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The Journal of Immunology, 2001, 166: 1748-1754.
Copyright © 2001 by The American Association of Immunologists

The Integrity of the Ball-and-Socket Joint Between V and C Domains Is Essential for Complete Activity of a Humanized Antibody

Nicholas F. Landolfi1, Archana B. Thakur, Helen Fu, Max Vásquez, Cary Queen and Naoya Tsurushita

Protein Design Labs, Fremont, CA 94555

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 VH framework residue at position 11. VH 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 CH1, which with residue 149 comprises the corresponding ball portion of the joint. Changing this single CH1 domain residue diminished the ability of the Ab to neutralize IFN-{gamma} to a level similar to that observed with the VH alteration. Thus, an intact ball-and-socket joint between the V 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.




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