Engineering Upper Hinge Improves Stability and Effector Function of ...

1. Boxu Yan^‡,¹,
2. Daniel Boyd^‡,
3. Timothy Kaschak^‡,
4. Joni Tsukuda^§,
5. Amy Shen^§,
6. Yuwen Lin^¶,
7. Shan Chung^¶,
8. Priyanka Gupta^‖,
9. Amrita Kamath^‖,
10. Anne Wong**,
11. Jean-Michel Vernes**,
12. Gloria Y. Meng**,
13. Klara Totpal^‡‡,
14. Gabriele Schaefer^‡‡,
15. Guoying Jiang^§§,
16. Bartek Nogal^‡,
17. Craig Emery^‡,
18. Martin Vanderlaan^§§,
19. Paul Carter^¶¶,
20. Reed Harris^§§ and
21. Ashraf Amanullah^‡

1. From the ^‡Department of Pharma Technical Development, Genentech, Oceanside, California 92056 and
2. the Departments of ^§Early Stage Cell Culture,
3. ^¶Bioanalytical Science,
4. ^‖Pharmacokinetics and Pharmacodynamics,
5. ^**Assay and Automation Technology,
6. ^‡‡Immunology,
7. ^§§Analytical Development & Quality Control, and
8. ^¶¶Antibody Engineering, Genentech, San Francisco, California 94080

1. ↵1 To whom correspondence should be addressed. E-mail: byan1027{at}gmail.com.

Background: Radical reactions result in breakage of the heavy-light chain linkage and hinge cleavage of an IgG1.

Results: The degraded products are generated by different reaction pathways and mechanisms.

Conclusion: A His²²⁹/Tyr substitution improves stability and effector function of an IgG1.

Significance: A mechanism based strategy to engineer the upper hinge to improve multiple properties of an IgG1 is feasible.

Abstract

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 Cys²³¹ directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His²²⁹-mediated hinge cleavage. On the other hand, the substitution of His²²⁹ 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.

• Antibodies
• Histidine
• Protein Degradation
• Protein Engineering
• Protein Stability
• Tyrosine
• Degradation
• Hinge
• His
• Radical Reactions

Footnotes

• ↵ This article contains supplemental Figs. S1 and S2.

• Received October 11, 2011.
• Revision received December 9, 2011.

• © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.