Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model

Abstract

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti-C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti-murine C5aR (anti-mC5aR) surrogate antibody in mouse collagen-induced arthritis (CIA). First, efficacy was demonstrated in a multiple-dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti-mC5aR-treated mice. Then, the mechanism of action was examined by looking for early effects of anti-mC5aR treatment in single-dose treatment studies. We found that 48 h after single-dose treatment with anti-mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose-setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti-C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.

Keywords: anti-C5aR; arthritis; macrophages; neutrophils.

Fig. 1

Effect of therapeutic treatment with multiple doses of anti-murine C5a receptor (mC5aR) in the collagen-induced arthritis (CIA) model. Mice (n = 17–18/group) were immunized for induction of arthritis at day 0 and boosted at day 21. Therapeutic treatment with anti-murine C5a receptor (anti-mC5aR) (0·5 mg/mouse) or etanercept (10 mg/kg) was initiated when mice reached a clinical score of 2–4 (individual treatment) corresponding to score day 1. Isotype controls for anti-mC5aR [anti-trinitrophenyl (TNP) mouse immunoglobulin (Ig)G2a] and etanercept (anti-TNP humanized IgG1) were included. (a) Mean clinical score (± standard error of the mean) per group over time. (b) AUC (area under the curve for individual clinical scores), black line represents the mean. Mice that were killed before all six doses were given due to severe arthritis symptoms (humane end-points) were included in the calculation with the last observed score. Significance was calculated by unpaired two-tailed t-test with Welch's correction. (c) Blood samples were analysed at termination and changes in polymorphonuclear (PMN) numbers were analysed by fluorescence activated cell sorter (FACS) in duplicate. Statistical significance calculated by unpaired two-tailed t-test. *P < 0·05; **P < 0·01; **P < 0·001.

Fig. 2

Histopathology of paws from mice treated with multiple doses of anti-murine C5a receptor (mC5aR). (a) Histology score index represents average score per mouse (two hind paws per mouse), and histology score of bone erosion and cartilage degradation represents average score of worst-affected hind paw per mouse. (b) Immunohistochemistry was used to detect infiltrating macrophages and neutrophils by staining for F4/80 and Ly6B.2, respectively. On a digital image of the worst-affected paw per mouse, a region-of-interest (ROI) was automatically defined of the entire paw, excluding bone marrow, and an analysis was run inside the ROI to detect the brown 3-3′-diamino-benzidine-tetrahydrochloride (DAB) immunoreactivity. The results were given as percentage of F4/80 or Ly6B.2 immunoreactive area within the ROI. Scatterplots of the automatized digital image analyses are shown. (c) Haematoxylin and eosin (H&E) stain, and F4/80 and Ly6B.2 immunohistochemical stains are shown to exemplify arthritic changes and cell influx in the metatarsal joint. In the images annotations represent: B, bone marrow; S, synovium; arrow heads, cartilage; arrows, brown immunoreactive cells; *immunororeactivity in bone marrow excluded from analysis. Scale bar is 200 μm and applies to all images. Analyses are presented as mean (± standard error of the mean). Statistical significance was calculated by unpaired two-tailed t-test with Welch's correction. *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 3

Single-dose therapeutic treatment with anti-murine C5a receptor (mC5aR) ameliorates arthritis. Mice (n = 22–26/group) were immunized for induction of arthritis at day 0 and boosted at day 21. Single-dose treatment (0·5 mg/mouse) with anti-mC5aR or anti-trinitrophenyl (TNP) mIgG2a were initiated individually when mice reached a clinical score of 2–4, corresponding to score day 1. Mice were terminated 48 h after the treatment. (a) Mean clinical score (± standard error of the mean) per group over time and (b) AUC (area under the curve for individual clinical scores); black line represents the mean. Significance was calculated by Student's t-test (two-tailed) with Welch's correction. Changes in polymorphonuclear (PMN) numbers and expression of activation stage markers (CD11b and FcγR) were analysed by fluorescence activated cell sorter (FACS) in duplicate (PMN number and CD11b) or single-sample analysis (FcgR); statistical significance was calculated by two-tailed unpaired t-test (data not shown).

Fig. 4

Single-dose treatment with anti-murine C5a receptor (mC5aR) mediates local changes of inflammatory markers. Mice were immunized for induction of arthritis at day 0 and boosted at day 21. Single-dose treatment (0·5 mg/mouse) with anti-mC5aR or anti-trinitrophenyl (TNP) mIgG2a were initiated individually when mice reached a clinical score of 2–4. Paw homogenate from both hind paws (n = 11–15 mice/group) were prepared 48 h after single-dose treatment. The RodentMAP 58 analyte assay was used to detect the level of the individual analytes in anti-mC5aR-treated mice compared to control-treated mice. The following analytes are shown: (a) the neutrophil marker myeloperoxidase (MPO), (b) the proinflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and IL-17A, (c) the chemoattractants keratinocyte chemoattractant (KC), monocyte chemoattractant parotein (MCP)-1, MCP-5 and (d) the tissue remodelling marker tissue inhibitor of metalloproteinases (TIMP)-1. The mean of the two hind paws per mouse is shown. Statistical significance calculated by Student's two-tailed t-test, *P < 0·05; **P < 0·01.

Fig. 5

Reduced macrophage and neutrophil infiltration after single-dose anti-murine C5a receptor (mC5aR) treatment. Mice (n = 15–16/group) were immunized for induction of arthritis at day 0 and boosted at day 21. Single-dose treatment (0·5 mg/mouse) with anti-mC5aR or anti-trinitrophenyl (TNP)mIgG2a were initiated individually when mice reached a clinical score of 2–7, corresponding to score day 1. Mice were terminated 48 h after the treatment. Histology score index and immunohistochemistry were used to determine the degree of (a) synovitis and destruction of joint architecture and to detect infiltrating (b) macrophages and (c) neutrophils as described previously in Fig. 2. *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 6

Dose–response relationship of anti-murine C5a receptor (mC5aR) in collagen-induced arthritis (CIA) mice. Arthritis symptoms were induced as described and assessed every day post-boosting. CIA mice were treated therapeutically starting when they presented with a clinical score of minimum 2. On the day of enrolment they received a loading dose of either 1, 6 or 30 mg/kg followed by five doses of either 0·75, 2 or 6 mg/kg of anti-mC5aR or 30 mg/kg + 6 mg/kg anti-trinitrophenyl (TNP)within 2 weeks; n = 12–13 mice per group. (a) Mean clinical scores ± standard error of the mean. (b) Area under the curve (AUC) of clinical scores. (c) C5aR occupancy. Blood samples were collected 72 h post-dosing and subjected to flow cytometric analysis for C5aR occupancy on circulating polymorphonuclear (PMN) numbers. The C5a receptor was only blocked by the highest dose of anti-mC5aR at this time-point. Dotted line represents mean of isotype-stained samples; n = 11–12 per group. (d) Target engagement; 72-h blood samples were stimulated with 100 nM or without recombinant mouse C5a and the effect on the CD11b expression level on PMN numbers was addressed by flow cytometry. Only the highest dose of anti mC5aR sufficiently blocked the C5aR at this time-point. Dotted line represents the mean of CD11 mean fluorescence intensity (MFI) of unstimulated PMNs from isotype control-treated animals; n = 11–12 per group. Statistical significance was calculated by an unpaired two-tailed t-test with Welch's correction; n.s., not significant; *P < 0·05; ***P < 0·001.

Fig. 7

The effect of supersaturating doses on clinical outcome. The collagen-induced arthritis (CIA) mice were treated therapeutically starting when they presented with a clinical score of minimum 2. On the day of enrolment they received a loading dose of either 10, 30 or 90 mg/kg followed by five doses of either 2, 6 or 18 mg/kg of anti-murine C5a receptor (mC5aR) or 90 mg/kg + five doses of 18 mg/kg anti-trinitrophenyl (TNP)within 2 weeks; n = 16–18 mice per group. (a) Mean clinical scores ± standard error of the mean. (b) Area under the curve (AUC) of clinical scores. Statistical significance was calculated by an unpaired two-tailed t-test with Welch's correction; n.s., not significant; *P < 0·05.

Fig. 8

C5a receptor (C5aR) occupancy and target engagement. Blood samples collected 4, 24 and 72 h post-dosing from the second-dose titration experiment were subjected to flow cytometric analysis for C5aR occupancy and target engagement on circulating polymorphonuclear (PMN) numbers. (a) Four h after dosing all three tested doses of anti-murine C5a receptor (mC5aR) resulted in complete occupancy of the target; (b) 24 h and (c) 72 h after dosing the C5a receptor was blocked by the intermediate and the highest dose of anti-mC5aR. Dotted line represents the mean of isotype stained samples; n = 11–18 per group. Blood samples were stimulated with 100 nM or without recombinant mouse C5a and the effect on the CD11b surface level on PMN numbers was addressed by flow cytometry. (d) The CD11b mean fluorescence intensity (MFI) was unchanged in PMNs from mice treated with all three doses of anti-mC5aR 4 h post-dosing; (e) 24 h and (f) 72 h after dosing the intermediate and the highest dose of anti-mC5aR sufficiently blocked C5a mediated up-regulation of CD11b on the PMNs. Dotted line represents the mean of CD11 MFI of unstimulated PMNs from isotype control-treated animals; n = 11–18 per group. Statistical significance was calculated by an unpaired two-tailed t-test with Welch's correction; n.s., not significant, *P < 0·05.