HEAVY-CHAIN CDR3 OPTIMIZATION OF A GERMLINE ENCODED RECOMBINANT ANTIBODY FRAGMENT PREDISPOSED TO BIND THE U1A PROTEIN

Previously, we described a DP-65 encoded heavy chain variable (V-H) ge ne restriction in anti-U1A antibodies, The U1A protein (a component of the U1 ribonucleoprotein particle) is an important autoantigenic targ et in certain systemic lupus erythematosus (SLE) patients, Here we exa mined the effect of randomizing amino acids in the heavy chain complem entarity determining region 3 (CDR3) of this germline encoded recombin ant antibody fragment on binding to the U1A protein, A phage display l ibrary was constructed using the DP-65 V-H domain with four randomized CDR3 residues and our results showed that a high frequency (10%) of t he randomized mutants in the unselected library were able to bind the U1A protein. This corroborates our previous finding that this V-H doma in provides an appropriate structure for U1A binding, although the nat ure of the CDR3 residues appears crucial in determining whether or not this V-H domain binds U1A, After two rounds of selection UIA binders show a consensus sequence in their randomized CDR3 residues i,e, SO(K, R,S)XG, in which X is an uncharged residue, This consensus is partiall y present in an antibody which was derived from an SLE patient indicat ing that this consensus, to some extent, is also followed in vivo, Clo nes which match the consensus sequence obtained up to 25-fold higher a ffinities compared with the original clones, illustrating the importan ce of the V-H CDR3 residues in determining the affinity of these antib odies.