Engineering upper hinge improves stability and effector function of a human IgG1.
Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.
Department of Pharma Technical Development, Genentech, Oceanside, California 92056, USA. byan1027@gmail.com
Boxu
B
Yan
Daniel
D
Boyd
Timothy
T
Kaschak
Joni
J
Tsukuda
Amy
A
Shen
Yuwen
Y
Lin
Shan
S
Chung
Priyanka
P
Gupta
Amrita
A
Kamath
Anne
A
Wong
Jean-Michel
JM
Vernes
Gloria Y
GY
Meng
Klara
K
Totpal
Gabriele
G
Schaefer
Guoying
G
Jiang
Bartek
B
Nogal
Craig
C
Emery
Martin
M
Vanderlaan
Paul
P
Carter
Reed
R
Harris
Ashraf
A
Amanullah
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005240:Feasibility Studies; D006801:Humans; D017665:Hydroxyl Radical / Q000494:pharmacology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D059748:Proteolysis / Q000187:drug effects
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D017665:Hydroxyl Radical