FIGURE 6.

Selective FcRn cross-species binding to the monoclonal Abs depends on His-161. A, close-up of the structural areas of hFcRn and rat FcRn encompassing the α1-domain loop with the tryptophan residues (red), hFcRn Val-52 and rat FcRn Ile-52 (cyan), and the α2-domain α-helix with hFcRn His-161 (blue), and rat FcRn Glu-163 (orange). The figures were made using the available crystal structures of hFcRn (25) and rat FcRn (24). B, an alignment of the stretch of amino acids of the α-helix containing His-166 in the α-2 domain of FcRn from 10 different species. A non-conserved amino acid variation is found at position 161 (human numbering). C, SDS-PAGE gel migration of recombinant GST-tagged hFcRn WT and mutants (V52I, V52M, H161E, and H161Q) showing the expected molecular sizes of the GST-fused HC and β2-microglobulin (β2m). Binding of WT hFcRn and mutants (V52I, V52M, W59A, H161A, H161E, and H161Q) to DVN1 (D), ADM31 (E), ADM32 (F), and DVN24 (G) at pH 7.4. Relative binding of the Abs to the hFcRn variants was calculated from an ELISA screen (data not shown), where binding of WT hFcRn to the Abs was set to 1.0 (n = 3). All data are presented as mean ± S.D.