Abstract
Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of ∼40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gerard, C. & Gerard, N.P. C5A anaphylatoxin and its seven transmembrane-segment receptor. Annu. Rev. Immunol. 12, 775–808 (1994).
Mackay, C.R. Chemokines: immunology's high impact factors. Nat. Immunol. 2, 95–101 (2001).
von Andrian, U.H. & Mackay, C.R. T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343, 1020–1034 (2000).
Luster, A.D., Alon, R. & von Andrian, U.H. Immune cell migration in inflammation: present and future therapeutic targets. Nat. Immunol. 6, 1182–1190 (2005).
Heath, H. et al. Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J. Clin. Invest. 99, 178–184 (1997).
Guo, R.F. & Ward, P.A. Role of C5a in inflammatory responses. Annu. Rev. Immunol. 23, 821–852 (2005).
Riedemann, N.C., Guo, R.F. & Ward, P.A. The enigma of sepsis. J. Clin. Invest. 112, 460–467 (2003).
Ji, H. et al. Arthritis critically dependent on innate immune system players. Immunity 16, 157–168 (2002).
Gerard, C. & Gerard, N.P. The pro-inflammatory seven-transmembrane segment receptors of the leukocyte. Curr. Opin. Immunol. 6, 140–145 (1994).
Sumichika, H. C5a receptor antagonists for the treatment of inflammation. Curr. Opin. Investig. Drugs 5, 505–510 (2004).
Carter, P.J. Potent antibody therapeutics by design. Nat. Rev. Immunol. 6, 343–357 (2006).
Reichert, J.M., Rosensweig, C.J., Faden, L.B. & Dewitz, M.C. Monoclonal antibody successes in the clinic. Nat. Biotechnol. 23, 1073–1078 (2005).
Hopkin, M. Can super-antibody drugs be tamed? Nature 440, 855–856 (2006).
Qin, S. et al. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101, 746–754 (1998).
Wu, L. et al. Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding. J. Exp. Med. 186, 1373–1381 (1997).
Gerard, N.P. et al. An anti-inflammatory function for the complement anaphylatoxin C5a binding protein, C5L2. J. Biol. Chem. 280, 39677–39680 (2005).
Soruri, A., Kim, S., Kiafard, Z. & Zwirner, J. Characterization of C5aR expression on murine myeloid and lymphoid cells by the use of a novel monoclonal antibody. Immunol. Lett. 88, 47–52 (2003).
Gerard, C. et al. Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor. J. Immunol. 149, 2600–2606 (1992).
Siciliano, S.J. et al. Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 91, 1214–1218 (1994).
Oppermann, M. et al. Probing the human receptor for C5a anaphylatoxin with site-directed antibodies. Identification of a potential ligand binding site on the NH2-terminal domain. J. Immunol. 151, 3785–3794 (1993).
Farzan, M. et al. Sulfated tyrosines contribute to the formation of the C5a docking site of the human C5a anaphylatoxin receptor. J. Exp. Med. 193, 1059–1066 (2001).
Shevchuk, N.A. et al. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res. 32, e19 (2004).
Klco, J.M., Wiegand, C.B., Narzinski, K. & Baranski, T.J. Essential role for the second extracellular loop in C5a receptor activation. Nat. Struct. Mol. Biol. 12, 320–326 (2005).
Grant, E.P. et al. Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis. J. Exp. Med. 196, 1461–1471 (2002).
Korganow, A.S. et al. From systemic T cell self-reactivity to organ-specific autoimmune disease via immunoglobulins. Immunity 10, 451–461 (1999).
Wang, Y., Rollins, S.A., Madri, J.A. & Matis, L.A. Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc. Natl. Acad. Sci. USA 92, 8955–8959 (1995).
Wipke, B. & Allen, P. Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis. J. Immunol. 167, 1601–1608 (2001).
Kontgen, F., Suss, G., Stewart, C., Steinmetz, M. & Bluethmann, H. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957–964 (1993).
Campbell, J.J., Qin, S., Bacon, K.B., Mackay, C.R. & Butcher, E.C. Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells. J. Cell Biol. 134, 255–266 (1996).
Lee, D.M. et al. Mast cells: a cellular link between autoantibodies and inflammatory arthritis. Science 297, 1689–1692 (2002).
Acknowledgements
This research was supported by grants from National Health and Medical Research Council, AusIndustry and the New South Wales Department of State and Regional Development.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
Work described in this paper is associated with an agreement between the Garvan Institute, G2 Therapies and Novo Nordisk. G2 Therapies and Novo Nordisk recently entered into an agreement in relation to this work, worth potentially US$102 million.
Supplementary information
Supplementary Fig. 1
Screening antibodies to C5aR for ability to inhibit C5a mediated chemotaxis or 125I-C5a binding to human neutrophils. (PDF 45 kb)
Supplementary Fig. 2
mAbs to hC5aR specifically block C5a-induced calcium flux in human neutrophils. (PDF 47 kb)
Supplementary Fig. 3
Profile of C5aR protein and transcript expression in human leukocytes. (PDF 180 kb)
Supplementary Fig. 4
Administration of anti-C5aR mAb 3C5 to mice does not deplete PMN (neutrophil) population. (PDF 587 kb)
Supplementary Fig. 5
Serum concentration of 3C5 and 7F3 over time following injection into mice. (PDF 48 kb)
Supplementary Table 1
Sequences of PCR primers used to construct the chimeric mouse/human C5aR. (PDF 45 kb)
Rights and permissions
About this article
Cite this article
Lee, H., Zahra, D., Vogelzang, A. et al. Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies. Nat Biotechnol 24, 1279–1284 (2006). https://doi.org/10.1038/nbt1248
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nbt1248
This article is cited by
-
Transgenic disruption of endogenous glucocorticoid signaling in osteoblasts does not alter long-term K/BxN serum transfer-induced arthritis
Arthritis Research & Therapy (2023)
-
Nesting behavior is associated with body weight and grip strength loss in mice suffering from experimental arthritis
Scientific Reports (2023)
-
Inactivation of the gene encoding procalcitonin prevents antibody-mediated arthritis
Inflammation Research (2023)
-
Carboxypeptidase B blocks ex vivo activation of the anaphylatoxin-neutrophil extracellular trap axis in neutrophils from COVID-19 patients
Critical Care (2021)
-
Production of a bispecific antibody targeting TNF-α and C5a in Pichia pastoris and its therapeutic potential in rheumatoid arthritis
Biotechnology Letters (2020)