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Efficacy of Targeted Complement Inhibition in Experimental C3 Glomerulopathy

† † † † Marieta M. Ruseva,* Tao Peng, Melissa A. Lasaro, Keith Bouchard, Susan Liu-Chen, † † † † Fang Sun, Zhao-Xue Yu, Andre Marozsan, Yi Wang, and Matthew C. Pickering*

*Centre for Complement and Inflammation Research, Imperial College, London, United Kingdom; and †Alexion Pharmaceuticals, Cheshire, Connecticut

ABSTRACT C3 glomerulopathy refers to renal disorders characterized by abnormal accumulation of C3 within the kidney, commonly along the glomerular basement membrane (GBM). C3 glomerulopathy is associated with complement alternative pathway dysregulation, which includes functional defects in complement regulator (FH). There is no effective treatment for C3 glomerulopathy. We investigated the efficacy of a recombinant mouse protein composed of domains from 2 (CR2) and FH (CR2-FH) in two models of C3 glomerulopathy with either preexisting or triggered C3 deposition along the GBM. FH-deficient mice spontaneously develop renal pathology associated with abnormal C3 accumu- BASIC RESEARCH lation along the GBM and secondary plasma C3 deficiency. CR2-FH partially restored plasma C3 levels in FH-deficient mice 2 hours after intravenous injection. CR2-FH specifically targeted glomerular C3 deposits, reduced the linear C3 reactivity assessed with anti-C3 and anti-/iC3b/C3c , and prevented further spontaneous accumulation of C3 fragments along the GBM. Reduction in glomerular C3d and C9/C5b-9 reactivity was observed after daily administration of CR2-FH for 1 week. In a second mouse model with combined deficiency of FH and , CR2-FH prevented de novo C3 deposition along the GBM. These data show that CR2-FH protects the GBM from both spontaneous and triggered C3 deposition in vivo and indicate that this approach should be tested in C3 glomerulopathy.

J Am Soc Nephrol 27: 405–416, 2016. doi: 10.1681/ASN.2014121195

C3 glomerulopathy (C3G) is characterized by ab- progression in C3G are poorly defined, but include normal accumulation of C3 within the glomeruli male sex in CFHR5N,8 DDD subtype,9 and crescen- and includes dense deposit disease (DDD) and C3 tic glomerulonephritis at presentation.9 glomerulonephritis.1–3 C3G can be associated with There is no specific treatment for C3G. While inherited or acquired defective regulation of the nonspecific measures such as antiproteinuric ther- complement alternative pathway (AP).4 Genetic apy were associated with better renal survival in the factors include loss of function mutations in the large French series,3 immunosuppressive treatment AP-negative regulator, complement factor H was not. Complement C5 inhibition, using the (FH).5,6 Acquired causes include (C3 nephritic factors) that potentiate AP activa- 4,7 tion. The clinical course of C3G is variable, but Received December 9, 2014. Accepted April 1, 2015. overall it has a poor prognosis and frequently recurs 1 Published online ahead of print. Publication date available at in the transplant kidney. In a French series that www.jasn.org. included 29 DDD and 56 C3 glomerulonephritis cases, overall renal survival at 10 years following Correspondence: Prof. Matthew Pickering, Centre for Comple- ment and Inflammation Research, Division of Immunology and 3 presentation was 63.5%. In a study of 91 patients Inflammation, Imperial College London, Hammersmith Campus, with complement FH-related protein 5 nephropathy Du Cane Road, London W12 0NN, UK. Email: matthew.pickering@ (CFHR5N), 31% developed chronic renal failure, and imperial.ac.uk 20% developed end stage renal failure.8 Predictors of Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 405–416, 2016 ISSN : 1046-6673/2702-405 405 BASIC RESEARCH www.jasn.org monoclonal eculizumab, showed variable efficacy after injection, but not at later time points (Figure 1, A and B). in an open label study10,11 and case reports.12–19 In the FH- Plasma C3 levels increased (median=59.15 mg/l, range 56– deficient mouse model of C3G, coexistent C5 deficiency ame- 59.9, n=3, Figure 1C) in CR2-FH–treated animals at the liorated spontaneous glomerular inflammation but did not 2-hour time point compared with either PBS-injected or alter the degree of C3 staining or electron-dense changes along FH(1–5)-injected animals, but plasma C3 levels were compa- the glomerular basement membrane (GBM).20 The lack of a rable between the experimental groups at both 24 and 48 uniform response to eculizumab perhaps indicates that there hours. These data were confirmed by western blot analysis are both C3- and C5-dependent mechanisms of renal injury, (Supplemental Figure 3). Plasma C5 levels were analyzed using which may be different between patients and over time western blotting under nonreducing conditions (Figure 1D). within a given patient. Arguably the most logical therapy In the CR2-FH group, C5 was detectable at all tested time would be that which prevented or ameliorated uncontrolled points, but the greatest intensity was 24 hours after injection. C3 regulation. In the FH(1–5)-injected animals, C5 was only present 2 hours Strategies to reduce C3 activation specifically at sites of after injection and was absent at 24 and 48 hours (Figure 1D). complement activation include CR2-FH,21 a fusion protein 2/2 comprised of the complement regulatory domains of FH CR2-FH Reduced Glomerular C3 in Cfh Mice 2/2 (FH1–5) linked to the C3 fragment-binding domains of com- Cfh mice spontaneously develop C3 and C9 accumulation 29 plement receptor 2 (CR21–4). Using these components, CR2- along the GBM. After a single intravenous injection of CR2- FH can interact with C3 fragments at sites of complement FH, there was a reduction in the linear glomerular C3 at the 2, activation and then prevent further C3 activation. CR2-FH 24, and 48 hour time points, together with the appearance of has demonstrated efficacy in models of AP-mediated tissue mesangial C3 staining at 24 and 48 hours (Figure 2, A and B). injury including paroxysmal nocturnal hemoglobinuria,22 We confirmed that the decrease in C3 reactivity was not due to ischemia/reperfusion injury,21,23 -induced airway CR2-FH masking on C3 and preventing binding of hyperresponsiveness,24 laser-induced choroidal neurovascu- the anti-C3 antibody (Supplemental Figure 4). Glomerular C3 larization,25,26 and collagen antibody–induced arthritis.27 staining did not change following administration of either We investigated the efficacy of CR2-FH in two animal models FH(1–5) or PBS. We used two additional anti-C3 antibodies of C3G, one with preexisting and one with triggered C3 de- that react with either C3b/iC3b/C3c or C3d. Using the position along the GBM. Our data indicate that CR2-FH ef- C3b/iC3b/C3c , significantly reduced fectively interacts with GBM-bound C3 and prevents further linear staining was apparent in the CR2-FH group compared C3 activation within the kidney. with the mice injected with FH(1–5) or PBS at all time points (Figure 3). The staining observed with the anti-C3d polyclonal antibody was linear in distribution with no mesangial reactivity. RESULTS In contrast to both the anti-C3 polyclonal and anti-C3b/iC3b/C3c monoclonal antibody data, the intensity of the C3d (Supplemen- CR2-FH is Rapidly Cleared from the Circulation via tal Figure 5) and C9/C5b-9 staining (data not shown) did not the Kidney change after CR2-FH injection and remained unchanged between After a single intravenous bolus injection of 40 mg/kg CR2-FH all three experimental groups. in wild-type animals, plasma CR2-FH concentration reached 2/2 peak levels at 15 minutes (383.6644.82 mg/mL), dropping CR2-FH Colocalized with Glomerular C3 in Cfh Mice rapidly by 6 hours (2.9260.61 mg/ml, Supplemental Figure Using an Alexa 594-conjugated polyclonal anti-mouse FH 1). AP-mediated membrane attack complex formation was antibody, CR2-FH and not FH(1–5) was detectable within glo- inhibited at .90% at 15 minutes and 2 hours, but was restored meruli, and colocalized with the linear C3 reactivity. CR2-FH by 6 hours. The highest concentrations of CR2-FH were de- did not bind along the GBM in wild-type mice (Supplemental tected within the kidney in wild-type and C3-deficient ani- Figure 6). The interaction of CR2-FH with the linear glomer- mals. In wild-type mice almost 90% of the CR2-FH had ular C3 progressively reduced in intensity following injection cleared from the kidney at 2 hours postinjection (Supplemen- but was still detectable at 48 hours (Figure 2A). Glomerular tal Figure 2), indicating rapid clearance from the circulation CR2-FH fluorescence intensity at 2 hours (median=1001.7 via the kidney. arbitrary units, range 767.7–1451.2, n=3) was higher than that at 24 hours (median=384.7 arbitrary units, range CR2-FH Partially Restored Plasma C3 and C5 Levels in 358.9–432.9, n=3) and 48 hours (median=358.7 arbitrary 2/2 FH-Deficient (Cfh ) Mice units, range 352.9–440.9, n=3, Supplemental Figure 7A). We Molar equivalent doses of CR2-FH (0.8 mg) and FH(1–5) (0.44 did not detect an interaction between CR2-FH and mesangial 2 2 mg) were administered intravenously to Cfh / mice and the C3 that developed in animals injected with CR2-FH (Figures animals were sacrificed 2, 24, and 48 hours postinjection. In 2A and 3A). To determine if this reflected a lack of availability 2 2 the Cfh / mice there is depletion of both C3 and C5.28,29 of CR2-FH at the time that the mesangial C3 developed, we 2 2 Both reagents were detected by western blot in plasma 2 hours incubated kidney sections taken from Cfh / mice 48 hours

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Figure 1. Plasma complement profile. Plasma samples collected 2, 24, and 48 hours after injection with recombinant CR2-FH, FH(1–5) and PBS were subjected to western blot and ELISA analysis. Both (A) CR2-FH and (B) FH(1–5) were detected by western blot in plasma samples 2 hours after injection, but were absent at 24 and 48 hours. High molecular weight background bands were observed in all plasma samples including those collected from PBS-injected mice, but not in the recombinant protein preparations. (C) C3 levels, measured by ELISA, were increased in the CR2-FH group 2 hours after injection compared with either FH(1–5)- or PBS-injected mice. No difference between the groups was observed at other time points. Unmanipulated wild-type C3 levels in this ELISA are shown for reference (n=3). Horizontal bars denote median values. *P,0.05 versus FH(1–5) group and P,0.01 versus PBS group, Bonferroni’s Multiple Comparison Test. (D) Plasma C5 was analyzed by western blot under nonreducing conditions using 1 ml of mouse plasma. Under these conditions, C5 (band indicated by arrow) was readily detectable in mice injected with CR2-FH at all analyzed time points.

C5 was detected in the FH(1–5) group only at 2 hours, and absent at all time points in PBS-injected animals.

after CR2-FH injection with CR2-FH ex vivo. The exogenous with anti-C3 polyclonal and anti-C3b/iC3b/C3c monoclonal CR2-FH reacted with the C3 along the GBM ex vivo but not antibodies at the 48-hour and all later time points appeared with the mesangial C3 (Supplemental Figure 7B), indicating reduced compared with that observed in nontreated animals that the lack of reactivity was not a consequence of CR2-FH (Figure 4). No difference in the intensity of C3d staining was availability. observed between treated and nontreated mice, even at the 120-hour time point (Figure 4). Although the staining intensity The Reduction in Glomerular C3 Reactivity Persists progressively reduced, CR2-FH was present in the glomeruli 2/2 after Single Injection of CR2-FH in Cfh Mice 120 hours after the single injection (Figure 4). We next determined how long the reduction in linear C3 2 2 persists following a single injection of CR2-FH. Cfh / mice Multiple Injections of CR2-FH Ameliorated C3d and 2/2 were injected with a single dose of CR2-FH and kidneys were C9/C5b-9 Reactivity in Cfh Mice 2 2 harvested at 48 hours (n=2), 72 hours (n=4), 96 hours (n=4), To determine the effects of repeated CR2-FH dosing, Cfh / mice and 120 hours (n=4). The C3 staining observed with the three received CR2-FH (0.8 mg, n=5) or PBS (n=6) daily for 7 days anti-C3 antibodies at the earliest time point (48 hours) was and were then culled at day 8, 24 hours after the last injection. similar in distribution pattern and intensity to that obtained at Plasma C3 levels in the CR2-FH group (median=37.28 mg/L, the same time point (48 hours) in the previous experiment range 33.53–42.7, n=5) were slightly higher than those in the (Figures 2A and 3A). The glomerular C3 reactivity assessed PBS-injected group (median=32.85 mg/L, range 28.26–35.67,

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2 2 Figure 2. CR2-FH colocalized with and reduced glomerular C3 in Cfh / mice. (A) Representative images of glomerular C3, CR2-FH, 2/2 and FH(1–5) in Cfh mice injected with a single dose of CR2-FH, FH(1–5), or PBS. C3 was visualized using FITC-conjugated goat anti- mouse C3 polyclonal antibody (green). Alexa 594-conjugated rabbit anti-mouse FH polyclonal antibody was used to detect either CR2-

FH or FH(1–5) (red). CR2-FH (red) colocalized with C3 (green) along the GBM. No staining was observed with the Alexa 594-conjugated

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polyclonal (median=253.5 arbitrary units, range 215.6–306.6, n=5 and median=2085.1, range 1889.2–2276.8, n=6, CR2-FH–treated versus PBS-treated, respectively) and anti- C3b/iC3b/C3c monoclonal antibodies (me- dian=253.3, range 199.7–301.5, n=5 and median=1028.1, range 857.9–1117.6, n=6, CR2-FH–treated versus PBS-treated, respec- tively; Figure 5, C and D). However, repeated doses of CR2-FH were associated with a significant decrease in glomerular C3d (median=305.6 arbitrary units, range 262.1– 374.9, n=5 and median=496.7 arbitrary units, range 426.4–561.5, n=6, CR2-FH– treated versus PBS-treated, respectively; Fig- ure 5, C and D) and C9/C5b-9 reactivity (median=366.8 arbitrary units, range 346.6–416.1, n=5 and median=741.5 arbi- trary units, range 603.4–870.6, n=6, CR2- FH–treated versus PBS-treated, respectively; Figure 5, C and D). If there is less ligand for CR2-FH (C3d) within the glomeruli, then we would expect less of the reagent to target the kidney. Consistent with a reduction in C3d, we observed significantly reduced glo- merular staining for CR2-FH in the mice that 2 2 Figure 3. CR2-FH colocalized with and decreased glomerular C3b/iC3b/C3c in Cfh / received multiple doses of CR2-FH com- 2 2 mice. (A) Representative images of glomerular C3b/iC3b/C3c in Cfh / mice injected pared with those that received a single dose with a single dose of CR2-FH, FH(1–5), or PBS. C3b/iC3b/C3c was visualized using a rat (Supplemental Figure 8). anti-mouse C3b/iC3b/C3c monoclonal antibody (green). Alexa 594-conjugated rabbit anti-mouse FH polyclonal antibody was used to detect CR2-FH (red). CR2-FH (red) FH(1–5) Reduced, but Did Not colocalized with the linear C3b/iC3b/C3c (green) staining. This linear staining was Colocalize with, Glomerular C3 in reduced in intensity in CR2-FH-injected animals compared with either the FH(1–5) or Cfh2/2 PBS group. Granular C3b/iC3b/C3c staining was demonstrable 24 and 48 hours after Mice CR2-FH injection. Original magnification, 340. (B) Quantitative immunofluorescence Exogenous mouse and human FH restored demonstrated a reduction in glomerular C3b/iC3b/C3c in the CR2-FH group com- plasma C3 levels and reduced GBM C3 2/2 30,31 pared with either the FH(1–5)- or PBS-injected mice at 2, 24, and 48 hours. Horizontal deposition in Cfh mice. After a sin- 2/2 bars denote median values. *P,0.05 versus PBS. **P,0.001 versus FH(1–5) and PBS. gle injection of 0.44 mg of FH(1–5) in Cfh , , ’ ***P 0.001 versus FH(1–5) and P 0.01 versus PBS. Bonferroni s Multiple Comparison mice, we detected a faint band of intact Test. AFU, arbitrary fluorescent units. C3 alpha chain in serum at 2 hours but no changes in glomerular C3. At a higher dose, a single injection of 1.6 mg FH(1–5) n=6), but remained significantly lower than C3 levels in unma- transiently increased plasma C3 levels at 2 hours (Supplemen- nipulated wild-type mice (median 278.84 mg/L, range 277.08– tal Figure 9A), but did not alter glomerular C3 reactivity at 2 280.59, n=2, Figure 5A). Plasma C5 levels were comparable to hours (FH(1–5): median=1149.7 arbitrary units, range 748.2– those of nontreated wild-type animals, and strikingly different to 1300.3, n=4; PBS: median=1091.1 arbitrary units, range 2/2 the almost undetectable plasma C5 seen in PBS-injected Cfh 973.9–1209.1, n=2), or at 24 hours (FH(1–5): median=1524.3 mice (Figure 5B). CR2-FH–treated animals had significantly arbitrary units, range 1466.0–1582.7, n=2; PBS: median=1143.8 reduced glomerular C3 reactivity using both the anti-C3 arbitrary units, range 1095.4–1192.1, n=2). However, following

rabbit anti-mouse FH polyclonal antibody in mice injected with either FH(1–5) or PBS. Granular C3 was detected 48 hours after CR2-FH injection. Original magnification, 340. (B) Quantitative immunofluorescence demonstrated a reduction in glomerular C3 in the CR2-FH group compared with either the FH(1–5) or PBS-injected mice at 2, 24, and 48 hours. Horizontal bars denote median values. **P,0.001 versus FH(1–5) and P,0.01 versus PBS. *P,0.01 versus FH(1–5) and P,0.001 versus PBS, Bonferroni’s Multiple Comparison Test. AFU, arbitrary fluorescent units.

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2 2 Figure 4. Representative images of glomerular C3 in Cfh / mice injected with a single dose of CR2-FH. The reduction in glomerular 2 2 C3 and C3b/iC3b/C3c intensity at 48 hours following a single injection of CR2-FH in Cfh / mice persisted to at least 96 hours. In contrast, no change in glomerular C3d reactivity was demonstrable (see also Supplemental Figure 5). CR2-FH reactivity, detected using the Alexa 594-conjugated rabbit anti-mouse FH polyclonal antibody, was demonstrable at all time points after injection and no re- 2 2 activity was seen in nontreated in Cfh / mice. Original magnification, 340.

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Figure 5. Plasma and glomerular complement profile in mice injected with multiple doses of either CR2-FH or PBS. (A) Plasma C3 2 2 remained low in Cfh / mice despite repeated injections of CR2-FH. Horizontal bars denote median values. (B) Plasma C5 was analyzed by western blot under nonreducing conditions using 1 ml of mouse plasma. Under these conditions, C5 (band indicated by arrow) was readily detectable in mice injected with CR2-FH and unmanipulated wild-type mice, but absent in PBS-injected and unmanipulated 2 2 Cfh / mice. (C) Representative images of glomerular C3 and C9/C5b-9. Original magnification, 340. (D) Quantitative immunofluo- rescence demonstrated a reduction in glomerular C3, C3b/iC3b/C3c, C3d, and C9/C5b-9 in the CR2-FH-injected mice compared with the PBS group. Horizontal bars denote median values. *P=0.004 versus PBS group. Mann–Whitney test. AFU, arbitrary fluorescent units.

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three injections of 1.6 mg of FH(1–5) over 24 hours, there was a reduced glomerular C3 reactivity after a single injection for at reduction in C3 reactivity and the appearance of mesangial C3 least 48 hours, and daily administration for 7 days resulted in reactivity (Supplemental Figure 9B). Unlike CR2-FH, which was reduction in glomerular C3d reactivity. Following injection, readily detectable and colocalized with glomerular C3, we were CR2-FH rapidly localized to glomerular C3 deposits along the not able to detect FH(1–5) in association with glomerular C3, GBM and remained detectable for up to 120 hours after a suggesting that the effect was either indirect (e.g., control of single injection. Despite a long half-life within the GBM, the C3 activation in plasma and consequent reduction of C3 depo- agent was rapidly cleared from the circulation. It was detect- sition along the GBM), or that the interaction with glomerular able in plasma 2 hours after injection, but absent at 24 hours C3 was very rapid (e.g., binding to C3b and rapid release follow- and later time points. Consistent with this rapid plasma clear- ing factor I [FI]-mediated cleavage of C3b to iC3b). ance, plasma C3 levels rose only transiently and partially fol- lowing administration. This is in contrast to the circulating CR2-FH Prevented Experimentally Triggered C3 half-life of FH following single administration: exogenous hu- Accumulation on the GBM man FH had an estimated half-life of 6 days in human FH 2 2 2 2 Mice deficient in both FI and FH (Cfh / .Cfi / )develop deficiency,33 was detectable in circulation 4 days after injection mesangial but not GBM accumulation of C3.32 Administra- in FH-deficient mice,31 and was associated with prolonged tion of a source of FI (serum from mice with a combined increase in plasma C3 levels. Our data showed that CR2-FH 2 2 2 2 deficiency of C3 and FH, denoted C3 / .Cfh / serum) to was rapidly cleared from the plasma both in wild-type and these animals results in the appearance of GBM C3 deposition.32 factor H–deficient mice. So, if C3G was caused by a systemic We investigated whether CR2-FH could influence the develop- defect in the regulation of C3 activation, we would predict that 2 2 2 2 ment of GBM C3 by administering CR2-FH to Cfh / .Cfi / CR2-FH would not correct the systemic defect. However, 2 2 2 2 mice (n=4) prior to injection of C3 / .Cfh / serum. As pre- based on our data, it would protect the GBM from C3 activa- viously demonstrated,32 plasma C3b (alpha prime chain) was tion, which may be sufficient to ameliorate or prevent renal 2 2 2 2 detected in control Cfh / .Cfi / mice while, following in- injury. We did not detect an interaction between CR2-FH and 2 2 2 2 jection of C3 / .Cfh / serum, plasma C3 alpha chain frag- the mesangial C3 that appeared after administration of CR2- ments were evident (Figure 6A), together with the appearance of FH. This remains unexplained, but could reflect the nature of linear staining along the GBM (Figure 6C). The appearance of the mesangial C3; for example, if this was C3c or intact C3, we the plasma C3 profile did not change with prior administration would not expect an interaction with CR2-FH. of CR2-FH at the 24-hour time point. No C5 was detected in CR2-FH was also able to prevent the de novo appearance of mice injected with PBS irrespective of whether or not they re- glomerular C3 in a triggered model of C3G. In this setting, 2 2 2 2 ceived C3 / .Cfh / serum (Figure 6B). However, C5 became the administration of a source of FI results in proteolytic cleav- 2 2 2 2 detectable in Cfh / .Cfi / mice following the injection of CR2- age of C3b and generation of C3b metabolites together with the FH and this was independent of the administration of mouse appearance of GBM C3 reactivity.32 Our data show that the serum containing FI. As previously reported, linear glomerular pre-administration of CR2-FH completely prevented the de- 2 2 2 2 C3 staining developed in Cfh / .Cfi / mice following injection velopment of GBM C3 reactivity, but did not influence the of mouse serum containing FI (Figure 6C).32 In marked con- metabolism of C3b. We speculate that CR2-FH interacted trast, this linear glomerular C3 staining was not seen in the with C3b metabolites, preventing their association with the animals pre-injected with CR2-FH. In these mice, there was GBM, and/or interacted with any C3 that did associate with mesangial C3 reactivity only and this was less intense than that the GBM and prevented further amplification. seen in animals treated with CR2-FH or PBS that did not CR2-FH differentially affected plasma C3 and C5 levels in 2 2 2 2 receive the C3 / .Cfh / serum (Figure 6D). Using the anti- the experimental models. Potential explanations include re- FH antibody, CR2-FH was found to colocalize with the duction of C5 activation in fluid-phase or along surfaces within mesangial C3 in all mice injected with the reagent (Figure 6C). the kidney or both. C5 activation in fluid-phase is inefficient in In summary, a single CR2-FH injection increased plasma C5 vitro.34 We looked at the rate of C3 and C5 activation in human 2 2 2 2 levels in Cfh / .Cfi / mice, colocalized with mesangial C3, FH-depleted sera in vitro. C3 was rapidly cleaved (within 20 and prevented the appearance of linear glomerular C3 following minutes) on addition of cations (Supplemental Figure 10). In injection of mouse serum containing FI. contrast, C5 levels did not change over 24 hours as assessed by both western blotting and the ability of the test sera to recon- stitute hemolytic activity to human C5-depleted sera. This DISCUSSION indicates that C3 activation is likely to be rapid in the setting of FH deficiency in vivo, while the activation of C5 will be CR2-FH localized to C3 along the GBM in the FH-deficient slower and depend on the availability of C3b. We speculate mouse. The targeting domain (CR2) of CR2-FH was required that the C5 convertase in complete FH deficiency is ineffi- 35,36 for this effect because a single injection of FH1–5, unlike a single cient, and is therefore particularly sensitive to inhibition injection of CR2-FH, did not localize to the kidney or alter by exogenous regulators or the presence of . Pro- glomerular C3 reactivity. In the FH-deficient animal, CR2-FH perdin deficiency differentially affected C3 and C5 levels in

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2 2 2 2 Figure 6. CR2-FH prevented triggered C3 accumulation on the GBM. Mice with combined deficiency of FH and FI (Cfh / .Cfi / ) were treated with CR2-FH or PBS and 1 hour later either did (+) or did not (–) receive an injection of serum deficient in both C3 and FH (as a source of FI) to trigger GBM C3 deposition. Animals were culled 24 hours later and (A) plasma C3, (B) plasma C5, and (C,D) glomerular C3, 2 2 2 2 and (C) CR2-FH assessed. (A) The C3 alpha prime chain of C3b was present in the Cfh / .Cfi / mice irrespective of prior treatment with CR2-FH or PBS. C3 alpha chain fragments were detected in all mice that received C3- and FH-deficient mouse serum irrespective of pretreatment with CR2-FH or PBS. (B) C5 became detectable in animals that had received CR2-FH irrespective of subsequent injection with C3- and FH-deficient serum. (C) Representative images of glomerular C3 (green) and CR2-FH (red), original magnification 340, and (D) quantitative glomerular C3 immunofluorescence, horizontal bars denote median values. *P,0.001, Bonferroni’s Multiple Comparison Test. NS, not significant. GBM C3 reactivity was detected in mice injected with C3- and FH-deficient serum alone, but not in those who had been pretreated with CR2-FH with consequent significant reduction in glomerular C3 staining intensity. The intensity of the mesangial C3 staining pattern seen in mice that had not received C3- and FH-deficient serum was not altered by pretreatment with CR2-FH.

FH-deficient mice,37,38 and here we could detect increases in changes in C3 and C5 levels derive from the different conver- C5 2 hours after injection of either FH1–5 or CR2-FH to FH- tase efficiencies. It is also possible that C5 activation within the deficient mice. It is possible that these inhibitors transiently mesangium or along the GBM contributes to plasma C5 de- regulate both the C3 and C5 convertases, but the discordant pletion and that this is influenced by CR2-FH and FH1–5.

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However, we could not detect an interaction between FH1–5 Animals and Treatment Protocols and glomerular C3. Notably, glomerular C9 reactivity was not Mice were housed in specific pathogen-free conditions and animal 2 2 influenced by properdin deficiency in Cfh / mice.37 procedures were performed in accordance with institutional guide- 2/2 lines and approved by the United Kingdom government. The gener- Repeated administration of FH1–5 to Cfh mice over a 24- 2/2 2/2 2/2 2/2 2/2 hour period was associated with a reduction in glomerular C3 ation of Cfh ,29 Cfi .Cfh , and C3 .Cfh 29,32 mice has staining. Notably, when a mutant FH protein that lacked the been described previously. Reagents were administered intravenously terminal five short consensus repeat (SCR) domains (FHD16–20) at the doses indicated (CR2-FH 0.8 mg; FH(1–5) 0.44 or 1.6 mg) and 2 2 was expressed in the Cfh / mice, abnormal GBM C3 deposition samples and tissue collected at various time points. In the experi- did not develop, and plasma C3 levels increased in proportion ment where we administered three injections of 1.6 mg FH(1–5), the to the circulating FHD16–20 level.39 These data indicate that second injection was given intraperitoneally. To trigger the C3 2/2 2/2 restoration of systemic C3 dysregulation by FH proteins that accumulation along the GBM in Cfi .Cfh mice, we injected 2/2 2/2 lack surface recognition domains is sufficient to ameliorate (ex- C3 .Cfh mouse serum as described previously,32 and mice were sacrificed 24 hours later. In these experiments the injection ogenous FH1–5) or prevent (endogenous FHD16–20) glomeru- 2 2 2/2 2/2 lar C3 accumulation in Cfh / mice. However, the inability of of C3 .Cfh mouse serum was preceded 1 hour earlier by in- travenous injection of either 0.8 mg CR2-FH or PBS. FH1–5 to target to complement within the kidney indicated that this would not be an effective therapeutic approach in situations where C3 dysregulation is due to local activation. It would also, CR2-FH Biodistribution like FH, not be efficient in C3G associated with systemic C3 Wild-type C57BL/6 mice received a single intravenous injection of dysregulation that is driven by factors other than FH deficiency. 40 mg/kg CR2-FH. Animals were anesthetized, perfused with PBS, This is important, as C3G associated with FH deficiency is ex- and sacrificed at 15 minutes, 2, 6, and 24 hours after injection. Organ tremely rare. More commonly, fluid-phase C3 dysregulation in homogenates were isolated and plasma collected for quantitative C3G is due to the presence of C3 nephritic factors and normal analysis of CR2-FH levels. CR2-FH concentrations were determined levels of FH. In these settings, CR2-FH, by virtue of its ability to by ELISA using anti-CR2 7E9 (Hycult, The Netherlands) as capture localize to glomerular-bound C3, would be the definitive antibody and biotinylated rabbit anti-mouse FH polyclonal antibody method of specifically protecting glomerular components for detection (Alexion Pharmaceuticals, Cheshire, CT). (GBM and/or mesangium) from the deleterious consequences of complement activation. Complement Inhibition Assay In summary, we have utilized two models of C3G and Complement activity in mouse sera was determined by its ability demonstrated that CR2-FH: (1) rapidly associates with glo- to lyse rabbit erythrocytes.40 CR2-FH at 1000, 62.5, and 3.91 nM in merular C3 irrespective of whether it is within the mesangium gelatin veronal-buffered saline (0.1% gelatin, 141 mM NaCl, 2.5 mM or along the GBM; (2) ameliorated preexisting GBM-bound MgCl2,0.15mMCaCl2, 1.8 mM sodium barbital, 10 mM EGTA), was C3; and (3) prevented triggered GBM-bound C3. Our data used as low, medium, and high lysis controls, respectively. Water and indicate that CR2-FH could be efficacious in human C3G re- 42 mM EDTA were used as 100% lysis and negative controls, respec- gardless of whether the C3 accumulation derives from sys- tively. Samples were prepared in 1:2 dilutions in triplicate in gelatin temic or renal C3 dysregulation. Ideally, this would require veronal-buffered saline. Rabbit erythrocytes (1.53106) were added to validation in models of C3G not driven by complete FH de- control and test samples and incubated at 37°C for 30 min. The absor- ficiency. Unlike C5 inhibition, from experimental data, C3 bance of the supernatant was measured at 415 nm, and percent hemolysis inhibition is the definitive therapeutic target in C3G and our was calculated by standard methods.41 data validate exploring the efficacy of CR2-FH in C3G. C3 Quantification by ELISA Mouse blood was collected by cardiac puncture in the presence of CONCISE METHODS EDTA, chilled on ice and the plasma separated. Coating antibody wasagoat anti-mouseC3polyclonalantibody(MPBiomedicals,Cedex, France) used at a dilution of 1:8000 in 0.1 M carbonate buffer, pH 9.6. Generation of CR2-FH and FH(1–5) The recombinant fusion protein CR2-FH, a hybrid mouse protein Captured mouse C3 was detected using a horseradish peroxidase– comprised of mouse CR2(1–4) and mouse FH(1–5), was generated as conjugated goat anti-mouse C3 polyclonal antibody (MP Biomedicals) previously described.21 Subscript numbers denote SCR domains used at a dilution of 1:25,000 PBS/0.2% Tween. Plates were developed within CR2 and FH, with the first amino-terminal domain num- using TMB substrate (Sigma-Aldrich, Dorset, UK). The concentration bered one. The construct was prepared with a linker sequence be- of plasma C3 was estimated by reference to a calibration curve con- tween CR2 and FH encoding KEIL, a portion of the natural linker structed using reference sera containing a known amount of mouse C3

SCR4–5 of mouse CR2. The mouse FH(1–5) recombinant protein (Serum Amyloid P mouse standard; Calbiochem, Darmstadt, Germany). was purified from the supernatant of transiently transfected

EXP1293 cells by cation exchange, and complement activity eval- Western Blot for CR2-FH, FH(1–5), C3, and C5 uated using a complement alternative pathway hemolysis assay One microliter of EDTA plasma was used for western blot analysis. (data not shown). Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene

414 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 405–416, 2016 www.jasn.org BASIC RESEARCH

fluoride membrane. CR2-FH and FH(1–5) were detected with rabbit anti- ACKNOWLEDGMENTS mouse FH (Alexion Pharmaceuticals) and horseradish peroxidase– conjugated anti-rabbit immunoglobulins (DAKO). Western blotting We thank Prof. B. Paul Morgan, Cardiff University, United Kingdom formouseC3andC5wasperformedasdescribedpreviously.37 for the anti-rat C9 antibody. We acknowledge the valuable con- tributions of Drs. Michael Holers (University of Colorado), Steve Immunostaining Tomlinson (Medical University of South Carolina) and Danielle Kidney samples were frozen in O.C.T. compound and 5 mm cryosec- Paixao-Cavalcante (previously Imperial College) in experimental tions were fixed in acetone for 10 minutes. C3 was visualized using a work leading up to this study. FITC-conjugated goat anti-mouse C3 polyclonal antibody (MP M.C.P. is a Wellcome Trust Senior Fellow in Clinical Science Biomedicals), a rat anti-mouse C3b/iC3b/C3c monoclonal antibody (WT082291MA) and M.M.R. is funded by this fellowship. (Hycult, The Netherlands), and a biotinylated goat anti-mouse C3d (R&D System, Abingdon, UK). The latter was visualized using strep- tavidin Alexa Fluor 488 (Life Technologies, Renfrew, UK). CR2-FH DISCLOSURE was visualized using an Alexa 594-conjugated rabbit anti-mouse FH Alexion Pharmaceuticals provided CR2-FH reagent and funded the antibody (Alexion Pharmaceuticals). Mouse C9 was visualized study. M.A.L., K.B., S.L.C., F.S., Z.X.Y., A.M., and Y.W. are employees of Alexion usingarabbitanti-ratC9polyclonalantibodythatcross-reacts Pharmaceuticals. T.P. is a former employee of Alexion Pharmaceuticals. M.C.P. with mouse C9 (a gift from Prof. B. Paul Morgan, Cardiff University, has received fees from Alexion Pharmaceuticals for invited lectures. Cardiff, UK), and an Alexa Fluor 594-conjugated F(abʹ)2 of goat anti-rabbit IgG polyclonal antibody (Life Technologies). Quantitative immunofluorescence staining was performed as previously described.37 REFERENCES Ten glomeruli were assessed per section and the fluorescence intensity expressed in arbitrary units. 1. 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