This disclosure provides polypeptides comprising
antibody heavy chain variable domain (VH) variants, the VH variants comprising multiple
antigen-binding regions and a framework comprising
framework region 1,
framework region 2, and
framework region 3 (FWR3), wherein the VH variants have an
amino acid sequence satisfying at least eight of the following criteria i)-x): i) the residue at Kabat position 19 is R, S, K, or T; ii) the residue at Kabat position 23 is T, V, A, or S; iii) the residue at Kabat position 24 is I, V, or S; iv) the residue at Kabat position 40 is selected from R, I, T, or K; v) the residue at Kabat position 43 is R, K, Q, E, or G; vi) the residue at Kabat position 44 is E, Q, A, D, or G; vii) the residue at Kabat position 45 is R, I, or L; viiii) the residue at Kabat position 76 is N or Q; ix) the residue at Kabat position 79 is Y, W, or F; and x) the residue at Kabat position 89 is V or I. The VH variant has improved basic stability and can be used as a backbone for
affinity binding agents for
in vitro applications, such as detecting, analyzing or separating analytes based on affinity interactions.