Rational engineering of antibodies with irreversible binding: Antibodies with infinite affinity

This dissertation describes a new methodology for the rational engineering of a natural ligand/receptor pair into one with infinite binding affinity. As an example of this methodology the anti chelate antibody CHA255 was engineered to increase its affinity for its ligand, indium benzyl-EDTA. This methodology involves two separate processes, derivativization of the ligand to introduce a weakly electrophilic function and cloning and expression of the antibody Fab domain, its cognate receptor, with an artificially introduced nucleophile. These steps are undertaken separately but each is influenced by the other such that they work in tandem.; In this reduction to practice, a scheme was developed based on the three dimensional structure of the antibody/hapten complex. Close inspection of the binding domain using computer visualization lead to a plan where an electrophile would be introduced in the ligand in a position distant from structural features know from biochemical and structural evidence to be important for recognition and binding. Within the binding domain a nucleophilic amino acid would be substituted such that it was in close proximity to electrophile in the ligand upon forming of the ligand/receptor complex. To this end, ABE (ABE: (S)-1-(para-aminobenzyl)ethylenediaminetetracetic acid) chelates were synthesized and derivatized to introduce a selection of electrophilic groups and tested in vitro and in vivo to determine the one best suited for use. To produce antibody fragments bearing introduced cystienes in the binding domain a system was developed using Drosophila Meg. S2 cells to express human/mouse chimeric Fab domains. With both halves of the system in place the capabilities of the engineered ligands were tested versus a small family of engineered Fabs in terms of binding ability, covalent bond formation rate and specificity. The engineered Fab domains retained comparable selectivity and affinity for there native ligand, ABE and showed a half-life for reaction with the weakly electrophilic ligand AABE (AABE: (S)-1-p-acrylamidobenzyl-ethylenediaminetetraacetic acid) of ten minutes, less than half the bound lifetime. These results provide the foundation for a new generation of engineered proteins possessing the specificity characteristic of monoclonal antibodies along with unprecedented strength of binding to their specific targets.