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 (VH) gene restriction in anti-U1A antibodies. The U1A protein (a component of the U1 ribonucleoprotein particle) is an important autoantigenic target in certain systemic lupus erythematosus (SLE) patients. Here we examined the effect of randomizing amino acids in the heavy chain complementarity determining region 3 (CDR3) of this germline encoded recombinant antibody fragment on binding to the U1A protein. A phage display library was constructed using the DP-65 VH domain with four randomized CDR3 residues and our results showed that a high frequency (10%) of the randomized mutants in the unselected library were able to bind the U1A protein. This corroborates our previous finding that this VH domain provides an appropriate structure for U1A binding, although the nature of the CDR3 residues appears crucial in determining whether or not this VH domain binds U1A. After two rounds of selection U1A binders show a consensus sequence in their randomized CDR3 residues i.e. S(K,R,S)XG, in which X is an uncharged residue. This consensus is partially present in an antibody which was derived from an SLE patient indicating that this consensus, to some extent, is also followed in vivo. Clones which match the consensus sequence obtained up to 25-fold higher affinities compared with the original clones, illustrating the importance of the VH CDR3 residues in determining the affinity of these antibodies.
de Wildt
R M
RM
Department of Biochemistry, University of Nijmegen, The Netherlands.
Ruytenbeek
R
R
van Venrooij
W J
WJ
Hoet
R M
RM
D000595:Amino Acid Sequence; D000916:Antibody Diversity; D001324:Autoantigens / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism*; D008180:Lupus Erythematosus, Systemic / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015202:Protein Engineering; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000276:immunology* / Q000378:metabolism*
D001324:Autoantigens; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein