A Randomized Clinical Trial of Anti–IL-6 Antibody Clazakizumab in Late Antibody-Mediated Kidney Transplant Rejection

CLINICAL RESEARCH www.jasn.org

Konstantin Doberer ,1 Michael Duerr,2 Philip F. Halloran,3 Farsad Eskandary,1 Klemens Budde,2 Heinz Regele,4 Jeff Reeve,3 Anita Borski,1 Nicolas Kozakowski ,4 Roman Reindl-Schwaighofer ,1 Johannes Waiser ,2 Nils Lachmann,5 Sabine Schranz,6 Christa Firbas,6 Jakob Mühlbacher,7 Georg Gelbenegger,6 Thomas Perkmann ,8 Markus Wahrmann ,1 Alexander Kainz,1 Robin Ristl,9 Fabian Halleck,2 Gregor Bond,1 Edward Chong,10 Bernd Jilma,6 and Georg A. Böhmig1

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Background Late antibody-mediated rejection (ABMR) is a leading cause of transplant failure. Blocking IL-6 has been proposed as a promising therapeutic strategy. Methods We performed a phase 2 randomized pilot trial to evaluate the safety (primary endpoint) and efficacy (secondary endpoint analysis) of the anti–IL-6 antibody clazakizumab in late ABMR. The trial included 20 kidney transplant recipients with donor-specific, antibody-positive ABMR $365 days post-transplantation. Patients were randomized 1:1 to receive 25 mg clazakizumab or placebo (4-weekly subcutaneous injections) for 12 weeks (part A), followed by a 40-week open-label extension (part B), during which time all participants received clazakizumab. Results Five (25%) patients under active treatment developed serious infectious events, and two (10%) developed diverticular disease complications, leading to trial withdrawal. Those receiving clazakizumab dis- played significantly decreased donor-specific antibodies and, on prolonged treatment, modulated rejection-related gene-expression patterns. In 18 patients, allograft biopsies after 51 weeks revealed a negative molecular ABMR score in seven (38.9%), disappearance of capillary C4d deposits in five (27.8%), and resolution of morphologic ABMR activity in four (22.2%). Although proteinuria remained stable, the mean eGFR decline during part A was slower with clazakizumab compared with placebo (20.96; 95% confidence interval [95% CI], 21.96 to 0.03 versus 22.43; 95% CI, 23.40 to 21.46 ml/min per 1.73 m2 per month, respectively, P50.04). During part B, the slope of eGFR decline for patients who were switched from placebo to clazakizumab improved and no longer differed significantly from patients initially allocated to clazakizumab. Conclusions Although safety data indicate the need for careful patient selection and monitoring, our preliminary efficacy results suggest a potentially beneficial effect of clazakizumab on ABMR activity and progression.

JASN 32: 708–722, 2021. doi: https://doi.org/10.1681/ASN.2020071106

Received July 31, 2020. Accepted November 10, 2020. Late antibody-mediated rejection (ABMR) is a common cause of kidney allograft failure.1 Its mo- Published online ahead of print. Publication date available at lecular mechanisms are increasingly understood, www.jasn.org. and diagnostic criteria have been refined continu- Correspondence: Dr. Georg A. Böhmig, Division of Nephrology ously.2,3 Treating late ABMR, however, remains a and Dialysis, Department of Medicine III, Medical University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria, or Dr. Bernd Jilma, fi 4,5 signi cant challenge. Systematic trials have failed Department of Clinical Pharmacology, Medical University of Vienna, to demonstrate any benefit of therapies widely Währinger Gürtel 18–20, A-1090 Vienna, Austria. E-mail: georg. used for desensitization and acute ABMR, such as [email protected] or [email protected] rituximab plus intravenous Ig6 or bortezomib.7 Copyright © 2021 by the American Society of Nephrology

708 ISSN : 1046-6673/3203-708 JASN 32: 708–722, 2021 www.jasn.org CLINICAL RESEARCH

Consequently, there is a high unmet need for an effective Significance Statement treatment.4,5 One promising therapeutic target is IL-6, a cytokine that is There is no proven effective treatment for a major cause of graft critically involved in the regulation of inflammation and im- failure, late antibody-mediated rejection, but IL-6, a cytokine known mune cell differentiation.8 Over the last decade, the concept of to promote B cell immunity, may be a promising therapeutic target. The authors describe the results of a phase 2 randomized clinical targeting IL-6 or its receptor (IL-6R) has entered clinical rou- trial involving 20 patients, designed to evaluate the safety (primary – tine, and is well established in rheumatoid arthritis.9 11 More endpoint) and efficacy (secondary endpoint analysis) of an anti–IL-6 recently, IL-6/IL-6R interference has also become of interest in antibody, clazakizumab, versus placebo in late antibody-mediated organ transplantation.8 An observational study evaluating rejection. Although the occurrence of serious infections and di- anti–IL-6R antibody tocilizumab in chronic ABMR has sug- verticulitis presented important safety signals, clazakizumab was associated with an early decrease in donor-specific antibody levels, gested stabilization of allograft function, presumably resulting modulated antibody-mediated rejection activity, and slowed the from reduced levels of donor-specific antibody (DSA).12,13 decline of renal function. Preliminary efficacy results suggest a po- The occurrence of graft losses in four recipients in whom to- tentially beneficial effect of clazakizumab and may therefore sup- cilizumab was prematurely stopped,12 however, suggested a port the design of larger trials with a longer duration of follow-up. rebound effect, triggered by IL-6 accumulated on treatment.14 Thus, the use of antibodies that directly neutralize IL-6 may be inclusion, and the study was conducted in accordance with of particular interest. International Council for Harmonisation of Technical Require- Clazakizumab is a humanized monoclonal IgG1 antibody ments for Pharmaceuticals for Human Use Good Clinical Prac- fi with high af nity for IL-6 and a long t1/2 of approximately 30 tice requirements, Good Laboratory Practice, the principles of days. This antibody has been systematically evaluated in rheu- the Declaration of Helsinki 2008, and the Declaration of Istan- 10,15 matoid and psoriatic arthritis, but has not yet been ap- bul. The trial was registered on the European Union Drug Reg- proved for clinical use. We designed this phase 2 pilot trial, the ulating Authorities Clinical Trials Database (2017–001604–30) first randomized controlled trial evaluating IL-6 signaling and ClinicalTrials.gov (NCT03444103). blockade in transplantation, to assess the safety and tolerabil- ity (primary endpoint) as well as efficacy (secondary endpoint Participants analysis) of clazakizumab in late ABMR. The trial was designed to include 20 patients (Figure 1, Supplemental Figure 1). Eligible participants were adult kid- . METHODS ney transplant recipients ( 18 years) with late active or chronic active ABMR $365 days after transplantation (with or without C4d deposits along the peritubular capillaries), Trial Oversight and Design associated with a molecular pattern of ABMR in gene array This investigator-driven, randomized, double-blind, placebo- analysis, preformed or de novo HLA class I and/or II DSA, and controlled, parallel-group phase 2 pilot trial was conducted at an eGFR (CKD Epidemiology Collaboration equation) two sites (Medical University of Vienna, Austria; Charité Uni- .30 ml/min per 1.73 m2.Exclusioncriteriawereage#18 versitätsmedizin Berlin, Germany) from January 2018 to April years, participation in another clinical trial, pregnancy or 2020 (recruitment period: January 2018 to April 2019). Details – fi of the protocol have been described previously.16 We hypoth- breastfeeding, T cell mediated rejection classi ed Banff grade $ esized that clazakizumab is a safe treatment to counteract I, de novo or recurrent severe thrombotic microangiopathy, ABMR progression. The study consisted of two parts polyoma virus nephropathy, de novo or recurrent GN, acute , (Supplemental Figure 1); a 12-week randomized placebo- rejection treatment 3 months before screening, acute dete- – controlled phase to decipher the short-term effects of treat- rioration of graft function (eGFR decline within 1 3 months . . ment (part A), followed by a 40-week open-label extension 25%), nephrotic range proteinuria 3500 mg/g protein/ where all participants received clazakizumab (part B). The creatinine ratio, active viral, bacterial, or fungal infection pre- fi rationale behind this design was to offer all participants the cluding intensi ed immunosuppression, active malignant fi option of a potentially effective treatment. Furthermore, two disease precluding intensi ed immunosuppressive therapy, follow-up biopsies to analyze short- and intermediate-term abnormal liver-function tests (alanine aminotransferase, treatment effects were considered unacceptable for patients aspartate aminotransferase, bilirubin .1.53 upper limit of on long-term placebo treatment. The study was approved by normal), other significant liver disease, latent or active tuber- the institutional review board of the Medical University of culosis (positive QuantiFERON-TB-Gold test, chest x-ray), Vienna (EK1428/2017), the Berlin State Ethics Committee administration of a live vaccine within 6 weeks of screening, (17/0485– EK 15), and the regulatory authorities in Austria neutropenia (,1 G/L) or thrombocytopenia (,100 G/L), his- (Federal Office for Safety in Health Care, Austrian Agency for tory of gastrointestinal perforation, diverticulitis, or inflam- Health and Food Safety) and Germany (Federal Institute for matory bowel disease, history of alcohol or illicit substance Vaccines and Biomedicines, Paul-Ehrlich Institute). All pa- abuse, or a serious medical or psychiatric illness likely to in- tients provided written informed consent before study terfere with participation in the study.

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22 Donor-specific antibody-positive kidney transplant recipients were assessed for eligibility 2 Were not eligible 1 Had immune complex glomerulonephritis 1 Had a negative molecular ABMR pattern 20 Were eligible for inclusion and underwent randomization (Medical University of Vienna: n=16; Charité Berlin: n=4)

10 Were assigned to receive 10 Were assigned to receive clazakizumab, subcutaneously placebo, subcutaneously (25 mg, day 0, week 4, week 8) (day 0, week 4, week 8)

2 Received only 2 doses in

part A (adverse events) Part A

20 Underwent a follow-up biopsy at week 11 1 Was withdrawn (serious adverse event) 19 Entered part B of the study (week 12 to week 52) to receive clazakizumab in 4-weekly intervals 1 Was withdrawn (serious adverse event) 9 Received less than10 doses in part B 8 Had adverse events 1 Had personal reasons Part B

18 Completed the trial and had a follow-up biopsy at week 51

Safety was evaluated in 19 patients Efficacy endpoints were evaluated in 18 patients

Figure 1. Trial ﬂowchart.

Randomization subjects did not receive all 13 scheduled clazakizumab injec- Patients were randomized 1:1 to one of the two study arms tions (12 doses: n53; 11 doses: n52; ten doses: n52; nine (clazakizumab versus placebo) in part A using a web-based doses: n51; eight doses: n51; four doses: n51; two doses: randomization platform (www.meduniwien.ac.at/randomizer). n51). In accordance with a July 2019 trial amendment after Permuted block randomization with block sizes of two was two patients had serious gastrointestinal complications (di- stratiﬁed by study site (Vienna versus Berlin) and ABMR cat- verticulitis; one patient requiring open surgery because of co- egory (active ABMR versus chronic/active ABMR). Study par- lon perforation), the dose of clazakizumab was reduced to ticipants, care providers, and those assessing outcomes were 12.5 mg per injection in two other active patients with docu- unaware of the randomization sequence. The allocation se- mented diverticulosis (last injection before completion of the quence was generated, and medication or placebo was prepared study). by independent nonblinded study pharmacists. Study physicians and nurses were provided with blinded subcutaneous Immunosuppression medication. Subjects were enrolled and assigned to interven- Tacrolimus, cyclosporin A, and everolimus doses were ad- tions by study physicians. The participating investigators, staff justed to trough levels of 5–10 ng/ml, 80–120 ng/ml, and with medical interaction, and the study participants were blin- 3–8 ng/ml, respectively. In total, 18 recipients were on calci- ded to group allocation until the last patient had completed neurin- or mTOR inhibitor–based triple immunosuppressive part A. Treatment code envelopes were securely stored to be therapy, and two on dual therapy without steroids. The single opened in case of emergency. patient on everolimus achieved levels within the target range throughout the study period (not shown). In one patient off Study Medication steroids, prednisolone was initiated on trial inclusion. Dosages Clazakizumab (25 mg in 1 ml single-dose vials; Vitaeris Inc., of steroids were not adjusted during the trial (prednisolone, 16 Vancouver, Canada) and placebo (0.9% saline) were admin- patients: 5 mg/d, n515; 2.5 mg/day, n51; methylprednisolone, istered via subcutaneous injection in 4-weekly intervals. Due four patients: 4 mg/d). The following medications were prohibi- to adverse events (n510) or personal reasons (n51), 11 (55%) ted and not applied during the study: rituximab, eculizumab,

710 JASN JASN 32: 708–722, 2021 www.jasn.org CLINICAL RESEARCH proteasome inhibitors, intravenous Ig, plasma exchange, immu- Serum samples were incubated with EDTA (10 mM) to pre- noadsorption, and other investigational drugs or treatments in- vent complement interference. Data acquisition was cluding commercially available anti–IL-6/IL-6R mAbs. performed via a LABScan 200 flow analyzer (Luminex Corpo- ration, Austin, TX). For longitudinal analysis of DSA levels, Endpoints bead assays were performed retrospectively (centralized anal- As effect sizes were unknown, the design of this pilot study— ysis) to avoid influences of day-by-day variations in test results the first randomized trial evaluating clazakizumab in patients (test batches including samples from four to six patients each). who had undergone a transplant—did not include sample size Donor specificity was defined according to serological and/or estimations. Trial visits were conducted at day 0 and weeks 1, low- or high-resolution donor/recipient HLA typing (HLA-A, 2, 3, 4, 8, 11, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 51, and 52. -B, -Cw, -DR, -DQ, -DP on availability) provided either by the The primary endpoint was the assessment of safety and toler- local HLA lab or the Eurotransplant database. Test results ability. Key secondary endpoints were the evolution of mor- were documented as mean fluorescence intensity (MFI) of phologic and molecular rejection patterns (index biopsy, week the immunodominant DSA. An MFI threshold .1000 was 11, and week 51 follow-up biopsies); DSA and non-DSA levels, considered as positive. To estimate the effect of clazakizumab IgG, IgM, and IgG subclass concentrations and Torque Teno treatment on DSA levels, we documented the percent change virus (TTV) load (day 0, week 12, and week 52); the pharma- in MFI. In an effort to quantify changes in DSA levels more codynamics of clazakizumab (reduction of C-reactive protein, accurately, we additionally performed dilution experiments CRP); and the course of eGFR (CKD Epidemiology Collabo- after an earlier described protocol.17 In brief, nonlinear stan- ration), and protein/creatinine ratio (all visits). dard curves on the basis of raw DSA MFI levels (immunodominant DSA) were obtained by serial dilution of individual Safety patient sera collected before the start of treatment (all samples Adverse events were closely monitored throughout the study were incubated with EDTA). According to computed standard and classified using the Medical Dictionary for Regulatory curves, the fold change of antibody levels was then calculated Activities Version 23.0. The study was monitored by an inde- from DSA MFI levels detected in the same experiment for pendent data and safety monitoring board (DSMB), and in- undiluted week 12 and week 52 samples. cluded two interim analyses, the first after 10 and the second Total IgG, IgM, and IgG subclasses were assessed in serum after 20 subjects had completed part A of the study. The DSMB applying immunonephelometry on a BN II analyzer (Siemens was instructed to consider stopping the trial if the pattern of Healthineers, Erlangen, Germany). related serious adverse events or safety laboratory results strongly supported a major safety signal. Exact statistical def- Transplant Biopsies initions of criteria for premature study termination were not The study included 20 index, 20 11-week follow-up, and defined. Stopping rules were defined as follows. Part A: if six 18 51-week follow-up biopsies. None of the patients under- subjects experience related (definitely and possibly) serious went additional indication biopsies. Biopsies were performed adverse events (common toxicity criteria . Grade 3 or se- using ultrasound-guided percutaneous techniques (1–2 cores vere/medically significant), and/or substantially elevated levels per biopsy, 16-gauge needle). Histomorphology and C4d of liver parameters (alanine aminotransferase, aspartate ami- staining was evaluated on formalin-fixed paraffin-embedded notransferase, and/or bilirubin .33 upper limit of normal) sections. C4d in peritubular capillaries was scored as 0 (nega- or neutropenia (below 0.5 G/L), the DSMB would unblind the tive), 1 (minimal), 2 (focal), and 3 (diffuse), and a score $1 safety results. If five or all subjects were to be in the clazaki- was considered positive. In total, 35 of the 58 study biopsies zumab group, then the study would be stopped. Similarly, if six were additionally evaluated using electron microscopy to de- related serious adverse events and/or substantial abnormali- tect microcirculation injury. Morphologic results were read ties in liver enzymes or neutrophil counts occurred in the same locally (Medical University of Vienna, Charité Universitäts- system organ class, the DSMB would unblind these serious medizin Berlin) in a blinded fashion, following the rules of adverse events, and if five or all serious adverse events were the Banff 2017 scheme. In addition, all biopsies were analyzed in the clazakizumab group, then the study would be stopped. using a thoroughly validated molecular method (MMDx).18,19 Part B: if ten or more subjects experienced drug-related seri- For each biopsy, a 3 mm portion of one core was immediately ous adverse events or ten or more related serious adverse placed in RNAlater, stored at 220°C, and shipped either at events occurred in the same system organ class, then the trial ambient temperature or on dry ice to the Alberta Transplant would be stopped (the total number of related serious adverse Applied Genomics Centre (University of Alberta, Edmonton, events should also include the related events occurring in the AB, Canada) for gene array analysis. Molecular scores on the clazakizumab-treated subjects during part A). basis of lesion-based classifiers related to rejection (ABMR, T cell–mediated rejection, all rejection), inflammation (global Antibody Detection disturbance score), or chronic injury (atrophy/fibrosis score) For HLA antibody detection, LABscreen single-antigen flow- were generated using a reference set of 1529 biopsies. After the bead assays (One Lambda, Canoga Park, CA) were applied. 2017 update of the Banff classification,20 ABMR was defined

JASN 32: 708–722, 2021 Clazakizumab in Late Rejection 711 CLINICAL RESEARCH www.jasn.org and categorized on the basis of morphologic, immunohisto- recipients had anti-HLA class II DSA (with or without HLA chemical (C4d), ultrastructural (transplant glomerulopathy, class I DSA), 15 (75%) against HLA-DQ. The immunodomi- multilayering of the peritubular capillary basement mem- nant DSA MFI was 11,708 (median; interquartile range [IQR]: branes), serological (DSA detection), and thoroughly vali- 1947–17,709). Two (10%) recipients had active and 18 (90%) dated molecular criteria (molecular ABMR score $0.2), chronic/active ABMR. Median eGFR and protein/creatinine ra- respectively. tios were 39.3 (IQR 33.6–49.7) ml/min per 1.73 m2 and 962 (IQR, 310–1863) mg/g, respectively. Important baseline vari- TTV Quantification ables relating to the severity of rejection, such as eGFR or mor- For TTV analysis, DNA was extracted from plasma (200 ml) phologic results obtained in index biopsies were well balanced using the NucliSENS easyMAG platform (bioMeriéux, between patient groups. Despite stratified randomization, how- France), and eluted in 50 ml of elution buffer. TTV DNA ever, recipients allocated to placebo showed numerically higher was quantitated by TaqMan real time PCR, according to earlier median levels of DSA MFI, CRP levels, and proteinuria, whereas described protocols.21,22 The quantitative PCR reactions were molecular rejection-related scores were higher in the clazakizu- performed in a volume of 25 ml using 23 TaqMan Universal mab arm (Supplemental Tables 1 and 2, Table 1). PCR Master Mix, containing 5 ml of extracted DNA, 400 nM of At study inclusion, 18 (90%) recipients were on triple and each primer, and 80 nM of the probe. Thermal cycling was two (10%) were on dual immunosuppressive therapy started for 3 minutes at 50°C, followed by 10 minutes at 95°C, (Supplemental Table 2). Immunosuppressant trough levels and then by 45 cycles at 95°C for 15 seconds, at 55°C for 30 and doses are shown in Supplemental Figure 2. In total, 11 seconds, and at 72°C for 30 seconds, using the CFX96 Real- (55%) patients did not receive all scheduled clazakizumab time System (Bio-Rad, Hercules, CA). Results were recorded injections, mostly because of adverse events. These patients as copies per ml. were included in all endpoint analyses. Two (10%) patients were withdrawn from the study due to diverticular disease Statistical Methods complications, one after completion of part A, and one shortly For group comparisons we used Fisher’sexact,Mann– after initiation of part B (Figure 1). Whitney U, or Wilcoxon tests. GFR trajectories were analyzed using a linear mixed model with eGFR values from 0 to Safety 12weeks(day0,week1,2,3,4,8,12)andfrom12to52weeks In part A, overall incidences of adverse events were 50 in the (week 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52) as the dependent clazakizumab and 44 in the placebo arm (Supplemental Table 3, variable. Time, treatment, and their interaction were used as Table 2). The most frequent events were infections and gastro- fixed effects. Furthermore, patient-specific random effects for intestinal disorders (Supplemental Table 3). Serious adverse intercept and slope were specified. Each patient’s trajectories events occurred in four (20%) patients, three of whom received were modeled as a hockey-stick spline with a fixedknotat12 clazakizumab (Table 2). One patient on clazakizumab developed weeks, which is change of therapy for the placebo group. Due diverticulitis, leading to withdrawal from the trial (stable graft to the unevenly spaced time points, a spatial covariance matrix function until the end of the trial). Diverticulitis resolved after (power structure) was used. Intergroup differences were tested percutaneous abscess drainage and antibiotic therapy. at a two-sided significance level of 5%. For statistical analysis, Part B had 129 adverse events overall, of which nine were IBM SPSS Statistics version 24 (IBM Corporation, Armonk, serious. Three (15.8%) patients reported mild injection site NY) and SAS version 9.4. (The SAS Institute Inc., Cary, North reactions. Serious adverse events included a case of compli- Carolina) were applied. cated diverticulitis with colon perforation requiring surgery. This patient (clazakizumab in part A) was withdrawn from the study before the second visit in part B (return to dialysis RESULTS 9 months after trial withdrawal). Other serious events included pneumonia (n52), pyelonephritis (n51), ovarian ab- Patient Disposition and Characteristics scess (n51), Coxsackie virus–associated meningitis (n51), In total, 20 kidney transplant recipients (Vienna, n516; and recurrent pleural effusion requiring pleurodesis, and, sub- Berlin, n54) with DSA-positive ABMR after a median of sequently, permanent thorax cavity drainage. 10.6 years post-transplantation were randomly assigned to After the second case of diverticulitis, the DSMB requested clazakizumab or placebo (12-week randomized, placebo- a careful re-evaluation of all included participants and a re- controlled part A). In a subsequent 40-week open-label exten- duced dose of clazakizumab in two active patients diagnosed sion (part B), all participants received clazakizumab (Figure 1). with diverticulosis (12.5 mg, last injection in part B). Overall, Baseline characteristics are provided in Supplemental Tables 1 nine (45%) patients had colon diverticulosis, among them and 2, and Table 1. Ten (50%) patients were female, seven the two patients who developed diverticulitis. Additional (35%) were retransplant recipients, and five (25%) had been predisposing risk factors were polycystic kidney disease and subjected to desensitization because of preformed DSA and/or a long history of immunosuppression and steroid exposure broad HLA reactivity.23 At study inclusion, 18 (90%) (Supplemental Table 4).

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Table 1. Demographics and baseline characteristics Parameter Total (n520) Clazakizumab (n510) Placebo (n510) Variables recorded at transplantation Female sex, n (%) 10 (50) 3 (30) 7 (70) Recipient age (yr), median (IQR) 34.2 (24.6–47.6) 37.4 (27.1–57.9) 31.4 (22.3–42.3) Living donor, n (%) 6 (30) 3 (30) 3 (30) ABO-compatible transplant, n (%) 20 (100) 10 (100) 10 (100) Prior kidney transplant, n (%) 7 (35) 4 (40) 3 (30) Current CDC panel reactivity $10%, n (%)a 6 (33.3) 3 (33.3) 3 (33.3) Preformed anti-HLA DSA, n (%)b 5 (45.5) 3 (42.9) 2 (50) Donor age (yr), median (IQR)c 49.0 (21.8–57.3) 51.0 (21.8–57.3) 44.0 (23.3–66.0) HLA mismatch (A, B, DR), median (IQR)d 3(2–3) 3 (3–3) 3 (2–3) Cold ischemia time (h), median (IQR)e 13.0 (4.8–17.3) 10.4 (3.4–14.6) 15.5 (6.4–20.0) Variables recorded at trial inclusion Age of study patients (yr), median (IQR) 41.5 (36.4–60.1) 47.2 (38.7–62.1) 39.6 (30.2–59.6) Yr to inclusion in the trial 10.6 (4.4–16.2) 9.7 (4.1–16.7) 11.4 (5.9–16.1) eGFR (ml/min per 1.73 m2), median (IQR) 39.3 (33.6–49.7) 40.5 (33.3–49.8) 39.2 (32.9–51.7) Protein/creatinine ratio (mg/g), median (IQR) 962 (310–1863) 727 (197–1311) 1387 (532–3575) DSA characteristics HLA class I DSA only, n (%) 2 (10) 1 (10) 1 (10) HLA class II DSA only, n (%) 14 (70) 7 (70) 7 (70) HLA class I and II DSA, n (%) 4 (20) 2 (20) 2 (20) Anti-DQ DSA, n (%) 15 (75) 7 (70) 8 (80) No. of DSA, median (IQR) 1 (1–2) 1 (1–2) 1 (1–2) MFI of the peak DSA, median (IQR) 11,708 (1947–17,709) 10,789 (3092–15,437) 14,207 (1252–19,144) MFI sum of detected DSA, median (IQR) 13,130 (2137–18,962) 10,789 (4244–18,102) 16,126 (1252–19,302) Banff 2017 rejection categories ABMR, n (%) 20 (100) 10 (100) 10 (100) Active ABMR, n (%) 2 (10) 2 (20) 0 Chronic/active ABMR, n (%) 18 (90) 8 (80) 10 (100) C4d-positive ABMR, n (%) 7 (35) 4 (40) 3 (30) Banff borderline lesion, n (%) 1 (5) 0 1 (10) Markers of inﬂammation and immunosuppressive load CRP (mg/dl), median (IQR) 0.20 (0.05–0.44) 0.13 (0.04–0.26) 0.42 (0.08–0.48) TTV load (copies/ml), median (IQR)f 1.93105 (3.53104–9.13105)7.23104 (3.53104–2.13105)6.03105 (7.63104–1.73108) CDC, complement-dependent cytotoxicity. aCDC panel reactivity was not recorded for one recipient in the clazakizumab arm and one in the placebo arm. bPretransplant DSA data were available for seven recipients in the clazakizumab arm and four in the placebo arm (solid-phase HLA antibody screening on the waitlist was implemented at the Vienna transplant unit in July 2009).23 cDonor age was not recorded for two recipients in the placebo arm. dHLA mismatch was not recorded for one recipient in the placebo arm. eCold ischemia time was not recorded for one recipient in the clazakizumab arm and two recipients in the placebo arm. fOne patient had TTV levels below the detection threshold.

Laboratory ﬁndings are provided in Supplemental HLA Antibody and Ig Levels Figure 3 and Table 3. Mild liver parameter elevations were Within 12 weeks (part A), clazakizumab decreased DSA MFI noted in a few patients; none met Hy’slaw.Anemia,mostly to a median of 77% (IQR, 63%–101%) from baseline (pla- graded I or II, was already prevalent in both arms before cebo: 103% [IQR, 94%–104%]; P50.035). Extension of treat- study inclusion, with no relevant differences in part A ment in part B led to a further decrease of DSA levels and stable hemoglobin levels in part B. There were also no (P,0.001) (Figure 2A). Similar results were obtained for levels relevant changes in leukocyte and platelet counts. Lipid ab- of DSA interpolated from dilution experiments, nondonor- normalities were frequent in both study arms, with peak speciﬁc HLA reactivity (Figure 2A), total IgG (but not IgM), (unfasted) triglyceride levels higher under clazakizumab and, among IgG subclasses, most prominently IgG4 (Table 3). (Figure 3).

Efﬁcacy Evolution of Rejection Treatment with clazakizumab effectively suppressed CRP lev- The 11-week biopsies failed to demonstrate signiﬁcant inter- els and slightly increased TTV viral load (Supplemental group differences in rejection-related molecular and morpho- Figure 4). logic scores (Figure 2, B and C), and there were no meaningful

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Table 2. Serious treatment emergent adverse events by system organ class a Part A Part B Serious Adverse Events, n (%) Clazakizumab (n510) Placebo (n510) Clazakizumab (n519)b Infections and infestations 0 0 5 (26.3) Pneumonia 0 0 2 (10.5) Pyelonephritis 0 0 1 (5.3) Ovarian abscess 0 0 1 (5.3) Aseptic meningitis 0 0 1 (5.3) Gastrointestinal disorders 1(10) 0 1(5.3) Diverticulitis 1 (10)c 0 1 (5.3)d General disease and administration site conditions 0 1 (10) 0 Pyrexia 0 1 (10) 0 Respiratory, thoracic, and mediastinal disorders 1(10) 0 1(5.3) Pleural effusion 1 (10) 0 1 (5.3) Surgical and medical procedures 0 0 1 (5.3) Pleurodesis 0 0 1 (5.3) Permanent thorax cavity drainage 0 0 1 (5.3) Renal and urinary disorders 1(10) 0 0 Acute renal injury 1 (10) 0 0 Bold text indicates the number (%) of adverse events for individual system organ classes. aDifferences between groups in part A were NS. bOne patient was withdrawn from the trial in part A and was not included in the safety analysis of part B. cDiverticulitis resolved after percutaneous abscess drainage and antibiotic therapy. dDiverticulitis was complicated by colon perforation requiring open surgery (Hartmann’s procedure). changes in ABMR phenotypes (Figure 2D). In contrast, the showed significant improvement in the eGFR slope compared 51-week biopsies performed after prolonged clazakizumab with the slope calculated for part A (P,0.001), and differences treatment in part B demonstrated a significant decrease in to patients initially allocated to clazakizumab (no signifi- molecular ABMR (P50.020) and “all rejection” scores cant change on extension of treatment in part B) became NS (P50.037), whereas T cell–mediated rejection scores re- (20.29, 95% CI, 20.85 to 0.26 versus 20.64, 95% CI, 21.13 mained negative at all time points (Figure 2B). After 51 weeks, to 20.14 ml/min per 1.73 m2 per month, P50.37) (Figure 3). seven of 18 patients (38.9%) had a negative ABMR score Similar results were obtained in a separate analysis, including (,0.2) (not shown). Although microcirculation inflamma- the two patients who were prematurely withdrawn from the tion did not change significantly (Figure 2C), we found a res- trial (Supplemental Figure 6). Levels of proteinuria did not olution of ABMR activity in four (22.2%) and disappearance change over time (Supplemental Figure 7). of capillary C4d deposits in five (27.8%) patients (Figure 2D). As shown in Supplemental Figure 8, on 6 months follow-up Supplemental Table 5 details serial biopsy results obtained in after the end of the trial, we observed a slight increase in CRP the four patients who showed transition of active ABMR to an levels to ranges detected before trial initiation. None of the inactive phenotype (cg in the absence of evidence of current/ patients developed acute graft dysfunction (or underwent in- recent antibody interaction with the endothelium). At base- dication biopsies), and as illustrated in Supplemental Figure 8, line, three of these recipients were C4d negative, with rather there was no major change in the mean slope of eGFR. One low levels of microcirculation inflammation (g1ptc sum score patient returned to dialysis 3 months after the last visit. #3). On treatment, g1ptc scores decreased to ,2, paralleled by a marked reduction in molecular ABMR scores (Supplemental Table 5). Levels of interstitial fibrosis and tubular atrophy increased DISCUSSION significantly from week 11 to week 51, but transplant glomerulopathy or a molecular classifier reflecting atrophy/fibrosis This phase 2 trial evaluated the safety (primary endpoint) and remained unchanged (Supplemental Figure 5). efficacy (secondary endpoint analysis) of anti–IL-6 antibody clazakizumab in late ABMR. Major safety signals were the Clinical Outcomes occurrence of serious infectious events and diverticular dis- In part A, the mean slope of eGFR differed significantly be- ease complications. Key results of secondary endpoint analysis tween clazakizumab and placebo (20.96; 95% confidence in- were an early decrease in DSA levels, a slowed eGFR decline, terval [95% CI], 21.96 to 0.03 versus 22.43; 95% CI, 23.40 and, after extended treatment, modulation of rejection- to 21.46 ml/min per 1.73 m2 per month, P50.04) (Figure 4). associated gene expression patterns, reduction of C4d scores, In part B, patients switched from placebo to clazakizumab and, in some patients, resolution of ABMR activity.

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Table 3. Laboratory variables Part A Part B Variables Clazakizumab (n510) Placebo (n510) P Value Clazakizumab (n518) Liver parameters ALT levels (U/L) Highest level, median (IQR) 29 (23–39) 24 (18–36) 0.39 30 (23–43) .1–3 ULN, n (%) 1 (10) 1 (10) 1 (5.6) .3–5 ULN, n (%) 0 0 2 (11.1) .5–8 ULN, n (%) 0 0 0 AST levels (U/L) Highest level, median (IQR) 26 (22–34) 28 (25–34) 0.68 28 (24–35) .1–3 ULN, n (%) 1 (10) 0 2 (11.1) .3–5 ULN, n (%) 0 0 0 .5–8 ULN, n (%) 0 0 0 Total bilirubin levels (mg/dl) Highest level, median (IQR) 0.69 (0.41–0.94) 0.38 (0.31–0.55) 0.052 0.56 (0.43–1.0) .1–3 ULN, n (%) 0 0 3 (16.7) .3–5 ULN, n (%) 0 0 0 .5–8 ULN, n (%) 0 0 0 Blood count Hemoglobin levels (g/dl) Level at nadir, median (IQR) 11.4 (9.8–12.2) 9.4 (8.4–10.7) 0.023 9.8 (8.5– 11.0) Anemia grade at nadir, n (%)a 0 1 (10) 0 1 (5.6) I 7 (70) 3 (30) 8 (44.4) II 1 (10) 6 (60) 6 (33.3) IIII 1 (10) 1 (10) 3 (16.7) IV 0 0 0 Leukocyte count (x109/L) Level at nadir, median (IQR) 5.3 (3.8–6.7) 4.7 (3.3–8.9) 0.80 4.6 (3.3–6.1) Leukopenia grade at nadir, n (%)a 0 8 (80) 5 (50) 10 (55.6) I 2 (20) 5 (50) 8 (44.4) II 0 0 0 IIII 0 0 0 IV 0 0 0 Platelet count (x109/L) Level at nadir, median (IQR) 172 (131–207) 226 (130–284) 0.14 162 (136–213) Thrombocytopenia grade at nadir, na (%) 0 6 (60) 7 (70) 10 (55.6) I 4 (40) 3 (30) 7 (38.9) II 0 0 1 (5.6) IIII 0 0 0 IV 0 0 0 Lipids (unfasted) Cholesterol (mg/dl) LDL Level above threshold, n (%) 4 (40) 4 (40) .0.99 12 (66.7) Highest level, median (IQR) 149 (132–166) 142 (122–209) 0.97 205 (142–253) HDL Level below threshold, n (%) 8 (80) 8 (80) .0.99 17 (94.4) Level at nadir, median (IQR) 45 (30–59) 49 (39–61) 0.48 47 (41–52) Triglycerides (mg/dl) Level above threshold, n (%) 9 (90) 6 (60) 0.30 18 (100) Highest level, median (IQR) 313 (213–655) 205 (118–254) 0.029 246 (201–352) ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. aFor grading of hematologic toxicities we used the National Cancer Institute Common Terminology Criteria for Adverse Events.

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A Peak DSA MFI Interpolated peak DSA level Non-DSA MFI P = 0.006 200 200 200 P < 0.001 P = 0.002 175 175 175 P = 0.35 P = 0.035 P = 0.023 150 150 150 125 125 125 100 100 100 75 75 75 % Baseline % Baseline % Baseline 50 50 50 25 25 25 0 0 0 12 52 12 52 12 52 Week Week Week

B ABMR TCMR All rejection P = 0.02 P = 0.037 P = 0.80 P = 0.58 1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 P = 0.93 0.6

Score 0.4 Score 0.4 P = 0.97 Score 0.4

0.2 0.2 0.2

0.0 0.0 0.0 01151 01151 01151 Week Week Week

C Glomerulitis Peritubular capillaritis Capillary C4d P = 0.26 4 P = 0.76 4 P = 0.49 4 P = 0.63 P = 0.28 P = 0.57 3 3 3

2 2 2 Score Score Score

1 1 1

0 0 0 01151 01151 01151 Week Week Week

D Banff 2017 category Capillary C4d deposition Clazakizumab Placebo Clazakizumab Placebo Index biopsy Index biopsy 28 10 2116 1117

11-week biopsy 11-week biopsy 28 10 181136 Clazakizumab Clazakizumab Clazakizumab Clazakizumab

51-week biopsy 51-week biopsy 62822 12710

ABMR category C4d score active chronic/inactive C4d3 C4d1 no biopsy chronic/active no biopsy C4d2 C4d0

Figure 2. Antibody and biopsy results. Percentages (in relation to baseline values) of (A) peak DSA MFI, DSA levels interpolated from dilution experiments, and the median MFI detected for non-DSA, (B) rejection-associated molecular classiﬁers (ABMR, T cell–mediated rejection [TCMR], all rejection), and (C) morphologic single lesion scores (g, ptc, C4d) are shown in relation to treatment allocation (clazakizumab, part A/B: red closed boxplots; placebo, part A: open boxplots; placebo, part B [switch to clazakizumab]: red hatched

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The role of IL-6 in transplantation is supported by its already in part A of the trial, and a slowed progression of renal marked upregulation on rejection,24,25 with HLA class II dysfunction in part B. Our results are promising and in con- DSA being a potent trigger of endothelial IL-6 secretion.26 trast to previous trials conducted in late ABMR, where other IL-6 is known to contribute to plasma cell formation, which treatments, such as bortezomib7 or rituximab plus intrave- may promote DSA formation.8 In line with an observational nous Ig,6 failed to slow the progression of renal dysfunction. study evaluating tocilizumab,12,13 clazakizumab led to an early Nevertheless, the outcome data in our trial, which was not decrease in DSA, non-DSA, and total IgG levels. We found a primarily powered to detect differences in graft function, pronounced reduction of IgG4, an IgG subclass previously have to be interpreted with caution. Calculated eGFR slopes, postulated to play a critical role in chronic rejection.27 Inter- both in the placebo and clazakizumab arms, were within a preting our DSA results, we want to point out potential caveats range earlier reported for a cohort of 91 recipients with late associated with using MFI as a measure of antibody levels.28 It ABMR (most of them treated with intravenous Ig or ste- is well established that MFI levels detected in bead arrays do roids),31 and it remains unclear whether the slope differences not simply reflect antibody concentrations in serum, but also in our trial would result in differences in graft survival. In this depend on antibody avidity/affinity and the density or confor- context, we want to mention a recently initiated large multi- mation of HLA antigens coated to individual microbeads. Re- center, randomized, placebo-controlled phase 3 trial evaluat- porting numerical changes in MFI may be problematic and ing clazakizumab in chronic ABMR (IL 6 Blockade Modifying not accurately reflect quantitative changes in antibody levels, Antibody-Mediated Graft Injury and eGFR Decline trial; especially in the case of bead saturation. In this respect, titer ClinicalTrials.gov identifier: NCT03744910). The design of studies may be more informative. In an attempt to indirectly this trial, which aims to recruit 350 patients to detect differ- quantify changes in antibody levels, we applied dilution ex- ences in 5-year graft survival, will include an early evaluation periments creating standard curves for baseline sera. Another of eGFR slope differences as an interim surrogate endpoint point is the earlier reported spontaneous fluctuation of DSA (after approximately 200 subjects have received at least 1 year levels (independent of specific therapeutic interventions),29 of treatment). which has to be taken into account when interpreting antibody Our trial did not include follow-up biopsies to investigate a changes in the open-label part of our trial. potential role of rebound phenomena after stopping clazaki- Early follow-up biopsies did not show any effect of claza- zumab treatment. Nevertheless, a 6-month follow-up after the kizumab on morphologic and molecular results, despite an last visit revealed only a slight increase in CRP levels to values early decline in CRP and DSA levels. After prolonged treat- detected before trial initiation, and none of the patients de- ment in part B (biopsies after 51 weeks), however, biopsy re- veloped acute graft dysfunction. Together with our finding of sults suggested amelioration of rejection, at least in some of no major change in eGFR slope after the last visit, this may the included patients. These findings may relate to the ob- argue against major clinically relevant rebound phenomena. served decrease in DSA levels, but one may also argue that In line with earlier studies,10,15 clazakizumab was associ- other factors have potentially influenced ABMR activity, ated with mild injection site reactions, increases in lipid levels, such as the load of maintenance immunosuppression. There and mild abnormalities of liver enzymes or blood cell count. were, however, no meaningful adjustments in the composition When combining clazakizumab with baseline immunosup- and dose of immunosuppression, which, together with the pression, aggravated infectious complications may become a outcome differences observed in placebo-controlled part A, significant concern. Under prolonged treatment in part B, supports a specific therapeutic effect of IL-6 signaling block- five serious infectious events were recorded. In parallel, we ade. Our failure to detect any improvement in atrophy/fibrosis observed a significant reduction in serum IgG and a slight in- and transplant glomerulopathy (and proteinuria) was expec- crease in TTV load as a surrogate marker of intensified immu- ted, as advanced chronic injury may not be reversible. Inter- nosuppression.32 In trials performed in rheumatoid arthritis, preting morphologic biopsy results, however, we are aware of IL-6 interference was reported to be associated with increases the inherent limitation of poor reproducibility of Banff his- in serious infection incidence,9,10,33,34 although in a compre- tology scores, which may support the use of more objective hensive analysis of available trials and their extension phases, analysis of gene expression patterns, especially in patients with intergroup differences were NS.35 The most important safety ambiguous histology.30 signal was the occurrence of diverticulitis in two patients with The slope of eGFR is a useful surrogate predicting graft pre-existing diverticulosis, one event resulting in colon perfo- survival, and in late ABMR, a 30% deterioration in eGFR slope ration. The risk of gastrointestinal perforation is known to be was reported to be associated with a 10% increase in graft loss significant under IL-6 blockade.11,36 In a large US real-world rates.31 We found a significant intergroup slope difference dataset of patients with rheumatoid arthritis, tocilizumab was

boxplots). We applied unpaired Mann–Whitney U tests for group comparisons (clazakizumab versus placebo) at the end of part A, and paired Wilcoxon test to evaluate changes under clazakizumab (overall cohort) in the open-label extension (part B). (D) shows pro- portions of ABMR categories and C4d scoring results obtained in index, 11-week and 51-week biopsies.

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A IgG IgM P = 0.019 P = 0.12 150 P = 0.031 250 P = 0.98 200

100 150

100 %Baseline %Baseline 50

0 0 12 52 12 52 Week Week

B IgG1 IgG2 P = 0.043 150 P = 0.019 150 P = 0.085 P = 0.11

100 100 %Baseline %Baseline 50 50

0 0 12 52 12 52 Week Week

IgG3 IgG4 P = 0.007 P = 0.002 200 P = 0.085 150 P = 0.063 150 100

100 %Baseline %Baseline 50 50

0 0 12 52 12 52 Week Week

Figure 3. Serum Ig concentrations. Shown are the percent levels (in relation to values at baseline) of (A) IgG, IgM, and (B) IgG subclasses for patients randomized to clazakizumab (red closed boxplots) or placebo (part A: open boxplots; part B: red hatched boxplots). We used unpaired Mann–Whitney U test for group comparisons (clazakizumab versus placebo) at the end of part A and paired Wilcoxon test to evaluate changes under clazakizumab (overall cohort) in the open-label extension (part B).

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A Part A Part B B Part A Part B 80 80 ) ) 2 60 2 60

40 40

20 20 eGFR (ml/min/1.73 m eGFR (ml/min/1.73 m

Clazakizumab Placebo 0 0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 0 4 8 1216202428323640444852 Weeks Weeks

C Part A Part B D Part A Part B 80 60 ) 2

) 50 2 60

40 40 30

20 eGFR (ml/min/1.73 m 20 Predicted eGFR (ml/min/1.73 m 0 10 0 4 8 1216202428323640444852 0 4 8 12 16 20 24 28 32 36 40 44 48 52 Weeks Weeks

Figure 4. Kidney allograft function. The individual course of eGFR is shown for patients allocated to receive clazakizumab (A) versus placebo (B). (C) shows median, IQR, and individual levels of eGFR in relation to treatment allocation in part A (clazakizumab: red line, bars and asterisks; placebo: black lines, bars and circles). (D) shows individual (dashed lines) and mean eGFR slopes (solid lines; shaded areas represent 95% CIs) in relation to treatment in part A (clazakizumab: red lines; placebo: black lines). associated with a lower gastrointestinal perforation rate of 1.26 evaluation of the patient’s history, including documented colon per 1000 patient-years.37 A study by Choi et al.12 showed zero diverticulosis and, at least for patients deemed to be at partic- of 36 patients receiving tocilizumab for ABMR experiencing ular risk, such as those with polycystic kidney disease, use of diverticular disease complications; one case of colon perfora- diagnostic tests (e.g., colonoscopy or computer tomography) tion, however, was recorded in a small trial of tocilizumab for to rule out significant colon diverticulosis. Of note, the results recipient desensitization.38 Given that none of the trials evalu- of our safety analysis have significantly influenced the design of ating clazakizumab in rheumatologic disease (.500 treated an ongoing pivotal phase 3 trial (IL 6 Blockade Modifying patients) reported any patients with gastrointestinal perfora- Antibody-Mediated Graft Injury and eGFR Decline; tion (as in our study, patients with a history of diverticulitis or ClinicalTrials.gov identifier: NCT03744910), including a re- gastrointestinal perforation were not included),10,15 one may duction in monthly clazakizumab doses (from 25 to 12.5 mg assume that additional immunosuppression, in particular per month) and a strict definition of exclusion criteria (history long-term steroid use, and the background of certain predis- of gastrointestinal perforation; diverticular disease or divertic- posing conditions, including diverticulosis and polycystic kid- ulitis, except if disease has been fully excised; or inflammatory ney disease, have increased the risk significantly.39 Our data bowel disease). suggest the need for careful evaluation of transplant patients A major limitation of our trial is its small sample size. A considered for IL-6 signaling blockade, for example, in the strength, however, may be the granularity of analyzed end- context of future trials. This should include a thorough points, including a detailed work-up of three sequential

JASN 32: 708–722, 2021 Clazakizumab in Late Rejection 719 CLINICAL RESEARCH www.jasn.org biopsies. Although the trial was not primarily powered to de- European Societies for Pathology, 2018–2020 French Speaking Club of Renal tect efficacy outcome differences, the double-blind phase of Pathology, 2012–2020 Austrian, French, and European Societies for Trans- – the trial revealed significant differences in DSA levels and plantation, 2018 2020 Member then Co-chair of the Banff Working group for Thrombotic Microangiopathy, and 2020 Co-chair of the Banff Working progression of graft dysfunction, and observed changes in sec- group for Peritubular Capillaritis. N. Lachmann reports consultancy agree- ondary outcomes were pronounced upon treatment in the ments with BmT GmbH. P. Halloran reports consultancy agreements with open-label extension. One may argue that intergroup differ- Astellas, CSL Behring; ownership interest in Transcriptome Sciences Inc., a ences in the course of renal function in part A could partly University of Alberta research company with an interest in molecular diag- relate to a bias by chance due to imbalances in baseline nostics, has given lectures for Thermo Fisher, and is a consultant for CSL Behring; reports receiving research funding from Transcriptome Sciences variables associated with the severity of rejection or immuno- Inc.; reports receiving honoraria from Astellas, One Lambda; is a scientific suppressive therapy (despite stratified randomization), in advisorormemberofCEO,TranscriptomeSciencesInc.,Editor-in-Chief particular, higher numerical levels of DSA MFI, CRP, and pro- emeritus, American Journal of Transplantation; Speakers Bureau for Astellas teinuria and a lower dose of enteric-coated mycophenolic acid and One Lambda. All remaining authors have nothing to disclose. in the placebo arm. In contrast, however, baseline eGFR and morphologic features of rejection and/or chronic injury were similar between groups, and molecular rejection-related FUNDING scores were even higher in the clazakizumab arm. In conclusion, this phase 2 trial suggests the efficacy of The trial was funded by an investigator-initiated unrestricted grant from clazakizumab in late ABMR after kidney transplantation. Vitaeris Inc., Vancouver, Canada (to G. Böhmig and B. Jilma). Recorded safety outcomes—the primary trial endpoint— warrant careful patient selection and monitoring to minimize ACKNOWLEDGMENTS the risk of diverticular disease complications and serious infections. The results of secondary endpoint analysis, including fi first systematic data on the size of expected outcome effects, The authors wish to thank Prof. Josef Smolen for his scienti c advice, and Susanne Haindl, Sarah Ely, Dr. Christa Drucker, Rene Nadolny, and Silke may provide a valuable basis for larger phase 3 trials with Kasbohm for excellent assistance. longer duration of follow-up to systematically evaluate the concept of IL-6/IL-6R blockade in transplantation (in com- parison to placebo and/or other treatment concepts). SUPPLEMENTAL MATERIAL

This article contains the following supplemental material online at http:// DISCLOSURES jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2020071106/-/ DCSupplemental. Supplemental Table 1. Single lesions and molecular classifiers at baseline. B. Jilma reports having consultancy agreements with Guardian Therapeu- Supplemental Table 2. Baseline immunosuppression. tics; and Speakers Bureau from Sanofi. E. Chong is employed by and has Supplemental Table 3. Adverse events by system organ class. ownership interest in Vitaeris Inc., Vancouver, Canada. G. Böhmig and Supplemental Table 4. Diverticular disease and complications. K. Budde are members of the steering committee for an ongoing pivotal phase Supplemental Table 5. Resolution of ABMR activity in four recipients: serial 3 trial evaluating clazakizumab in chronic active ABMR (ClinicalTrials.gov biopsy results. number, NCT03744910; sponsored by Vitaeris Inc.). G. Böhmig reports con- Supplemental Figure 1. Summary of trial protocol. sultancy agreements from Vitaeris Inc., Vancouver, Canada; research funding Supplemental Figure 2. Immunosuppression levels and dosage. from Vitaeris Inc., Vancouver, Canada; honoraria from Astellas, Fresenius Supplemental Figure 3. Safety lab. Medical Care, OneLambda, Sandoz; and being a scientific advisor or member- Supplemental Figure 4. CRP and Torque Teno virus (TTV) levels. ship of Vitaeris Inc, Vancouver, Canada. G. Bond reports receiving research Supplemental Figure 5. Morphologic and molecular features of chronic funding from Chiesi. H. Regele reports receiving honoraria from Roche, injury. Menarini, and other interests/relationships with Gerson Lehrman Group. Supplemental Figure 6. Analysis of renal function including two patients K. Budde reports consultancy agreements with Abbvie, Alexion, Astellas, withdrawn from the trial. Bristol-Myers Squibb, Chiesi, CSL-Behring, Fresenius, Hansa, Hexal, Novar- Supplemental Figure 7. Spot urine protein/creatinine ratio. tis, MSD, Otsuka, Pfizer, Quark, Roche, Sandoz, Shire, Veloxis, Vifor, and Supplemental Figure 8. CRP and renal function after the end of the trial. Vitaeris; reports receiving research funding from Abbvie, Alexion, Astellas, Bristol-Myers Squibb, Chiesi, CSL-Behring, Fresenius, Hansa, Hexal, Novar- fi tis, MSD, Otsuka, P zer, Quark, Roche, Sandoz, Shire, Veloxis, Vifor, and REFERENCES Vitaeris; receiving honoraria from Abbvie, Alexion, Astellas, Bristol-Myers Squibb, Chiesi, Fresenius, Hexal, Novartis, Otsuka, Pfizer, Quark, Roche, San- doz, Shire, and Veloxis Pharma; being a scientific advisor or membership of 1. Loupy A, Lefaucheur C: Antibody-mediated rejection of solid-organ Astellas, Bristol-Myers Squibb, Chiesi, Hansa, Hexal, MSD, Novartis, Pfizer, allografts. NEnglJMed379: 1150–1160, 2018 Roche, and Veloxis. M. Duerr reports receiving research funding from Bristol- 2. Halloran PF, Venner JM, Madill-Thomsen KS, Einecke G, Parkes MD, Myers Squibb; honoraria from Shire, Novartis; being a scientific advisor or Hidalgo LG, et al.: Review: The transcripts associated with organ allo- member of Novartis. N. Kozakowski reports receiving research funding from graft rejection. Am J Transplant 18: 785–795, 2018 the Austrian Science Fund—Erwin Schrödinger Fellowship J-4377; being a 3. Haas M, Loupy A, Lefaucheur C, Roufosse C, Glotz D, Seron D, et al.: scientific advisor or member as Associate Editor—BMC Nephrology; other The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for interests/relationships with 2012–2020 Austrian, German, French, and chronic active T cell-mediated rejection, antibody-mediated rejection,

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AFFILIATIONS

1Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria 2Department of Nephrology, Charité Universitätsmedizin Berlin, Berlin, Germany 3Alberta Transplant Applied Genomics Centre, University of Alberta, Edmonton, Alberta, Canada 4Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria 5Centre for Tumor Medicine, Histocompatibility & Immunogenetics Laboratory, Charité Universitätsmedizin Berlin, Berlin, Germany 6Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria 7Department of Surgery, Medical University of Vienna, Vienna, Austria 8Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria 9Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Vienna, Austria 10Vitaeris Inc., Vancouver, Canada

722 JASN JASN 32: 708–722, 2021 A Randomized Trial of Anti-Interleukin-6 Antibody Clazakizumab in

Late Antibody-Mediated Kidney Transplant Rejection

Konstantin Doberer, Michael Duerr, Philip F. Halloran, Farsad Eskandary, Klemens

Budde, Heinz Regele, Jeff Reeve, Anita Borski, Nicolas Kozakowski, Roman Reindl-

Schwaighofer, Johannes Waiser, Nils Lachmann, Sabine Schranz, Christa Firbas,

Jakob Mühlbacher, Georg Gelbenegger, Thomas Perkmann, Markus Wahrmann,

Alexander Kainz, Robin Ristl, Fabian Halleck, Gregor Bond, Edward Chong, Bernd

Jilma, and Georg A. Böhmig

Table of contents Page

Table S1 2-3

Table S2 4-5

Table S3 6-10

Table S4 11

Table S5 12

Figure S1 13

Figure S2 14

Figure S3 15

Figure S4 16

Figure S5 17

Figure S6 18

Figure S7 19

Figure S8 20

References 21

Supplementary Table 1. Single lesions and molecular classifiers at baseline. Total Clazakizumab Placebo Parameter (n=20) (n=10) (n=10) Morphological ABMR lesions and scores Glomerulitis (g score ≥1), n (%)a 17 (89.5) 8 (80) 9 (100) g score, median (IQR) 2 (1-3) 2 (1-3) 3 (1-3) Peritubular capillaritis (ptc score ≥1), n (%)b 13 (68.4) 6 (60) 7 (77.8) ptc score, median (IQR) 2 (0-2) 2 (0-2) 2 (1-3) Transplant glomerulopathy (cg score ≥1), n (%) 17 (89.5) 8 (80) 9 (100) cg score, median (IQR) 3 (2-3) 3 (1-3) 3 (3-3) C4d in peritubular capillaries (C4d score ≥1), n (%) 7 (35) 4 (40) 3 (30) C4d score, median (IQR) 0 (0-2) 0 (0-2) 0 (0-1) MLPTC, n (%)c 6 (42.9) 2 (25) 4 (66.7) High-grade MLPTC, n (%)c 2 (14.3) 1 (12.5) 1 (16.7) Interstitial fibrosis (ci score ≥1), n (%) 17 (85) 8 (80) 9 (90) ci score, median (IQR) 2 (1-3) 2 (1-2) 2 (1-3) Tubular atrophy (ct score ≥1), n (%) 17 (85) 8 (80) 9 (90) ct score, median (IQR) 1 (1-2) 1 (1-2) 2 (1-2) Vascular fibrous intimal thickening (cv score ≥1), n (%)d 15 (88.2) 6 (75) 9 (100) cv score, median (IQR) 1 (1-2) 1 (0-2) 2 (1-2)

Molecular classifiers of rejection and injury ABMR score, median (IQR) 0.65 (0.35-0.81) 0.70 (0.48-0.81) 0.44 (0.29-0.82) TCMR score, median (IQR) 0.01 (0.01-0.18) 0.01 (0.01-0.01) 0.01 (0.01-0.02) ‘all Rejection’ score, median (IQR) 0.69 (0.43-0.83) 0.73 (0.44-0.84) 0.53 (0.42-0.80) Atrophy/Fibrosis score, median (IQR) 0.68 (0.35-0.84) 0.39 (0.24-0.78) 0.79 (0.67-0.87) ABMR, antibody-mediated rejection; DSA, donor-specific antibody; IQR, interquartile range; MFI, mean fluorescence intensity; MLPTC, multilayering of peritubular capillary basement membranes; TCMR, T cell mediated rejection. aFor 1 patient (placebo group), biopsy material was not sufficient for g and cg scoring. bFor 1 patient (placebo group), biopsy material was not adequate for ptc scoring. cMaterial for ultrastructural analysis of peritubular capillaries was available for 14 recipients (clazakizumab: n=8, placebo: n=6). dcv score was not available for 2 recipients in the clazakizumab arm and 1 in the placebo arm (biopsy material inadequate for complete lesion scoring).

Supplementary Table 2. Baseline immunosuppression. Total Clazakizumab Placebo Parameter (n=20) (n=10) (n=10) Initial immunosuppression Induction with antithymocyte globulin, n (%) 6 (30) 3 (30) 3 (30) Induction with Interleukin-2 receptor antibody, n (%) 8 (40) 5 (50) 3 (30) Tacrolimus, n (%) 9 (45) 4 (40) 5 (50) Cyclosporine A, n (%) 10 (50) 5 (50) 5 (50) Everolimus, n (%) 1 (5) 1 (10) 0 Azathioprine, n (%) 2 (10) 1 (10) 1 (10) Mycophenolic acid derivatives, n (%) 17 (85) 8 (80) 9 (90) Peri-transplant immunoadsorption, n (%)a 5 (25) 3 (30) 2 (20) CDC crossmatch conversion before transplantation, n (%) 3 (15) 2 (20) 1 (10) Immunsosuppression at the time of study inclusion Triple immunosuppression 18 (90) 9 (90) 9 (90) Dual immunosuppression without steroids 2 (10) 1 (10) 1 (10) Immunosuppressants Tacrolimus, n (%) 13 (65) 6 (60) 7(70) Trough level (ng/mL), median (IQR) 6.0 (5.2-7.1) 5.6 (4.3-7.4) 6.0 (5.6-7.0) Cyclosporine A, n (%) 6 (30) 4 (40) 2 (20) Trough level (ng/mL), median (IQR) 123 (103-152) 138 (115-170) 85, 114

Everolimus, n (%) 1 (5) 0 1(10) Trough level (mg/mL) 5.4 - 5.4 MMF, n (%) 10 (50) 6 (60) 4 (40) Daily dose (mg), median (IQR) 1,250 (938-2,000) 1,250 (938-2,000) 1,250 (625-1,875) EC-MPA, n (%) 10 (50) 4 (40) 6 (60) Daily dose (mg), median (IQR) 720 (360-1,170) 1,260 (810-1,440) 450 (315-720) Prednisolone, n (%) 15 (75) 8 (80) 7 (70) Daily dose (mg), median (IQR) 5 (5-5) 5 (5-5) 5 (5-5) Methylprednisolone, n (%) 3 (15) 1 (10) 2 (20) Daily dose (mg), median (IQR) 4 (4-4) 4 4, 4 CDC, complement-dependent cytotoxicity; EC-MPA, enteric-coated mycophenolic acid; IQR, interquartile range; MMF, mycophenolate mofetil. aFollowing our local standard, sensitized patients (until 2009: ≥40% CDC-PRA; since 2009: preformed DSA) were subjected to an earlier detailed protocol of peri-transplant immunoadsorption1.

Supplementary Table 3. Adverse events by system organ class. Part Aa Part B Adverse events Clazakizumab Placebo Clazakizumab (n=10) (n=10) (n=19)b Number of adverse events 50 44 129 Number of serious adverse events 3 1 9 Number (%) of patients with one or more adverse events 10 (100) 10 (100) 18 (90) Number (%) of patients with one or more serious adverse events 3 (30) 1 (10) 7 (35) Adverse events, number (%) of patients Infections and infestations 5 (50) 8 (80) 13 (68.4) Upper respiratory tract infection 5 (50) 6 (60) 8 (42.1) Urethritis and/or cystitis 1 (10) 2 (20) 3 (15.8) Bronchitis 0 0 4 (21.1) Herpes simplex 0 0 3 (15.8) Pneumonia 0 0 2 (10.5) Aseptic meningitis 0 0 1 (5.3) Coxsackie viral infection 0 0 1 (5.3) Ovarian abscess 0 0 1 (5.3) Pyelonephritis 0 0 1 (5.3) Skin papilloma 0 0 1 (5.3) Gastrointestinal disorders 5 (50) 7 (70) 11 (57.9)

Diarrhoea 4 (40) 3 (30) 6 (31.6) Abdominal pain 2 (20) 3 (30) 0 Gastroenenteritis 2 (20) 0 2 (10.5) Nausea and/or vomiting 0 2 (20) 2 (10.5) Diverticulitis 1 (10) c 0 1 (5.3) d Dyspepsia 0 0 2 (10.5) Aphtous ulcer 1 (10) 0 1 (5.3) Dry mouth 0 0 1 (5.3) Gastritis 0 1 (10) 0 Haemorrhoids 0 0 1 (5.3) Noninfective gingivitis 0 0 1 (5.3) Pancreatic enzyme abnormality 0 0 1 (5.3) General disease and administration site conditions 3 (30) 5 (50) 10 (52.6) Oedema 2 (20) 3 (30) 6 (31.6) Injection site reactions 1 (10) 0 3 (15.8) Fatigue 0 1 (10) 2 (10.5) Malaise 1 (10) 0 1 (5.3) Pyrexia 0 1 (10) 0 Skin and subcutaneous disorders 1 (10) 2 (20) 8 (42.1) Erysipela 0 0 2 (10.5) Alopecia 0 1 (10) 1 (5.3)

Eczema 0 1 (10) 1 (5.3) Pruritus 1 (10) 1 (10) 0 Blister 0 0 1 (5.3) Dry skin 0 0 1 (5.3) Hirsutism 0 0 1 (5.3) Impetigo 0 0 1 (5.3) Intertrigo 0 0 1 (5.3) Rash 1 (10) 0 0 Vascular disorders 2 (20) 4 (40) 5 (26.3) Accelerated hypertension 2 (20) 3 (30) 3 (15.8) Hypotension 0 1 (10) 1 (5.3) Deep vein thrombosis 1 (10) 0 0 Thrombophlebitis 0 0 1 (5.3) Musculoskeletal and connective tissue disorders 4 (40) 2 (20) 4 (21.1) Musculoskeletal pain 2 (20) 1 (10) 3 (15.8) Muscle cramps 3 (30) 1 (10) 1 (5.3) Tenosynovitis 0 0 1 (5.3) Nervous system disorders 0 4 (40) 5 (26.3) Headache 0 3 (30) 4 (21.1) Numbness 0 1 (10) 1 (5.3) Respiratory, thoracic and mediastinal disorders 2 (20) 1 (10) 4 (21.1)

Dyspnea 1 (10) 1 (10) 2 (10.5) Cough 0 0 3 (15.8) Chest pain 0 0 1 (5.3) Epistaxis 0 0 1 (5.3) Nasal dryness 1 (10) 0 0 Pleural effusion 1 (10) 0 1 (5.3) Small airway disease 1 (10) 0 0 Blood and lymphatic system disorders 0 2 (20) 2 (10.5) Anemia 0 2 (20) 1 (5.3) Leukopenia 0 1 (10) 1 (5.3) Injury poisoning and procedural complications 1 (10) 2 (20) 1 (5.3) Transplant biopsy complicatione 1 (10) 2 (20) 0 Traumatic bone or joint injury 0 0 1 (5.3) Cardiac disorders 2 (20) 0 1 (5.3) Atrial fibrillation 1 (10) 0 1 (5.3) Bradycardia 1 (10) 0 0 Palpitations 1 (10) 0 0 Psychiatric disorders 0 0 3 (15.8) Sleep disturbance 0 0 3 (15.8) Renal and urinary disorders 1 (10) 1 (10) 1 (5.3) Acute renal injury 1 (10) 0 0

Aggravated proteinuria 0 1 (10) 0 Dysuria 0 0 1 (5.3) Surgical and medical procedures 1 (10) 0 2 (10.5) Mole excision 1 (10) 0 0 Nasal septum operation 0 0 1 (5.3) Permanent thorax cavity drainage 0 0 1 (5.3) Pleurodesis 0 0 1 (5.3) Eye disorders 0 0 2 (10.5) Ocular infection 0 0 2 (10.5) Ear and labyrinth disorders 0 1 (10) 0 Ear pain 0 1 (10) 0 Otitis media 0 1 (10) 0 Metabolism and nutrition disorders 0 0 1 (5.3) Folate deficiency 0 0 1 (5.3) Hyperkalemia 0 0 1 (5.3) Reproductive system and breast disorders 0 0 1 (5.3) Pelvic pain 0 0 1 (5.3) aDifferences between groups (part A) were nonsignificant. bOne patient was withdrawn from the trial in part A was not included in the safety analysis of part B. cDiverticulitis resolved after percutaneous abscess drainage and antibiotic therapy. dDiverticulitis was complicated by colon perforation requiring open surgery (Hartmann’s procedure). eTransplant biopsy complications included skin hematoma, peritransplant hematoma, and arteriovenous fistula.

Supplementary Table 4. Diverticular disease and complications Colon Yrs from Immunosuppression Screening ID Diverticulitis Clazakizumab Diverticulosis Assessment Gender Age Renal disease Tx No. perforation Tx after inclusion #001_Vienna yes 2 doses no yes CT male 39 IgA nephropathy 9.9 1 CyA, MMF, Pred #002_Vienna no - - no CT male 36 Postrenal cause 3.0 7 Tac, EC-MPA, Pred #003_Vienna no - - no MRI female 41 Unknown 11.4 1 Tac, EC-MPA, Pred #004_Vienna no - - no Colonoscopy female 37 Pyelonehritis 16.5 1 CyA, EC-MPA, Pred #005_Vienna no - - no CT female 38 Goodpasture 11.3 1 Everolimus, EC- MPA, Pred #008_Vienna no - - yes CT female 62 Unknown 18.9 1 CyA, EC-MPA, Pred #009_Vienna no - - yes CT male 43 FSGN 4.8 2 Tac, MMF, Pred #010_Vienna no - - yes Colonoscopy male 67 Unknown 17.4 4 CyA, EC-MPS, Pred #011_Vienna no - - yes CT female 59 Chronic GN 3.1 2 Tac, MMF, Pred #012_Vienna no - - yes CT female 60 Pyelonephritis 18.2 1 Tac, EC-MPA, Pred #013_Vienna no - - no CT female 62 Unknown 4.2 4 Tac, MMF, Pred #014_Vienna no - - yes CT male 60 Diabetic NP 1.6 1 Tac, MMF, Pred #001_Berlin no - - - no imaging female 29 RPGN 11.4 1 CyA, MMF, Methypred #015_Vienna no - - yes CT female 29 Postrenal cause 15.4 1 Tac, MMF, Pred #016_Vienna no - - no Colonoscopy female 27 RPGN 24.1 1 Tac, EC-MPA,Pred #002_Berlin no - - - no imaging male 41 IgA nephropathy 9.4 1 Tac, EC-MPA, Methypred #017_Vienna yes 4 doses yes yes CT male 62 PKD 3.9 2 Tac, MMF, Pred #018_Vienna no - - no Colonoscopy male 42 IgA nephropathy 7.1 3 Tac, EC-MPA, Pred #003_Berlin no - - - no imaging male 31 IgA nephropathy 6.8 1 Tac, MMF, Methypred #004_Berlin no - - - no imaging male 52 Unknown 14.8 1 CyA, MMF CT, computer tomography; CyA, cyclosporine A; EC-MPA, enteric-coated mycopehnoloc acid; GN, glomerulonephritis; IgA, immunoglobulin A; Methylpred, methylprednisolone; MMF, mycophenolate mofetil; MRI, magnetic resonance imaging; NP, nephropathy; PKD, polycystic kidney disease; Pred, prednisolone; RPGN, rapid progressive glomerulonephritis; Tac, tacrolimus.

Supplementary Table 5. Resolution of ABMR activity in four recipients – Serial biopsy results Screening ID Biopsy ABMR category Banff single lesion scores ABMR score (Randomization - Part A) (Banff 2017) g ptc cg c4d (MMDx) #004_Vienna Index Chronic active 2 0 3 2 0.35 (Clazakizumab) Week 11 Chronic active 2 0 3 0 0.21

Week 51 Chronica 1 0 3 0 0.17

#008_Vienna Index Chronic active -b 3 -b 0 0.29 (Placebo) Week 11 Chronic active 2 1 3 0 0.25

Week 51 Chronica 1 0 3 0 0.14

#0018_Vienna Index Chronic active 1 1 3 0 0.41 (Placebo) Week 11 Chronic active 0 2 2 0 0.34

Week 51 Chronica 1 0 2 0 0.08

#004_Berlin Index Chronic active 2 0 3 0 0.73 (Clazakizumab) Week 11 Chronic active 1 1 2 0 0.86

Week 51 Chronica 1 0 3 0 0.23 ABMR, antibody-mediated rejection; cg, transplant glomerulopathy; g, glomerulitis; MMDx, molecular microscope diagnostic system; ptc, peritubular capillaritis. aAccording to the Banff 2017 update2, the term “chronic ABMR” was applied for cg without evidence of current/recent antibody interaction with the endothelium, but with a prior documented diagnosis of chronic active ABMR. bFor one patient (#008_Vienna), index biopsy material was not sufficient for g and cg scoring.

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