The integrity of the ball-and-socket joint between V and C domains is essential for complete activity of a humanized antibody

AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-gamma. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-gamma that was slightly less than that of AF2, but exhibited IFN-gamma -neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-gamma-neutralizing activity was due to altering the V-H framework residue at position 11. V-H position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-gamma-neutralizing capacity, we altered residue 148 of C-H1, which with residue 149 comprises the corresponding ball portion of the joint. Changing this single C-H1 domain residue diminished the ability of the Ab to neutralize IFN-gamma to a level similar to that observed with the V, alteration. Thus, an intact ball-and-socket joint between the V-H and C domains in AF2 is required for potent neutralization of IFN-gamma, These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.