Analysis of the effects of individual amino acid substitutions in a failed attempt of humanization: the original mouse sequence, the human template sequence and the chimeric construct were modelled and the sequence differences analyzed as to their potential effect on antigen binding

red: extremly likely to have an influence on antigen binding (L35, L45)
yellow: residues likely to have an indirect influence on CDR conformation (L70,H49)
orange: sequence differences in the hydrophobic core of VL- coordinated changes indicative of different framework type in mouse and human sequence. Individual back mutations might have destabilizing effect, combined influence on antigen binding not predictable, but probably minor, since all changes occur below the extremely conserved core glutamin L6, tryptophane L35 and disulfide bridge, which divide the core in an upper and a lower halve.
green: substitutions on the molecular surface, away from the antigen binding site: most probably no influence on antigen binding
cyan: maybe an indirect influence, but rather unlikely