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Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies

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.

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Figure 1: Generation of human C5aR knock-in mice and anti-human C5aR mAbs.
Figure 2: Potent antagonistic anti-C5aR mAbs map to a specific region on the C5aR second extracellular loop.
Figure 3: Anti-human C5aR mAbs prevent and reverse K/BxN serum-induced inflammation in human C5aR knock-in mice.

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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.

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Correspondence to Charles R Mackay.

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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)

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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

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