Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb 17;287(8):5891-7.
doi: 10.1074/jbc.M111.311811. Epub 2011 Dec 27.

Engineering upper hinge improves stability and effector function of a human IgG1

Boxu Yan  1 , Daniel Boyd, Timothy Kaschak, Joni Tsukuda, Amy Shen, Yuwen Lin, Shan Chung, Priyanka Gupta, Amrita Kamath, Anne Wong, Jean-Michel Vernes, Gloria Y Meng, Klara Totpal, Gabriele Schaefer, Guoying Jiang, Bartek Nogal, Craig Emery, Martin Vanderlaan, Paul Carter, Reed Harris, Ashraf Amanullah
Affiliations

Engineering upper hinge improves stability and effector function of a human IgG1

Boxu Yan et al. J Biol Chem. .

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 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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Schematic illustration of the •OH radical induced hinge degradation in a human IgG1. The •OH radicals induce degradations of an IgG1 hinge to generate different products under different conditions. Under high oxygen tension, hinge cleavage releases Fab fragment and a partial IgG1 that is missing the Fab, with complementary ladders of C- and N-terminal hinge residues (Asp226, Lys227, Thr228, His229, and Thr230) of the upper hinge (panel A) (9); whereas under low oxygen tension, breakage of the heavy-light chain linkage leads to the peptide bond cleavage that releases light chain (LC) and Fab portion of the heavy chain (HC) (panel B), or the breakage of the heavy chain (HC) and light chain (LC) linkage without any cleavage of peptide bond (panel C) (11). The hinge degradation is initiated by radical formation at Cys231 resulting from breakage of the first hinge disulfide bond between two heavy chains by the •OH attacks, and followed by radical reactions via ET and localization onto the upper hinge or the inter-chain disulfide bond. The degraded products (circled) varies depending on the reaction conditions, as described previously (–11).
FIGURE 2.
FIGURE 2.
Substitutions in the upper hinge and related impacts to radical induced hinge cleavage. Nine mutants were designed to determine any roles of Asp225 and His229 in the hinge cleavage. These substitutions were constructed by introducing single or double substitutions in the upper hinge of the native sequence by site-directed mutagenesis. Substituted residues are underlined (panel A). These mutants were evaluated for their ability to inhibit the hinge cleavage by SEC to measure the degraded products (Fab and a partial IgG1), the SEC was performed after incubating the IgG1 with H2O2 in a molar ratio of 1:200 at 25 °C for 2, 4, and 6 days, respectively. The cleavage products (% LMW) are shown as % of the peak area of the products to the whole molecule in the UV signals (panel B). For clarity, only the results from Day 6 and Day 0 are shown here. Percent change was calculated by comparing the level of % LMW at Day 6 to Day 0. Percent inhibition was calculated by the equation: % = (% change of the native − % change of mutant)/% change of the native. LMW: low molecular weight species (Fab and a partial IgG1). The results are reported as an average of two replicates.
FIGURE 3.
FIGURE 3.
Characterization of mutants 4 (His/Ser+Asp/Ser) and 9 (His/Tyr) for in vivo PK profile. Pharmacokinetics of the native IgG1 and two mutants were characterized by single IV bolus dose in SCID beige mice using 2 or 25 mg/kg of the IgG1. Serum samples were collected at various time points up to 21 days post-dose and analyzed for the IgG1, Mutant 4, or Mutant 9 concentrations that were measured using an ELISA method as described under “Experimental Procedures.” Each data point was obtained using 3 mice. These results showed similar PK profiles between the native IgG1 and its mutants.
FIGURE 4.
FIGURE 4.
Effect of the substitutions 4 and 9 on ADCC activity of the IgG1. ADCC assays were carried out using peripheral blood mononuclear cells (PBMCs) from healthy human donors as effector cells and A431 cells as target cells (see “Experimental Procedures” for details). The native IgG1 and its mutants were tested using PBMCs from two donors and a E:T ratio of 25:1 against A431 cells. S.D. are indicated by error bars and represent triplicate measurements within the same experiment. Data shown are representative using PBMCs from 4 different individual donors. These results indicate that the substitution 9 is more potent than the native IgG1 in the ADCC function, while the mutant 4 behaves similarly to the native IgG1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nelson A. L., Dhimolea E., Reichert J. M. (2010) Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discov. 9, 767–774 - PubMed
    1. Carter P. J. (2006) Potent antibody therapeutics by design. Nat. Rev. Immunol. 6, 343–357 - PubMed
    1. Nelson A. L., Reichert J. M. (2009) Development trends for therapeutic antibody fragments. Nat. Biotechnol. 27, 331–337 - PubMed
    1. Beck A., Wurch T., Bailly C., Corvaia N. (2010) Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10, 345–352 - PubMed
    1. Jefferis R. (2009) Glycosylation as a strategy to improve antibody-based therapeutics. Nat. Rev. Drug Discov. 8, 226–234 - PubMed

LinkOut - more resources