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Soluble CR1 Therapy Improves Complement Regulation in C3 Glomerulopathy

†‡ | Yuzhou Zhang,* Carla M. Nester,* Danniele G. Holanda,§ Henry C. Marsh, | | † Russell A. Hammond, Lawrence J. Thomas, Nicole C. Meyer,* Lawrence G. Hunsicker, †‡ Sanjeev Sethi,¶ and Richard J.H. Smith*

*Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa, Iowa City, Iowa; †Division of Nephrology, Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa; ‡Division of Nephrology, Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa; §Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa; |Celldex Therapeutics Inc., Needham, Massachusetts; and ¶Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

ABSTRACT Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are widely recognized subtypes of C3 glomerulopathy. These ultra-rare renal diseases are characterized by fluid-phase dysregulation of the alternative complement pathway that leads to deposition of complement proteins in the renal glomerulus. Disease triggers are unknown and because targeted treatments are lacking, progress to end stage renal failure is a common final outcome. We studied soluble CR1, a potent regulator of complement activity, to test whether it restores complement regulation in C3 glomerulopathy. In vitro studies using sera from patients with DDD showed that soluble CR1 prevents dysregulation of the alternative pathway C3 con- vertase, even in the presence of C3 nephritic factors. In mice deficient in complement factor H and trans- genic for human CR1, soluble CR1 therapy stopped alternative pathway activation, resulting in normalization of serum C3 levels and clearance of iC3b from glomerular basement membranes. Short- term use of soluble CR1 in a pediatric patient with end stage renal failure demonstrated its safety and ability to normalize activity of the terminal complement pathway. Overall, these data indicate that soluble CR1 re-establishes regulation of the alternative complement pathway and provide support for a limited trial to evaluate soluble CR1 as a treatment for DDD and C3GN.

J Am Soc Nephrol 24: ccc–ccc, 2013. doi: 10.1681/ASN.2013010045

Dense deposit disease (DDD) and C3 glomerulone- in color, and are more often mesangial and/or sub- phritis (C3GN) are twowidely recognized subtypes of endothelial, intramembranous, and subepithelial in C3 glomerulopathy (C3G).1,2 These ultra-rare renal location.4 In both diseases, mass spectroscopy of la- diseases are caused by fluid-phase dysregulation of ser dissected glomeruli is highly enriched for proteins the C3 convertase of the alternative pathway (AP) of the AP and terminal complement cascade.4,5 of complement, with variable concomitant dysregu- lation of the C5 convertase. Consistent with comple- Received January 10, 2013. Accepted May 14, 2013. ment-mediated disease acting through the AP,C3G is Y.Z. and C.M.N are co-first authors. strongly positive for C3 and notably negative for Igs by immunofluorescence microscopy.2 Electron Published online ahead of print. Publication date available at www.jasn.org. microscopy distinguishes DDD from C3GN, with Correspondence: Dr. Richard J.H. Smith, Molecular Otolaryn- the former characterized by pathognomonic gology and Renal Research Laboratories, Carver College of electron-dense transformation of the lamina densa Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA of the glomerular basement membrane (GBM).3 In 52242. Email: [email protected] C3GN, the electron microscopy deposits are lighter Copyright © 2013 by the American Society of Nephrology

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Although long-term outcome data are not available for C3GN, it prevented classic pathway (CP) and AP complement activa- nearly half of all DDD patients progress to end stage renal failure tion (IC50 values of 2.5560.55 nM and 0.7160.08 nM, re- (ESRF) within 10 years of diagnosis.6,7 In virtually all cases of spectively; Figure 1A). Confirmatory hemolytic assays were DDD, transplantation is associated with histologic recurrence, performed with rabbit and sheep erythrocytes. Rabbit eryth- explaining the 5-year graft failure rate of 50%.7,8 There are no rocytes are a complement-activating surface in human sera; target-specific treatments for C3G; however, its pathophysiology however, lysis could be prevented by the addition of sCR1 suggests that therapeutic approaches to restore C3 convertase (IC50=29.4664.64 nM). In comparison, fH did not prevent control, impair C3 convertase activity, or remove C3 breakdown hemolysis even at high concentrations (Figure 1B). Sheep products from the circulation warrant consideration.1,9 erythrocytes do not activate complement in normal sera; Similar to human DDD, the complement factor H (Cfh)– however, hemolysis occurred when tested against DDD sera. deficient mouse, reclassified recently as a murine model of The addition of sCR1 restored AP control in a dose-dependent C3GN, accumulates C3 fragments within glomeruli as the first manner (Figure 1C) and prevented hemolysis even when DDD pathologic abnormality to develop.10 Systemic administration sera contained C3 convertase-stabilizing autoantibodies called of either murine or human complement factor H (fH) to the C3Nefs (Figure 1D). Cfh2/2 mouse rapidly reverses both the renal deposition of Wenext measured the systemic effect of sCR1 in vivo in both 2 2 2 2 C3 and its plasma depletion, suggesting that fH replacement Cfh / and Cfh / /huCR1-Tg mice (the latter express CR1 therapy may be an effective therapy to restore the underlying on erythrocytes only) by introducing sCR1 by the tail vein or protein deficiency and correct the disease process in C3G pa- intraperitoneally, respectively, after first verifying that it pre- tients with CFH mutations.11,12 However, it is unlikely that fH vented hemolysis of rabbit erythrocytes in mouse sera administration would be therapeutically successful in the ab- (IC50=6.1560.18 nM; Figure 2 and Supplemental Figure 3). 2 2 sence of CFH-inactivating mutations. For example, fH would After a single tail vein injection of sCR1 in Cfh / mice be ineffective in C3G patients carrying C3 mutations that ren- (50 mg/kg, n=5; Figure 2A, blue), serum C3 levels rose drama- der C3 convertase fH resistant or in the presence of autoanti- tically within 24 hours from 12.03612.29 mg/L to bodies to C3 convertase that block regulation by fH.13,14 219.77642.32 mg/L but fell to near preinjection levels by 48 Among the non-fH proteins that regulate C3 convertase is hours (41.84624.29 mg/L; Figure 2B, blue). Single-dose in- 2 2 soluble CR1. CR1 is a cell-surface glycoprotein expressed on traperitoneal injections in Cfh / /huCR1-Tg mice at concen- erythrocytes, monocytes, neutrophils, B cells, some T cells, trationsof5mg/kg,25mg/kg,or50mg/kgshowedthat follicular dendritic cells, and podocytes, and modulates the changes in serum C3 levels were sCR1 dose dependent (Figure complement cascade at multiple levels (Supplemental Figures 2, A and B, red). In all sCR1-treated animals, glomerular C3 1and2).15–18 It is composed of 30 short consensus repeats deposition was markedly reduced (Figure 2C, top panel) with (SCRs) linked to transmembrane and cytosolic domains.19 On only trace glomerular staining for sCR1 (Figure 2C, bottom the basis of SCR sequence homology, the 30 extracellular SCRs panel). In PBS-treated and unmanipulated animals, glomer- can be grouped into four long homologous repeats, which are ular C3 deposition was clearly evident (Figure 2C, top panel) sequentially lettered A through D. These long homologous and sCR1 was undetectable (Figure 2C, bottom panel). repeats give CR1 C3 and C5 convertase-controlling activity. We calculated the serum t1/2 of sCR1 in mice to be approx- CR1 is also the only cofactor of factor I (fI) to promote cleav- imately 18 hours. On the basis of the dose-response curves to 2 2 age of inactive C3b and inactive C4b into their smaller protein single intraperitoneal injections, we treated four Cfh / / fragments, C3dg and C4d.20–22 huCR1-Tg mice with three intraperitoneal injections The soluble form of CR1 (sCR1) is present in the circulation (25 mg/kg, n=2 mice; 50 mg/kg, n=2 mice) at 24-hour intervals at extremely low concentrations precluding any recognized (Figure 3A). In all sCR1-treated animals, serum C3 levels rose physiologic significance. However, super-physiologic single- to near normal by 12 hours and remained high throughout the dose treatment with sCR1 has been used in .500 patients experiment (Figure 3B). Glomerular immunofluorescence (infants and adults) undergoing a variety of cardiac surgeries studies showed that there was a significant decrease in new or after myocardial infarctions to arrest complement activa- C3 deposition, with clearance of old C3 demonstrated by the tion.23–26 These studies showed that sCR1 was well tolerated marked reduction in C3c staining. C3d staining also decreased with no adverse events clearly or consistently associated with and sCR1 staining was absent (Figure 3D). To verify that con- its use. Importantly, the possible development of anti-sCR1 trol of the C3 convertase had been restored and that it was an antibodies was monitored but never observed in .350 increase in serum C3 and not its degradation products that we patients.23,26 were measuring, we performed Western blotting under reduc- ing conditions with a C3a-chain–specific antibody to docu- ment the appearance of the 106-kD C3a chain. In PBS-treated RESULTS and untreated animals, serum C3 levels remained nearly un- detectable and the 106-kDa C3a chain was not seen (Figure We assessed the efficacy of sCR1 as a complement regulator in 3C). Thus, sCR1 treatment restored fluid-phase AP regulation vitro in normal and DDD sera. In normal pooled human sera, in these murine models of C3G.

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Figure 1. sCR1 prevents complement activation. (A) The ability of sCR1 to prevent activation of the alternative pathway (AP, red) and classical pathway (CP, blue) was measured by Wieslab complement assay using pooled normal human serum (PNHS). Results are expressed as percent activation by PNHS in presence of increasing concentrations of sCR1. Half-maximal inhibitory concentration (IC50) of sCR1 for the CP and AP are 2.55 6 0.55 nM and 0.71 6 0.08 nM, respectively. (B) Rabbit erythrocytes are normally an activating surface for the AP. sCR1 (red) protects rabbit erythrocytes from AP-mediated complement lysis (IC50 = 29.46 6 4.64 nM) while FH (blue) does not. (C) Sheep erythrocytes normally do not activate the human complement system, but DDD patient serum (20% v/v) causes hemolysis due to the presence of C3 convertase-stabilizing C3 nephritic factors. sCR1 suppresses C3Nef activity in a dose-dependent manner (serum from patient DDD-10). (D) In all DDD sera (20% v/v) tested, sheep erythrocyte hemolysis was reduced by sCR1. All patients except DDD-07 and 08 were positive for C3Nefs. Data represent mean 6 SD of triplicates (dark blue, 0nM sCR1; light blue, 160nM sCR1).

On the basis of these data, we sought permission from During treatment, serum C3 rose to a peak of 48 mg/dl but the US Food and Drug Administration (FDA) for a limited dropped to pretreatment levels by the end of the trial. Soluble multidose safety trial with sCR1 when presented with a C3G C5b-9 levels were elevated before treatment, normalized after patient in ESRF.The patient was an 8-year-old girl with biopsy- the sixth dose of sCR1, and became abnormal after terminating proven DDD (Figure 4A) who had presented 3 months earlier therapy (Figure 4D). Biopsies taken before and after treatment with lower extremity swelling, nephritic urine sediment, ne- were similar (Figure 4, B and C, and Table 1). The patient phrotic-range proteinuria, positive C3Nefs, and a low serum continues on dialysis at this time. C3 level (21.6 mg/dl; normal range, 90–180 mg/dl). She tran- Although the trial was very short, there were no adverse sitioned to rapidly progressive GN (eGFR ,10 ml/min per effects associated with the administration of sCR1 and im- 1.73 m2) necessitating dialysis. Approval for a seven-dose trial munogenicity was not detected. Assays for anti-sCR1 anti- of sCR1 was obtained from the FDA and from the University of bodies on plasma obtained before dosing and at days 2, 4, 6, 8, Iowa Institutional Review Board. 10, 12, and 14 and 1 month after dosing remained consistently The patient received a 10 mg/kg loading dose of sCR1 negative (A450 values #0.076 at dilutions of 1:50 at all time followed by six maintenance doses at 5 mg/kg every 48 hours. points; positive control, A450 1.10, 0.68, and 0.40 at 1:100,

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2/2 2/2 Figure 2. Human sCR1 restores serum C3 levels and reduces C3 glomerular staining in Cfh and Cfh /huCR1-Tg mice. (A) Plasma 2/2 levels of sCR1 dropped rapidly following a single tail-vein injection in 5 Cfh mice (blue lines; t1/2 approximately 18 hours). After 2/2 a single IP injection of sCR1 (5 mg/kg, 25 mg/kg or 50 mg/kg) in Cfh /huCR1-Tg mice, plasma levels rose and then fell slowly (red 2/2 lines). Each line represents a single mouse. (B) Serum C3 levels in Cfh mice (blue lines) increased 24 hours after a single tail-vein 2/2 injection of sCR1 (50 mg/kg) but returned to baseline 48 hours later. Serum C3 levels in Cfh /huCR1-Tg mice (red lines) also in- creased 12 hours after a single IP injection (5 mg/kg, 25 mg/kg, or 50 mg/kg) and closely followed the sCR1 concentration curves in A. 2/2 All data (A and B) represent the mean of triplicate assays. (C) C3 glomerular deposition decreased after 48 hours in sCR1-injected Cfh mice (50 mg/kg by tail-vein injection) but not in PBS-injected or non-injected controls (top panel). Trace glomerular staining for human CR1 (CD35) was seen in sCR1-injected mice 48 hours after treatment (bottom).

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Figure 3. Multi-dose sCR1 treatment clears iC3b glomerular deposition in mouse. (A) Daily dosing (arrows) maintained sCR1 serum 2/2 levels (blue, 25mg/kg; red, 50mg/kg, black, PBS). (B) Serum C3 levels in Cfh /huCR1-Tg mice increased and remained high with daily IP dosing (0, 24, and 48 hours) of 25 (blue) or 50 (red) mg/kg sCR1 (black, PBS; normal range: 300;1500 mg/L). Data represent the mean of triplicate assays. (C) Intact C3a (992 amino acids) was detected by Western blotting in reducing conditions using a murine C3a- specific antibody. The 106-kDa band (equivalent to the C3a chain in wild-type mice) appeared 12 hours after sCR1 treatment but was absent before treatment and in untreated controls. (D) IF of glomeruli harvested 60 hours after injection with relative fluorescence

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1:200, and 1:400, respectively; negative control, 0.05 at 1:50). in vitro and murine data and support a longer trial using sCR1 Pharmacokinetic data from measured sCR1 serum concentra- in a carefully selected patient population to evaluate the po- tions in this patient were in good agreement with dose-based tential of sCR1 as a specific therapy for C3G. Therapeutic simulations using pharmacokinetic data from adult patients un- entities that incorporate select domains of sCR1 also warrant dergoing high-risk cardiac surgery (Supplemental Figure 4).23 investigation.

DISCUSSION CONCISE METHODS

The in vitro activity of sCR1 in complement inhibition is de- Patient pendent on the specific conditions of the assay and varies with An 8-year-old patient with rapidly progressive DDD was enrolled the concentration and hemolytic potential of the serum com- under an FDA-approved compassionate-use investigational new drug plement source and the efficiency of the complement-activat- studydesignedtoadministersevendosesofsCR1 under IRB-approved ing mechanisms. In the assays we used, sCR1 prevented C3 guidelines. DDD was diagnosed by renal biopsy. convertase activity in normal (rabbit erythrocyte hemolytic assay) and pathologic conditions (sheep erythrocyte hemo- Patient and Normal Sera lytic assay with DDD sera). In vivo experiments in two mouse Ten patients with biopsy-proven DDD consented to provide sera models of C3G confirmed the in vitro data—sCR1 stopped AP under IRB-approved guidelines. All patients were positive for C3Nefs dysregulation and restored plasma C3 levels to normal. These except for patients DDD-07 and DDD-08. Patient DDD-07 carries a changes were accompanied by reduced deposition of new iC3b genetic rearrangement between CFHR2 and CFHR5,andpatient and clearance of old iC3b in the GBM. DDD-06 carries a genetic variant in CFHR5 (c.1541 T.G; p.Met514- Our results are consistent with the known role that CR1 Arg). No other genetic mutations were identified in CFH, CFI, CFB, plays as a central complement regulator of both the C3 and C5 MCP, or C3. Pooled normal human sera were obtained from Inno- convertases. In addition to regulating these convertases, CR1 is vative Research (Novo, MI); pooled murine sera were collected from the only cofactor of fI that can cleave iC3b into smaller five 2-month-old C57BL/6 animals by cardiac puncture. fragments (C3c and the thioester-containing fragment C3dg), thus explaining the rapid clearance of iC3b from the GBM. The sCR1 slower clearance of C3d is consistent with its surface-binding sCR1 (also called TP10 or CDX-1135) was produced by recombinant properties (Figure 3D). Thus, by bringing complement dysreg- DNA technology using Chinese hamster ovary cells and purified using ulation under control, the deposition of new iC3b is arrested standard filtration and chromatography methods as a single-chain (Figure 3). polypeptide of 1931 amino acids with a protein molecular mass of These observations provide strong evidence that iC3b is 212 kD (Celldex Therapeutics, Needham, MA). Due to N-linked glyco- deposited in the GBM and is an important component of the sylation, the final total molecular mass was 247 kD.28 glomerular dense deposits, a conclusion consistent with work done by Pickering and colleagues demonstrating that 2 2 Complement Activity Assays the presence of fI is an absolute requirement in Cfh / mice for AP and CP activity were evaluated using the appropriate Wieslab the development of a C3GN renal phenotype. In mice defi- complement assay (Wieslab AB, Lund, Sweden) following the 2 2 2 2 cient in both fH and fI (Cfh / .Cfi / mice), although excess manufacturer’s protocols (serum dilution 1:34 for AP and 1:100 for C3b is present in the circulation, no iC3b forms and GBM CP). To measure complement inhibitory effects, increasing amounts 2 2 dense deposits are not seen.27 The Cfh / /huCR1-Tg mouse of sCR1 were added to diluted sera before conducting these assays. also provides an excellent animal model in which to study long-term gene therapy to assess the effect of constitutive he- Hemolytic Assays patic expression of sCR1 on the renal phenotype. AP hemolytic activity was measured using rabbit or sheep erythro- The patient with DDD who we treated received only seven cytes. Rabbit erythrocytes activate AP-mediated lysis in human and doses of sCR1; however, we demonstrated that multidose mouse sera; sheep erythrocytes do not. For the rabbit erythrocyte treatment was safe and nonimmunogenic in this limited study. hemolytic assay, increasing concentrations of sCR1 (0.2 nM to We did not expect to, nor did we, see a histologic improvement 200 nM) or fH (0.2 nM to 2000 M) were mixed with normal human in the renal biopsy results. sCR1 administration was associated or mouse sera (20% v/v) and incubated for 30 minutes at 37°C with with a transient improvement in serum C3 and soluble C5b-9 13108 rabbit erythrocytes in the presence of 10 mM EGTA/0.15 mM normalized. These biomarker changes were expected based on Mg2+/gelation veronal buffer (GVB) or EDTA-GVB as a

quantitation (averaged control value set to 100% with the exception of CD35 where averaged C3 control was set to 100%; scale bar: average of 10 glomeruli 6 SD). From top to bottom: C3 was dramatically decreased; CD35 (CR1) was absent; C3d decreased significantly with the higher dose of sCR1; C3c was greatly reduced. *P,0.05, **P,0.001; controls versus experiments.

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Figure 4. sCR1 restores soluble MAC levels in a DDD patient. (A) Biopsy at presentation showed a proliferative-appearing glomerulus with cellular crescents, endocapillary and mesangial hypercellularity and capillary loop “double contours” (light microscopy [LM] 3400). No global glomerulosclerosis was noted, and interstitial fibrosis and tubular atrophy were minimal. Glomerular “ribbon-like” electron-dense deposits were present on electron microscopy (EM, 38200) with discrete, coarsely granular C3 immunofluorescence of the mesangium and

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Table 1. Patient biopsies at presentation and before and plates were coated with mAb 6B1.H12; the detection antibody was after treatment with sCR1 horseradish peroxidase–conjugated mAb 4D6.1.31 Murine total C3 Before After serum levels were measured using a two-site ELISA (Kamiya Biomed- Histology Presentation Treatment Treatment ical, Seattle, WA); human total C3 was measured by radial immuno- Crescents (n) 21 of 25 6 of 18 3 of 13 diffusion (The Binding Site, San Diego, CA). Data were collected in Glomerular 0 of 25 9 of 18 3 of 13 three independent assays and expressed as the mean for each group. sclerosis (n) Interstitial fibrosis Minimal Moderate Moderate Immunohistochemical Analyses C3 immunofluorescence 2–3+ 2–3+ 2–3+ Kidneys were harvested at the time of euthanasia and imbedded in Tissue-Tek OCT medium for routine processing (Sakura Finetek, nonhemolytic control. The reaction was stopped by adding 150 mlof Torrance, CA). For glomerular C3 deposition, sections were stained 20 mM EDTA-GVB. After centrifugation, the supernatant was trans- with FITC-conjugated mouse C3 antibody (MP Biomedicals, Solon, ferred to a clean 96-well plate and absorbance was recorded at 415 OH) at a dilution of 1:800 for 1 hour. For glomerular sCR1, C3c, nm. Percent lysis was calculated as a fraction of the maximum A415 and C3d staining, sections were treated with goat anti-human CR1 absorbance in the absence of sCR1 [(A415sCR1-EGTA- A415EDTA)/ antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit

(A415EGTA) 3 100]. anti-human C3c (1:8000; Abcam, Cambridge, MA), or goat anti- To test whether sCR1 prevents C3Nef stabilization of C3 con- mouse C3d (1:400; R&D Systems, Minneapolis, MN) for 1 hour, re- vertase, 10 ml of patient serum was added to 10 ml of sheep erythro- spectively, followed by staining with the corresponding Alexa Fluor cytes (13109/ml) coated with preformed C3 convertase in a total 50 488 (1:500; Molecular Probes, Eugene, OR) for 1 hour at room tem- ml (EGTA-Mg-GVB) reaction as described.29 The mixture was al- perature. Fluorescence images were acquired on a Leica model TCS lowed to decay at 30°C (water bath) for 20 minutes. Hemolysis was SP5 laser system (Leica Microsystems, Buffalo Grove, IL). Relative assayed by adding 50 ml of rat serum (1:9 diluted in GVB-EDTA fluorescence intensities (average of 10 glomeruli; 6 SD) were quan- buffer) as a source of C5-9. Sheep red blood cells were lysed in the tified by ImageJ software (http://rsbweb.nih.gov/ij/; National Insti- presence of nondecayed C3 convertase stabilized by C3Nefs. The re- tutes of Health, Bethesda, MD). action was stopped and the percentage of hemolysis was measured as described above. In parallel tests, varying concentrations of sCR1 Immunoblot Analyses (0 nM, 40 nM, 120 nM, 160 nM) were added to patient serum before Mouse sera were diluted 1:40 in Laemmli buffer, separated by 7.5% mixing with sheep erythrocytes and incubating on ice for 15 minutes. SDS-PAGE, and transferred to nitrocellulose membranes (Whatman Repetition of this experiment was done with sera from 10 patients Schleicher and Schuell Inc., Dassel, Germany). Membranes were with DDD. blocked with5% skim milk in PBS at room temperature for 30 minutes and incubated with anti-mouse C3 antibody (1:500; in-house Mice antibody targeting the fI cleavage site on C3). Each membrane was 2 2 The Cfh / mutant mouse, obtained from Dr. Matthew Pickering, washed three times with 0.05% Tris-buffered saline Tween-20 was generated as described.10 On this background, human CR1 was followed by incubation with horseradish peroxidase–conjugated 2 2 introduced to create the Cfh / /huCR1-Tg mutant.30 The latter anti-mouse secondary antibody (Jackson ImmunoResearch, Inc., mouse line expresses CR1 only on mouse erythrocytes. The line West Grove, PA). Specific protein bands were visualized with an En- was used to avoid any murine immunoresponse. All procedures hanced Chemiluminescent System (GE Healthcare, Piscataway, NJ). were approved by the Institutional Animal Care and Use Committee at the University of Iowa. sCR1 Dose Simulations Pharmacokinetic data from adult cardiac surgery patients treated with sCR1 and C3 Concentrations sCR1 were used to estimate sCR1 serum concentrations over time sCR1 concentrations were measured by sandwich ELISA in a micro- using empirical calculations that assumed concentrations from titer plate format using two mouse mAbs specific for CR1. Microtiter multiple doses to be additive because sCR1 pharmacokinetics has

capillary loops (IF, 3400). (B) The pre-sCR1 biopsy, 6 weeks after the initial biopsy, showed persistent glomerular hypercellularity and capillary loop “double contours” (first LM, 3400), and a glomerular fibrocellular crescent (second LM, 3400) consistent with disease progression to chronicity; moderate interstitial fibrosis and tubular atrophy were present. IF showed “chunky” C3 staining in the glo- merular mesangium and capillary loops (3400). (C) Post-sCR1 biopsy, 6 days after the final dose, showed glomerular fibrocellular and fibrous crescents with periglomerular fibrosis (LM, 3200). The ribbon-like capillary loop and mesangial electron dense deposits remained unchanged (EM, 38200). IF (3400) continued to show bright C3 staining because the biopsy was done 6 days after sCR1 was stopped. (D) Immediately before receiving sCR1 serum C3 was low and sMAC was high. Although the C3 level initially doubled during treatment it trended back to the pre-treatment level. Soluble MAC normalized after the sixth dose (,30 mg/cL) but within 96 hours after termination of therapy was abnormal (asterisks, levels measured at 48 and 96 hours after the last dose of sCR1; red dotted line, normal level of sMAC; green dotted line low normal of plasma C3; sMAC in mg/cl, C3 in mg/dl, sCR1 in mg/ml to allow representation on same graph; sCR1, 1 mg/ml = 4nM).

8 Journal of the American Society of Nephrology J Am Soc Nephrol 24: ccc–ccc,2013 www.jasn.org BASIC RESEARCH been shown to be dose proportional (dose versus area under the curve findings, complement abnormalities, glomerular proteomic profile, are linear).23 Averaged data from 26 cardiac surgery patients treatment, and follow-up. Kidney Int 82: 465–473, 2012 receiving a single 10 mg/kg bolus of sCR1 were used to simulate 5. Sethi S, Gamez JD, Vrana JA, Theis JD, Bergen HR 3rd, Zipfel PF, Dogan A, Smith RJ: Glomeruli of dense deposit disease contain components of the dosing regimen used in the single DDD patient. The dose regimen the alternative and terminal complement pathway. Kidney Int 75: 952– m was designed to keep trough levels of sCR1 above 20 g/ml through- 960, 2009 out the dosing period. 6. Lu DF, McCarthy AM, Lanning LD, Delaney C, Porter C: A descriptive study of individuals with membranoproliferative glomerulonephritis. Nephrol Nurs J 34: 295–302,quiz303,2007 Immunogenicity Assays 7. Lu DF, Moon M, Lanning LD, McCarthy AM, Smith RJ: Clinical features Patient plasma samples were screened for anti-sCR1 antibodies using and outcomes of 98 children and adults with dense deposit disease. a microtiter plate ELISA format. Microtiter plates were coated with Pediatr Nephrol 27: 773–781, 2012 sCR1 and blocked with BSA in PBS. Patient samples were diluted 1:50 8. Braun MC, Stablein DM, Hamiwka LA, Bell L, Bartosh SM, Strife CF: in PBS 10 mM EDTA 0.5% Tween and incubated in coated plates. Recurrence of membranoproliferative glomerulonephritis type II in re- Antibodies were detected using horseradish peroxidase-conjugated nal allografts: The North American Pediatric Renal Transplant Co- operative Study experience. JAmSocNephrol16: 2225–2233, 2005 F(ab)2 goat anti-human IgG antibody (KPL Inc., Gaithersburg, MD). 9. Nester CM, Smith RJH: Treatment options for C3 glomerulopathy. Curr Normal human serum was used as a negative control. Knops-McCoy Opin Nephrol Hypertens 22: 231–237, 2013 blood group antiserum, which is specific for CR1, served as a positive 10. Pickering MC, Cook HT, Warren J, Bygrave AE, Moss J, Walport MJ, control.32 Positive patient samples were confirmed specific for sCR1 Botto M: Uncontrolled C3 activation causes membranoproliferative by preincubating with excess sCR1 and rerunning the assay. glomerulonephritis in mice deficientincomplementfactorH.Nat Genet 31: 424–428, 2002 11. Fakhouri F, de Jorge EG, Brune F, Azam P, Cook HT, Pickering MC: Statistical Analyses Treatment with human complement factor H rapidly reverses renal IC50 values were calculated by GraphPad Prism 5 software (GraphPad complement deposition in factor H-deficient mice. Kidney Int 78: 279– Software, Inc., La Jolla, CA). Statistical significance was calculated 286, 2010 with the t test. 12. Paixão-Cavalcante D, Hanson S, Botto M, Cook HT, Pickering MC: Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase. Mol Immunol 46: 1942–1950, 2009 13. Martínez-Barricarte R, Heurich M, Valdes-Cañedo F, Vazquez-Martul E, ACKNOWLEDGMENTS Torreira E, Montes T, Tortajada A, Pinto S, Lopez-Trascasa M, Morgan BP, Llorca O, Harris CL, Rodríguez de Córdoba S: Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and pro- We are grateful to those patients with DDD whose participation vides insights into complement activation and regulation. JClinInvest 2/2 2/2 made this research possible. The Cfh and Cfh /huCR1-Tg mu- 120: 3702–3712, 2010 tant mice were kindly provided by Drs. Matthew Pickering and Rick 14. Zhang Y, Meyer NC, Wang K, Nishimura C, Frees K, Jones M, Katz LM, Quigg. 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10 Journal of the American Society of Nephrology J Am Soc Nephrol 24: ccc–ccc,2013 Zhang et al –JASN-supplemental data 1 Soluble CR1 in C3 Glomerulopathy

Soluble CR1 Therapy Improves Complement Regulation in C3 Glomerulopathy

Yuzhou Zhang,* Carla M. Nester,*†‡ Danniele G. Holanda, § Henry C. Marsh,‖ Russell A. Hammond,‖ Lawrence J. Thomas,‖ Nicole C. Meyer,* Lawrence G. Hunsicker, † Sanjeev Sethi,¶ Richard J. H. Smith*†‡

Supplementary Materials

Figure S1

Figure S1. Complement Receptor 1 (CR1) is a monomeric single-pass type I membrane glycoprotein. Its four long homologous repeats (LHRs) are each comprised of seven short consensus repeats (SCRs), represented by ovals, which are followed by two additional SCRs, a transmembrane sequence and a cytosolic domain. The SCRs are also called complement control protein-repeat modules (CCPs) or sushi domains, and have sequence homology that ranges from

60-99%. The clusters of complement-inhibitory SCRs are referred to as sites. Site 1 (SCR 1-3) binds to C4b while Site 2 (SCR 8-10 and SCR 15-17) binds to C3b and C4b. Large insertions and deletions of the LHRs give rise to four variants of CR1, the most common of which is CR1-A.

Soluble CR1 (sCR1) is similar to CR1-A, but lacks the transmembrane and cytoplasmic domains.

CR1 controls C3 convertase formation and is the only cofactor that enables cleavage of iC3b into

C3c and C3dg. Zhang et al –JASN-supplemental data 2 Soluble CR1 in C3 Glomerulopathy

Figure S2

Figure S2. C3G is caused by complement dysregulation. The amplification loop of the AP is dependent on the generation of C3b from C3, shown by the red arrows. Dysregulation of the C3 convertase in the fluid phase leads to both DDD and C3GN, the two major subtypes of C3G.

This dysregulation can also lead to C5 convertase dysregulation with consequent activation of the terminal complement cascade, shown by the green arrows. The most potent naturally occurring fluid-phase regulator of the AP C3 convertase is factor H. Other regulators include CR1, MCP and factor I. Targeted therapy for C3G might: 1) restore C3 convertase control; 2) replace deficiencies of regulatory proteins; 3) remove auto-antibodies that stabilize C3 convertase such as

C3Nefs; 4) remove mutant proteins; or 5) remove C3b breakdown products like iC3b. CR1 has many properties that warrant its consideration as a treatment for C3G. It controls C3 and C5 convertase activity by blocking the conversion of C3 to C3b, it mediates the conversion of C3b to iC3b (as do factor H and MCP), and it serves as a cofactor for the cleavage of iC3b to C3c and

C3dg.

Zhang et al –JASN-supplemental data 3 Soluble CR1 in C3 Glomerulopathy

Figure S3

Figure S3. Human sCR1 prevents activation of murine complement cascade. Under normal circumstances, rabbit erythrocytes are an activating surface for the murine AP complement cascade. However, as the concentration of sCR1 is increased, C3 convertase-mediated generation of C3b is prevented and the AP is not activated. As a result, hemolysis does not occur (IC50 =

6.15 ± 0.18 nM). Data represent mean ± SD of triplicate assays.

Zhang et al –JASN-supplemental data 4 Soluble CR1 in C3 Glomerulopathy

Figure S4

Figure S4. Simulated sCR1 concentrations are based on pharmacokinetic data from 26 cardiac surgery patients (blue line). These data compare favorably to measured serum sCR1 concentrations from the single DDD patient (red squares). Error bars included in the first 48 hours of the simulation represent standard deviations in mean concentrations at the indicated time points.