiple mutations. By contrast, in normal BALB/c mice, immunization of hapten ( 1 ) resulted in eliciting catAbs in lower yield and the majority were the non-catAbs, whose sequences were quite different from those of the catAbs from MRL/lpr mice. They were clonally related to one another and most of them originated from a single clone. The positions of the interacting key residues in the CDRs that interact with the phosphorus moiety strongly differ between our catAbs and other reported catAbs with esterase/amidase activity, which were elicited by the phosphonate/phosphonamidate haptens from normal mice. Further comparison of antibodies elicited by the phosphorus haptens, such as DNA, RNA, phosphocholine, and phosphotyrosine, indicated that none of them had sequence similarity in the basic amino acids and their positions in the CDRs, except for one example, which is anti-DNA antibody elicited from C3H-lpr mice. Analysis based on the classification of canonical structures of the antibodies again suggested that our catAbs derived from MRL/lpr mice belong to an unusual class that is not listed in the literature. Taken together, the above evidence suggests that the unique catalytic subsets that existed in the initial repertoire in the MRL/lpr mice could effectively be captured by the phosphonate haptens through the interaction with the Lys at H95. In the BALB/c mice, however, another noncatalytic subset with an ability to bind only to a moiety other than the phosphonate moiety alternatively evolved, because of the lowest abundance or elimination of the catalytic subsets.