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Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization

Abstract

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra)1, an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R2 suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.

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Figure 1: Surface plasmon resonance (SPR) sensorgrams of tocilizumab (TCZ), two variants with pH-dependent binding to hsIL-6R (PH1, PH2), TCZ and PH2 with increased affinity to FcRn (TCZ-FcRn, PH2-FcRn) and an affinity matured variant with increased affinity to FcRn (AM-FcRn).
Figure 2: In vivo characterization of pH-dependent binding variants in mice.
Figure 3: In vivo characterization of pH-dependent binding variants in cynomolgus monkeys.

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Acknowledgements

We thank T. Kishimoto at the Graduate School of Frontier Biosciences, Osaka University and T. Taga at the Kumamoto University Graduate School of Medical Sciences for kindly providing human IL-6R transgenic mice; colleagues in Chugai Research Institute for Medical Science, Inc., O. Ueda, T. Tachibe, M. Kakefuda and K. Jishage for breeding human IL-6R transgenic mice, T. Matsuura, M. Hiranuma, T. Koike, R. Takemoto, H. Azabu, T. Sakamoto, H. Sano and M. Kawaharada for carrying out in vivo experiments, and M. Fujii and A. Maeno for antibody vector construction, expression and purification; and colleagues in Chugai Pharmaceutical Co. Ltd., K. Kasutani, F. Mimoto and K. Esaki for carrying out in vitro experiments.

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T.I. led the overall pH-dependent binding antibody program, designed experiments, generated tocilizumab variants and wrote the manuscript. S.I. and A.M. generated tocilizumab variants. T.T., R.T., Y.H. and K. Haraya performed in vivo studies. S.S. led the anti-IL-6R antibody program. C.M. and A.H. performed affinity analysis of tocilizumab variants. T. Watanabe performed in vitro studies of tocilizumab variants. Y.D. and T. Wakabayashi performed purification of tocilizumab variants. S.K. and T.M. provided structural information for designing tocilizumab variants. Y.S., T.K., Y.N., Y.A., Y.K., and K. Hattori provided direction and guidance for the various functional areas.

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Correspondence to Tomoyuki Igawa.

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The authors are employees of Chugai Pharmaceutical Co. Ltd.

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Igawa, T., Ishii, S., Tachibana, T. et al. Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol 28, 1203–1207 (2010). https://doi.org/10.1038/nbt.1691

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