Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

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.

Accession codes


NCBI Reference Sequence


  1. Mircic, M. & Kavanaugh, A. The clinical efficacy of tocilizumab in rheumatoid arthritis. Drugs Today (Barc) 45, 189–197 (2009).

    Article  Google Scholar 

  2. Hirota, H., Yoshida, K., Kishimoto, T. & Taga, T. Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice. Proc. Natl. Acad. Sci. USA 92, 4862–4866 (1995).

    Article  CAS  Google Scholar 

  3. Chan, A.C. & Carter, P.J. Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol. 10, 301–316 (2010).

    Article  CAS  Google Scholar 

  4. Weiner, L.M., Surana, R. & Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–327 (2010).

    Article  CAS  Google Scholar 

  5. Ohsugi, Y. & Kishimoto, T. The recombinant humanized anti-IL-6 receptor antibody tocilizumab, an innovative drug for the treatment of rheumatoid arthritis. Expert Opin. Biol. Ther. 8, 669–681 (2008).

    Article  CAS  Google Scholar 

  6. Tabrizi, M.A., Tseng, C.M. & Roskos, L.K. Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discov. Today 11, 81–88 (2006).

    Article  CAS  Google Scholar 

  7. Tabrizi, M., Bornstein, G.G. & Suria, H. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. AAPS J. 12, 33–43 (2010).

    Article  CAS  Google Scholar 

  8. Ng, C.M., Stefanich, E., Anand, B.S., Fielder, P.J. & Vaickus, L. Pharmacokinetics/pharmacodynamics of nondepleting anti-CD4 monoclonal antibody (TRX1) in healthy human volunteers. Pharm. Res. 23, 95–103 (2006).

    Article  CAS  Google Scholar 

  9. Kelley, S.K. et al. Preclinical pharmacokinetics, pharmacodynamics, and activity of a humanized anti-CD40 antibody (SGN-40) in rodents and non-human primates. Br. J. Pharmacol. 148, 1116–1123 (2006).

    Article  CAS  Google Scholar 

  10. Bostrom, J., Lee, C.V., Haber, L. & Fuh, G. Improving antibody binding affinity and specificity for therapeutic development. Methods Mol. Biol. 525, 353–376 (2009).

    Article  CAS  Google Scholar 

  11. Dall'Acqua, W.F., Kiener, P.A. & Wu, H. Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn). J. Biol. Chem. 281, 23514–23524 (2006).

    Article  CAS  Google Scholar 

  12. Deng, R. et al. Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor-{alpha} antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys. Drug Metab. Dispos. 38, 600–605 (2010).

    Article  CAS  Google Scholar 

  13. Zalevsky, J. et al. Enhanced antibody half-life improves in vivo activity. Nat. Biotechnol. 28, 157–159 (2010).

    Article  CAS  Google Scholar 

  14. Beck, A., Wurch, T., Bailly, C. & Corvaia, N. Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10, 345–352 (2010).

    Article  CAS  Google Scholar 

  15. Rose-John, S., Scheller, J., Elson, G. & Jones, S.A. Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer. J. Leukoc. Biol. 80, 227–236 (2006).

    Article  CAS  Google Scholar 

  16. Sato, K. et al. Reshaping a human antibody to inhibit the interleukin 6-dependent tumor cell growth. Cancer Res. 15, 851–856 (1993).

    Google Scholar 

  17. Maxfield, F.R. & McGraw, T.E. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132 (2004).

    Article  CAS  Google Scholar 

  18. Sarkar, C.A. et al. Rational cytokine design for increased lifetime and enhanced potency using pH-activated “histidine switching”. Nat. Biotechnol. 20, 908–913 (2002).

    Article  CAS  Google Scholar 

  19. Maeda, K., Kato, Y. & Sugiyama, Y. pH-dependent receptor/ligand dissociation as a determining factor for intracellular sorting of ligands for epidermal growth factor receptors in rat hepatocytes. J. Control. Release 82, 71–82 (2002).

    Article  CAS  Google Scholar 

  20. Burmeister, W.P., Huber, A.H. & Bjorkman, P.J. Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372, 379–383 (1994).

    Article  CAS  Google Scholar 

  21. Mihara, M. et al. Anti-interleukin 6 receptor antibody inhibits murine AA-amyloidosis. J. Rheumatol. 31, 1132–1138 (2004).

    CAS  PubMed  Google Scholar 

  22. Finkelman, F.D. et al. Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes. J. Immunol. 151, 1235–1244 (1993).

    CAS  PubMed  Google Scholar 

  23. Shinkura, H. et al. In vivo blocking effects of a humanized antibody to human interleukin-6 receptor on interleukin-6 function in primates. Anticancer Res. 18, 1217–1221 (1998).

    CAS  PubMed  Google Scholar 

  24. Genovese, M.C. et al. Interleukin-6 receptor inhibition with tocilizumab reduces disease activity in rheumatoid arthritis with inadequate response to disease-modifying antirheumatic drugs: the tocilizumab in combination with traditional disease-modifying antirheumatic drug therapy study. Arthritis Rheum. 58, 2968–2980 (2008).

    Article  CAS  Google Scholar 

  25. Yasukawa, K. et al. Purification and characterization of soluble human IL-6 receptor expressed in CHO cells. J. Biochem. 108, 673–676 (1990).

    Article  CAS  Google Scholar 

  26. Okazaki, M., Yamada, Y., Nishimoto, N., Yoshizaki, K. & Mihara, M. Characterization of anti-mouse interleukin-6 receptor antibody. Immunol. Lett. 84, 231–240 (2002).

    Article  CAS  Google Scholar 

  27. Gerhartz, C. et al. Biosynthesis and half-life of the interleukin-6 receptor and its signal transducer gp130. Eur. J. Biochem. 223, 265–274 (1994).

    Article  CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Tomoyuki Igawa.

Ethics declarations

Competing interests

The authors are employees of Chugai Pharmaceutical Co. Ltd.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–6 and Supplementary Figs. 1–4 (PDF 514 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research