Anti‐D and anti‐i activities are inseparable in V4‐34‐encoded monoclonal anti‐D: the same framework 1 residues are required for both reactivities

Abstract

BACKGROUND: The heavy‐chain V4‐34 germline gene segment is mandatory for pathologic cold‐reacting autoantibodies with anti‐I/i specificity (cold agglutinins) and is also preferentially used by monoclonal immunoglobulin M alloantibodies against D and other Rh antigens. The use of the V4‐34 segment by monoclonal anti‐D has previously been shown to also confer anti‐I/i reactivity (cold agglutinin activity), which has implications for the use of such antibodies for Rh blood typing. V4‐34 framework 1 (FR1) sequence is believed to be critical for cold agglutinin activity of cold agglutinins.STUDY DESIGN AND METHODS: The aim of this investigation was to use site‐directed mutagenesis of a recombinant V4‐34‐encoded anti‐D to determine the contribution of V4‐34 FR1 sequence to anti‐D activity and whether mutational modifications in the FR1 region could separately alter anti‐D and anti‐i activities.RESULTS: The results show that amino acid changes in V4‐34 FR1 at W7, A23, and Y25 have a profound effect on anti‐D activity as well as on anti‐i activity. It was not possible to substantially reduce or remove anti‐i activity without reducing anti‐D activity to a comparable extent.CONCLUSIONS: The same nonpolar hydrophobic amino acids in FR1 are critical for maintaining both anti‐D and anti‐i activity. It is proposed that these residues influence the conformation of the antigen‐binding site.