A Randomized Clinical Trial of Anti–IL-6 AntibodyClazakizumab in Late Antibody-Mediated KidneyTransplant Rejection
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.
ABSTRACTBackground Late antibody-mediated rejection (ABMR) is a leading cause of transplant failure. BlockingIL-6 has been proposed as a promising therapeutic strategy.
MethodsWe 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 kidneytransplant recipientswithdonor-specific, antibody-positiveABMR$365dayspost-transplantation.Patientswererandomized1:1 to receive25mgclazakizumaborplacebo (4-weekly subcutaneous injections) for12weeks (partA),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%) de-veloped diverticular disease complications, leading to trial withdrawal. Those receiving clazakizumab dis-played significantly decreased donor-specific antibodies and, on prolonged treatment, modulatedrejection-related gene-expression patterns. In 18 patients, allograft biopsies after 51 weeks revealed a neg-ative molecular ABMR score in seven (38.9%), disappearance of capillary C4d deposits in five (27.8%), andresolution of morphologic ABMR activity in four (22.2%). Although proteinuria remained stable, the meaneGFR decline during part A was slower with clazakizumab compared with placebo (20.96; 95% confidenceinterval [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, re-spectively, P50.04). During part B, the slope of eGFRdecline for patients whowere switched fromplacebo toclazakizumab improved and no longer differed significantly from patients initially allocated to clazakizumab.
ConclusionsAlthoughsafetydata indicate theneed for carefulpatient selectionandmonitoring,ourpreliminaryefficacy results suggest a potentially beneficial effect of clazakizumab on ABMR activity and progression.
Late antibody-mediated rejection (ABMR) is acommon cause of kidney allograft failure.1 Its mo-lecular mechanisms are increasingly understood,and diagnostic criteria have been refined continu-ously.2,3 Treating late ABMR, however, remains asignificant challenge.4,5 Systematic trials have failedto demonstrate any benefit of therapies widelyused for desensitization and acute ABMR, such asrituximab plus intravenous Ig6 or bortezomib.7
Received July 31, 2020. Accepted November 10, 2020.
Published online ahead of print. Publication date available atwww.jasn.org.
Correspondence: Dr. Georg A. Böhmig, Division of NephrologyandDialysis, Department ofMedicine III, Medical University of Vienna,Währinger Gürtel 18–20, A-1090 Vienna, Austria, or Dr. Bernd Jilma,Department of Clinical Pharmacology, Medical University of Vienna,Währinger Gürtel 18–20, A-1090 Vienna, Austria. E-mail: [email protected] or [email protected]
Consequently, there is a high unmet need for an effectivetreatment.4,5
One promising therapeutic target is IL-6, a cytokine that iscritically involved in the regulation of inflammation and im-mune cell differentiation.8 Over the last decade, the concept oftargeting IL-6 or its receptor (IL-6R) has entered clinical rou-tine, and is well established in rheumatoid arthritis.9–11 Morerecently, IL-6/IL-6R interference has also become of interest inorgan transplantation.8 An observational study evaluatinganti–IL-6R antibody tocilizumab in chronic ABMR has sug-gested stabilization of allograft function, presumably resultingfrom reduced levels of donor-specific antibody (DSA).12,13
The occurrence of graft losses in four recipients in whom to-cilizumab was prematurely stopped,12 however, suggested arebound effect, triggered by IL-6 accumulated on treatment.14
Thus, the use of antibodies that directly neutralize IL-6 may beof particular interest.
Clazakizumab is a humanized monoclonal IgG1 antibodywith high affinity for IL-6 and a long t1/2 of approximately 30days. This antibody has been systematically evaluated in rheu-matoid and psoriatic arthritis,10,15 but has not yet been ap-proved for clinical use. We designed this phase 2 pilot trial, thefirst randomized controlled trial evaluating IL-6 signalingblockade in transplantation, to assess the safety and tolerabil-ity (primary endpoint) as well as efficacy (secondary endpointanalysis) of clazakizumab in late ABMR.
METHODS
Trial Oversight and DesignThis investigator-driven, randomized, double-blind, placebo-controlled, parallel-group phase 2 pilot trial was conducted attwo sites (Medical University of Vienna, Austria; Charité Uni-versitätsmedizin Berlin, Germany) from January 2018 to April2020 (recruitment period: January 2018 to April 2019). Detailsof the protocol have been described previously.16 We hypoth-esized that clazakizumab is a safe treatment to counteractABMR progression. The study consisted of two parts(Supplemental Figure 1); a 12-week randomized placebo-controlled phase to decipher the short-term effects of treat-ment (part A), followed by a 40-week open-label extensionwhere all participants received clazakizumab (part B). Therationale behind this design was to offer all participants theoption of a potentially effective treatment. Furthermore, twofollow-up biopsies to analyze short- and intermediate-termtreatment effects were considered unacceptable for patientson long-term placebo treatment. The study was approved bythe institutional review board of the Medical University ofVienna (EK1428/2017), the Berlin State Ethics Committee(17/0485– EK 15), and the regulatory authorities in Austria(Federal Office for Safety in Health Care, Austrian Agency forHealth and Food Safety) and Germany (Federal Institute forVaccines and Biomedicines, Paul-Ehrlich Institute). All pa-tients provided written informed consent before study
inclusion, and the study was conducted in accordance withInternational Council for Harmonisation of Technical Require-ments for Pharmaceuticals for Human Use Good Clinical Prac-tice requirements, Good Laboratory Practice, the principles ofthe Declaration of Helsinki 2008, and the Declaration of Istan-bul. The trial was registered on the European Union Drug Reg-ulating Authorities Clinical Trials Database (2017–001604–30)and ClinicalTrials.gov (NCT03444103).
ParticipantsThe trial was designed to include 20 patients (Figure 1,Supplemental Figure 1). Eligible participants were adult kid-ney transplant recipients (.18 years) with late active orchronic active ABMR $365 days after transplantation (withor without C4d deposits along the peritubular capillaries),associated with a molecular pattern of ABMR in gene arrayanalysis, preformed or de novoHLA class I and/or II DSA, andan eGFR (CKD Epidemiology Collaboration equation).30 ml/min per 1.73 m2. Exclusion criteria were age #18years, participation in another clinical trial, pregnancy orbreastfeeding, T cell–mediated rejection classified Banff grade$I, de novo or recurrent severe thrombotic microangiopathy,polyoma virus nephropathy, de novo or recurrent GN, acuterejection treatment ,3 months before screening, acute dete-rioration of graft function (eGFR decline within 1–3 months.25%), nephrotic range proteinuria .3500 mg/g protein/creatinine ratio, active viral, bacterial, or fungal infection pre-cluding intensified immunosuppression, active malignantdisease precluding intensified immunosuppressive therapy,abnormal liver-function tests (alanine aminotransferase,aspartate aminotransferase, bilirubin .1.53 upper limit ofnormal), other significant liver disease, latent or active tuber-culosis (positive QuantiFERON-TB-Gold test, chest x-ray),administration of a live vaccine within 6 weeks of screening,neutropenia (,1 G/L) or thrombocytopenia (,100 G/L), his-tory of gastrointestinal perforation, diverticulitis, or inflam-matory bowel disease, history of alcohol or illicit substanceabuse, or a serious medical or psychiatric illness likely to in-terfere with participation in the study.
Significance Statement
There is no proven effective treatment for a major cause of graftfailure, late antibody-mediated rejection, but IL-6, a cytokine knownto promote B cell immunity, may be a promising therapeutic target.The authors describe the results of a phase 2 randomized clinicaltrial involving 20 patients, designed to evaluate the safety (primaryendpoint) and efficacy (secondary endpoint analysis) of an anti–IL-6antibody, clazakizumab, versus placebo in late antibody-mediatedrejection. Although the occurrence of serious infections and di-verticulitis presented important safety signals, clazakizumab wasassociated with an early decrease in donor-specific antibody levels,modulated antibody-mediated rejection activity, and slowed thedecline of renal function. Preliminary efficacy results suggest a po-tentially beneficial effect of clazakizumab and may therefore sup-port the design of larger trials with a longer duration of follow-up.
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RandomizationPatients were randomized 1:1 to one of the two study arms(clazakizumab versus placebo) in part A using a web-basedrandomization platform (www.meduniwien.ac.at/randomizer).Permuted block randomization with block sizes of two wasstratified by study site (Vienna versus Berlin) and ABMR cat-egory (active ABMR versus chronic/active ABMR). Study par-ticipants, care providers, and those assessing outcomes wereunaware of the randomization sequence. The allocation se-quencewas generated, andmedication or placebowas preparedby independent nonblinded study pharmacists. Study physi-cians and nurses were provided with blinded subcutaneousmedication. Subjects were enrolled and assigned to interven-tions by study physicians. The participating investigators, staffwith medical interaction, and the study participants were blin-ded to group allocation until the last patient had completedpart A. Treatment code envelopes were securely stored to beopened in case of emergency.
Study MedicationClazakizumab (25 mg in 1 ml single-dose vials; Vitaeris Inc.,Vancouver, Canada) and placebo (0.9% saline) were admin-istered via subcutaneous injection in 4-weekly intervals. Dueto adverse events (n510) or personal reasons (n51), 11 (55%)
subjects did not receive all 13 scheduled clazakizumab injec-tions (12 doses: n53; 11 doses: n52; ten doses: n52; ninedoses: n51; eight doses: n51; four doses: n51; two doses:n51). In accordance with a July 2019 trial amendment aftertwo patients had serious gastrointestinal complications (di-verticulitis; one patient requiring open surgery because of co-lon perforation), the dose of clazakizumab was reduced to12.5 mg per injection in two other active patients with docu-mented diverticulosis (last injection before completion of thestudy).
ImmunosuppressionTacrolimus, cyclosporin A, and everolimus doses were ad-justed to trough levels of 5–10 ng/ml, 80–120 ng/ml, and3–8 ng/ml, respectively. In total, 18 recipients were on calci-neurin- or mTOR inhibitor–based triple immunosuppressivetherapy, and two on dual therapy without steroids. The singlepatient on everolimus achieved levels within the target rangethroughout the study period (not shown). In one patient offsteroids, prednisolone was initiated on trial inclusion. Dosagesof steroids were not adjusted during the trial (prednisolone, 16patients: 5 mg/d, n515; 2.5 mg/day, n51; methylprednisolone,four patients: 4mg/d). The followingmedications were prohibi-ted and not applied during the study: rituximab, eculizumab,
22 Donor-specific antibody-positive kidney transplantrecipients were assessed for eligibility
20 Were eligible for inclusion and underwent randomization(Medical University of Vienna: n=16; Charité Berlin: n=4)
2 Were not eligible1 Had immune complex glomerulonephritis1 Had a negative molecular ABMR pattern
Par
t AP
art B
1 Was withdrawn(serious adverse event)
10 Were assigned to receiveplacebo, subcutaneously(day 0, week 4, week 8)
20 Underwent a follow-up biopsy at week 11
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)
18 Completed the trial and had a follow-up biopsy at week 51
Safety was evaluated in 19 patientsEfficacy endpoints were evaluated in 18 patients
9 Received less than10 doses in part B8 Had adverse events
1 Had personal reasons
10 Were assigned to receiveclazakizumab, subcutaneously(25 mg, day 0, week 4, week 8)
proteasome inhibitors, intravenous Ig, plasma exchange, immu-noadsorption, and other investigational drugs or treatments in-cluding commercially available anti–IL-6/IL-6R mAbs.
EndpointsAs effect sizes were unknown, the design of this pilot study—the first randomized trial evaluating clazakizumab in patientswho had undergone a transplant—did not include sample sizeestimations. Trial visits were conducted at day 0 and weeks 1,2, 3, 4, 8, 11, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 51, and 52.The primary endpoint was the assessment of safety and toler-ability. Key secondary endpoints were the evolution of mor-phologic andmolecular rejection patterns (index biopsy, week11, andweek 51 follow-up biopsies); DSA and non-DSA levels,IgG, IgM, and IgG subclass concentrations and Torque Tenovirus (TTV) load (day 0, week 12, and week 52); the pharma-codynamics of clazakizumab (reduction of C-reactive protein,CRP); and the course of eGFR (CKD Epidemiology Collabo-ration), and protein/creatinine ratio (all visits).
SafetyAdverse events were closely monitored throughout the studyand classified using the Medical Dictionary for RegulatoryActivities Version 23.0. The study was monitored by an inde-pendent data and safety monitoring board (DSMB), and in-cluded two interim analyses, the first after 10 and the secondafter 20 subjects had completed part A of the study. The DSMBwas instructed to consider stopping the trial if the pattern ofrelated serious adverse events or safety laboratory resultsstrongly supported a major safety signal. Exact statistical def-initions of criteria for premature study termination were notdefined. Stopping rules were defined as follows. Part A: if sixsubjects experience related (definitely and possibly) seriousadverse events (common toxicity criteria . Grade 3 or se-vere/medically significant), and/or substantially elevated levelsof liver parameters (alanine aminotransferase, aspartate ami-notransferase, and/or bilirubin .33 upper limit of normal)or neutropenia (below 0.5 G/L), the DSMBwould unblind thesafety results. If five or all subjects were to be in the clazaki-zumab group, then the studywould be stopped. Similarly, if sixrelated serious adverse events and/or substantial abnormali-ties in liver enzymes or neutrophil counts occurred in the samesystem organ class, the DSMB would unblind these seriousadverse events, and if five or all serious adverse events werein the clazakizumab group, then the study would be stopped.Part B: if ten or more subjects experienced drug-related seri-ous adverse events or ten or more related serious adverseevents occurred in the same system organ class, then the trialwould be stopped (the total number of related serious adverseevents should also include the related events occurring in theclazakizumab-treated subjects during part A).
Antibody DetectionFor HLA antibody detection, LABscreen single-antigen flow-bead assays (One Lambda, Canoga Park, CA) were applied.
Serum samples were incubated with EDTA (10 mM) to pre-vent complement interference. Data acquisition wasperformed via a LABScan 200 flow analyzer (Luminex Corpo-ration, Austin, TX). For longitudinal analysis of DSA levels,bead assays were performed retrospectively (centralized anal-ysis) to avoid influences of day-by-day variations in test results(test batches including samples from four to six patients each).Donor specificity was defined according to serological and/orlow- or high-resolution donor/recipient HLA typing (HLA-A,-B, -Cw, -DR, -DQ, -DPon availability) provided either by thelocal HLA lab or the Eurotransplant database. Test resultswere documented as mean fluorescence intensity (MFI) ofthe immunodominant DSA. An MFI threshold .1000 wasconsidered as positive. To estimate the effect of clazakizumabtreatment on DSA levels, we documented the percent changein MFI. In an effort to quantify changes in DSA levels moreaccurately, we additionally performed dilution experimentsafter an earlier described protocol.17 In brief, nonlinear stan-dard curves on the basis of raw DSA MFI levels (immunodo-minant DSA) were obtained by serial dilution of individualpatient sera collected before the start of treatment (all sampleswere incubated with EDTA). According to computed standardcurves, the fold change of antibody levels was then calculatedfrom DSA MFI levels detected in the same experiment forundiluted week 12 and week 52 samples.
Total IgG, IgM, and IgG subclasses were assessed in serumapplying immunonephelometry on a BN II analyzer (SiemensHealthineers, Erlangen, Germany).
Transplant BiopsiesThe study included 20 index, 20 11-week follow-up, and18 51-week follow-up biopsies. None of the patients under-went additional indication biopsies. Biopsies were performedusing ultrasound-guided percutaneous techniques (1–2 coresper biopsy, 16-gauge needle). Histomorphology and C4dstaining was evaluated on formalin-fixed paraffin-embeddedsections. C4d in peritubular capillaries was scored as 0 (nega-tive), 1 (minimal), 2 (focal), and 3 (diffuse), and a score $1was considered positive. In total, 35 of the 58 study biopsieswere additionally evaluated using electron microscopy to de-tect microcirculation injury. Morphologic results were readlocally (Medical University of Vienna, Charité Universitäts-medizin Berlin) in a blinded fashion, following the rules ofthe Banff 2017 scheme. In addition, all biopsies were analyzedusing a thoroughly validatedmolecular method (MMDx).18,19
For each biopsy, a 3 mm portion of one core was immediatelyplaced in RNAlater, stored at 220°C, and shipped either atambient temperature or on dry ice to the Alberta TransplantApplied Genomics Centre (University of Alberta, Edmonton,AB, Canada) for gene array analysis. Molecular scores on thebasis of lesion-based classifiers related to rejection (ABMR,T cell–mediated rejection, all rejection), inflammation (globaldisturbance score), or chronic injury (atrophy/fibrosis score)were generated using a reference set of 1529 biopsies. After the2017 update of the Banff classification,20 ABMR was defined
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www.jasn.org CLINICAL RESEARCH
and categorized on the basis of morphologic, immunohisto-chemical (C4d), ultrastructural (transplant glomerulopathy,multilayering of the peritubular capillary basement mem-branes), serological (DSA detection), and thoroughly vali-dated molecular criteria (molecular ABMR score $0.2),respectively.
TTV QuantificationFor TTV analysis, DNA was extracted from plasma (200 ml)using the NucliSENS easyMAG platform (bioMeriéux,France), and eluted in 50 ml of elution buffer. TTV DNAwas quantitated by TaqMan real time PCR, according to earlierdescribed protocols.21,22 The quantitative PCR reactions wereperformed in a volume of 25 ml using 23 TaqMan UniversalPCRMasterMix, containing 5ml of extracted DNA, 400 nMofeach primer, and 80 nM of the probe. Thermal cycling wasstarted for 3 minutes at 50°C, followed by 10 minutes at 95°C,and then by 45 cycles at 95°C for 15 seconds, at 55°C for 30seconds, and at 72°C for 30 seconds, using the CFX96 Real-time System (Bio-Rad, Hercules, CA). Results were recordedas copies per ml.
Statistical MethodsFor group comparisons we used Fisher’s exact, Mann–Whitney U, or Wilcoxon tests. GFR trajectories were analyzedusing a linear mixed model with eGFR values from 0 to12 weeks (day 0, week 1, 2, 3, 4, 8, 12) and from 12 to 52 weeks(week 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52) as the dependentvariable. Time, treatment, and their interaction were used asfixed effects. Furthermore, patient-specific random effects forintercept and slope were specified. Each patient’s trajectorieswere modeled as a hockey-stick spline with a fixed knot at 12weeks, which is change of therapy for the placebo group. Dueto the unevenly spaced time points, a spatial covariancematrix(power structure) was used. Intergroup differences were testedat a two-sided significance level of 5%. For statistical analysis,IBM SPSS Statistics version 24 (IBM Corporation, Armonk,NY) and SAS version 9.4. (The SAS Institute Inc., Cary, NorthCarolina) were applied.
RESULTS
Patient Disposition and CharacteristicsIn total, 20 kidney transplant recipients (Vienna, n516;Berlin, n54) with DSA-positive ABMR after a median of10.6 years post-transplantation were randomly assigned toclazakizumab or placebo (12-week randomized, placebo-controlled part A). In a subsequent 40-week open-label exten-sion (part B), all participants received clazakizumab (Figure 1).Baseline characteristics are provided in Supplemental Tables 1and 2, and Table 1. Ten (50%) patients were female, seven(35%) were retransplant recipients, and five (25%) had beensubjected to desensitization because of preformed DSA and/orbroad HLA reactivity.23 At study inclusion, 18 (90%)
recipients had anti-HLA class II DSA (with or without HLAclass I DSA), 15 (75%) against HLA-DQ. The immunodomi-nant DSAMFIwas 11,708 (median; interquartile range [IQR]:1947–17,709). Two (10%) recipients had active and 18 (90%)chronic/active ABMR. Median eGFR and protein/creatinine ra-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-ables relating to the severity of rejection, such as eGFR or mor-phologic results obtained in index biopsies were well balancedbetween patient groups. Despite stratified randomization, how-ever, recipients allocated to placebo showed numerically highermedian levels of DSAMFI, CRP levels, and proteinuria, whereasmolecular rejection-related scores were higher in the clazakizu-mab arm (Supplemental Tables 1 and 2, Table 1).
At study inclusion, 18 (90%) recipients were on triple andtwo (10%) were on dual immunosuppressive therapy(Supplemental Table 2). Immunosuppressant trough levelsand doses are shown in Supplemental Figure 2. In total, 11(55%) patients did not receive all scheduled clazakizumabinjections, mostly because of adverse events. These patientswere included in all endpoint analyses. Two (10%) patientswere withdrawn from the study due to diverticular diseasecomplications, one after completion of part A, and one shortlyafter initiation of part B (Figure 1).
SafetyIn part A, overall incidences of adverse events were 50 in theclazakizumab and 44 in the placebo arm (Supplemental Table 3,Table 2). The most frequent events were infections and gastro-intestinal disorders (Supplemental Table 3). Serious adverseevents occurred in four (20%) patients, three of whom receivedclazakizumab (Table 2). One patient on clazakizumab developeddiverticulitis, leading to withdrawal from the trial (stable graftfunction until the end of the trial). Diverticulitis resolved afterpercutaneous abscess drainage and antibiotic therapy.
Part B had 129 adverse events overall, of which nine wereserious. Three (15.8%) patients reported mild injection sitereactions. Serious adverse events included a case of compli-cated diverticulitis with colon perforation requiring surgery.This patient (clazakizumab in part A) was withdrawn from thestudy before the second visit in part B (return to dialysis9 months after trial withdrawal). Other serious events in-cluded pneumonia (n52), pyelonephritis (n51), ovarian ab-scess (n51), Coxsackie virus–associated meningitis (n51),and recurrent pleural effusion requiring pleurodesis, and, sub-sequently, permanent thorax cavity drainage.
After the second case of diverticulitis, the DSMB requesteda careful re-evaluation of all included participants and a re-duced dose of clazakizumab in two active patients diagnosedwith diverticulosis (12.5 mg, last injection in part B). Overall,nine (45%) patients had colon diverticulosis, among themthe two patients who developed diverticulitis. Additionalpredisposing risk factors were polycystic kidney disease anda long history of immunosuppression and steroid exposure(Supplemental Table 4).
Laboratory findings are provided in SupplementalFigure 3 and Table 3. Mild liver parameter elevations werenoted in a few patients; none met Hy’s law. Anemia, mostlygraded I or II, was already prevalent in both arms beforestudy inclusion, with no relevant differences in part Aand stable hemoglobin levels in part B. There were also norelevant changes in leukocyte and platelet counts. Lipid ab-normalities were frequent in both study arms, with peak(unfasted) triglyceride levels higher under clazakizumab(Table 3).
EfficacyTreatment with clazakizumab effectively suppressed CRP lev-els and slightly increased TTV viral load (SupplementalFigure 4).
HLA Antibody and Ig LevelsWithin 12 weeks (part A), clazakizumab decreased DSA MFIto a median of 77% (IQR, 63%–101%) from baseline (pla-cebo: 103% [IQR, 94%–104%]; P50.035). Extension of treat-ment in part B led to a further decrease of DSA levels(P,0.001) (Figure 2A). Similar results were obtained for levelsof DSA interpolated from dilution experiments, nondonor-specific HLA reactivity (Figure 2A), total IgG (but not IgM),and, among IgG subclasses, most prominently IgG4(Figure 3).
Evolution of RejectionThe 11-week biopsies failed to demonstrate significant inter-group differences in rejection-related molecular and morpho-logic scores (Figure 2, B and C), and there were nomeaningful
Table 1. Demographics and baseline characteristics
Parameter Total (n520) Clazakizumab (n510) Placebo (n510)
Variables recorded at transplantationFemale 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 inclusionAge 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 characteristicsHLA 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 categoriesABMR, n (%) 20 (100) 10 (100) 10 (100)Active ABMR, n (%) 2 (10) 2 (20) 0Chronic/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 loadCRP (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 DSAdatawere available for seven recipients in the clazakizumab arm and four in the placebo arm (solid-phase HLA antibody screening on thewaitlistwas implemented at the Vienna transplant unit in July 2009).23cDonor 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.
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changes in ABMR phenotypes (Figure 2D). In contrast, the51-week biopsies performed after prolonged clazakizumabtreatment in part B demonstrated a significant decrease inmolecular ABMR (P50.020) and “all rejection” scores(P50.037), whereas T cell–mediated rejection scores re-mained negative at all time points (Figure 2B). After 51 weeks,seven of 18 patients (38.9%) had a negative ABMR score(,0.2) (not shown). Although microcirculation inflamma-tion did not change significantly (Figure 2C), we found a res-olution of ABMR activity in four (22.2%) and disappearanceof capillary C4d deposits in five (27.8%) patients (Figure 2D).Supplemental Table 5 details serial biopsy results obtained inthe four patients who showed transition of active ABMR to aninactive phenotype (cg in the absence of evidence of current/recent antibody interaction with the endothelium). At base-line, three of these recipients were C4d negative, with ratherlow levels ofmicrocirculation inflammation (g1ptc sum score#3). On treatment, g1ptc scores decreased to ,2, paralleledby a marked reduction in molecular ABMR scores(Supplemental Table 5).
Levels of interstitial fibrosis and tubular atrophy increasedsignificantly from week 11 to week 51, but transplant glomer-ulopathy or a molecular classifier reflecting atrophy/fibrosisremained unchanged (Supplemental Figure 5).
Clinical OutcomesIn part A, the mean slope of eGFR differed significantly be-tween clazakizumab and placebo (20.96; 95% confidence in-terval [95% CI], 21.96 to 0.03 versus 22.43; 95% CI, 23.40to21.46 ml/min per 1.73 m2 per month, P50.04) (Figure 4).In part B, patients switched from placebo to clazakizumab
showed significant improvement in the eGFR slope comparedwith the slope calculated for part A (P,0.001), and differencesto patients initially allocated to clazakizumab (no signifi-cant change on extension of treatment in part B) became NS(20.29, 95% CI,20.85 to 0.26 versus 20.64, 95% CI,21.13to20.14 ml/min per 1.73 m2 per month, P50.37) (Figure 3).Similar results were obtained in a separate analysis, includingthe two patients who were prematurely withdrawn from thetrial (Supplemental Figure 6). Levels of proteinuria did notchange over time (Supplemental Figure 7).
As shown in Supplemental Figure 8, on 6months follow-upafter the end of the trial, we observed a slight increase in CRPlevels to ranges detected before trial initiation. None of thepatients developed acute graft dysfunction (or underwent in-dication biopsies), and as illustrated in Supplemental Figure 8,there was no major change in the mean slope of eGFR. Onepatient returned to dialysis 3 months after the last visit.
DISCUSSION
This phase 2 trial evaluated the safety (primary endpoint) andefficacy (secondary endpoint analysis) of anti–IL-6 antibodyclazakizumab in late ABMR. Major safety signals were theoccurrence of serious infectious events and diverticular dis-ease complications. Key results of secondary endpoint analysiswere an early decrease in DSA levels, a slowed eGFR decline,and, after extended treatment, modulation of rejection-associated gene expression patterns, reduction of C4d scores,and, in some patients, resolution of ABMR activity.
Table 2. Serious treatment emergent adverse events by system organ class
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).
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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Figure 2. Antibody and biopsy results. Percentages (in relation to baseline values) of (A) peak DSA MFI, DSA levels interpolated fromdilution experiments, and the median MFI detected for non-DSA, (B) rejection-associated molecular classifiers (ABMR, T cell–mediatedrejection [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 itsmarked upregulation on rejection,24,25 with HLA class IIDSA being a potent trigger of endothelial IL-6 secretion.26
IL-6 is known to contribute to plasma cell formation, whichmay promote DSA formation.8 In line with an observationalstudy evaluating tocilizumab,12,13 clazakizumab led to an earlydecrease in DSA, non-DSA, and total IgG levels. We found apronounced reduction of IgG4, an IgG subclass previouslypostulated to play a critical role in chronic rejection.27 Inter-preting our DSA results, we want to point out potential caveatsassociated with using MFI as a measure of antibody levels.28 Itis well established that MFI levels detected in bead arrays donot simply reflect antibody concentrations in serum, but alsodepend on antibody avidity/affinity and the density or confor-mation of HLA antigens coated to individual microbeads. Re-porting numerical changes in MFI may be problematic andnot accurately reflect quantitative changes in antibody levels,especially in the case of bead saturation. In this respect, titerstudies may be more informative. In an attempt to indirectlyquantify changes in antibody levels, we applied dilution ex-periments creating standard curves for baseline sera. Anotherpoint is the earlier reported spontaneous fluctuation of DSAlevels (independent of specific therapeutic interventions),29
which has to be taken into account when interpreting antibodychanges in the open-label part of our trial.
Early follow-up biopsies did not show any effect of claza-kizumab on morphologic and molecular results, despite anearly decline in CRP and DSA levels. After prolonged treat-ment in part B (biopsies after 51 weeks), however, biopsy re-sults suggested amelioration of rejection, at least in some ofthe included patients. These findings may relate to the ob-served decrease in DSA levels, but one may also argue thatother factors have potentially influenced ABMR activity,such as the load of maintenance immunosuppression. Therewere, however, nomeaningful adjustments in the compositionand dose of immunosuppression, which, together with theoutcome differences observed in placebo-controlled part A,supports a specific therapeutic effect of IL-6 signaling block-ade. Our failure to detect any improvement in atrophy/fibrosisand transplant glomerulopathy (and proteinuria) was expec-ted, as advanced chronic injury may not be reversible. Inter-preting morphologic biopsy results, however, we are aware ofthe inherent limitation of poor reproducibility of Banff his-tology scores, which may support the use of more objectiveanalysis of gene expression patterns, especially in patients withambiguous histology.30
The slope of eGFR is a useful surrogate predicting graftsurvival, and in late ABMR, a 30% deterioration in eGFR slopewas reported to be associated with a 10% increase in graft lossrates.31 We found a significant intergroup slope difference
already in part A of the trial, and a slowed progression of renaldysfunction in part B. Our results are promising and in con-trast to previous trials conducted in late ABMR, where othertreatments, such as bortezomib7 or rituximab plus intrave-nous Ig,6 failed to slow the progression of renal dysfunction.Nevertheless, the outcome data in our trial, which was notprimarily powered to detect differences in graft function,have to be interpreted with caution. Calculated eGFR slopes,both in the placebo and clazakizumab arms, were within arange earlier reported for a cohort of 91 recipients with lateABMR (most of them treated with intravenous Ig or ste-roids),31 and it remains unclear whether the slope differencesin our trial would result in differences in graft survival. In thiscontext, we want to mention a recently initiated large multi-center, randomized, placebo-controlled phase 3 trial evaluat-ing clazakizumab in chronic ABMR (IL 6 Blockade ModifyingAntibody-Mediated Graft Injury and eGFR Decline trial;ClinicalTrials.gov identifier: NCT03744910). The design ofthis trial, which aims to recruit 350 patients to detect differ-ences in 5-year graft survival, will include an early evaluationof eGFR slope differences as an interim surrogate endpoint(after approximately 200 subjects have received at least 1 yearof treatment).
Our trial did not include follow-up biopsies to investigate apotential role of rebound phenomena after stopping clazaki-zumab treatment. Nevertheless, a 6-month follow-up after thelast visit revealed only a slight increase in CRP levels to valuesdetected before trial initiation, and none of the patients de-veloped acute graft dysfunction. Together with our finding ofno major change in eGFR slope after the last visit, this mayargue against major clinically relevant rebound phenomena.
In line with earlier studies,10,15 clazakizumab was associ-ated with mild injection site reactions, increases in lipid levels,and mild abnormalities of liver enzymes or blood cell count.When combining clazakizumab with baseline immunosup-pression, aggravated infectious complications may become asignificant concern. Under prolonged treatment in part B,five serious infectious events were recorded. In parallel, weobserved a significant reduction in serum IgG and a slight in-crease in TTV load as a surrogate marker of intensified immu-nosuppression.32 In trials performed in rheumatoid arthritis,IL-6 interference was reported to be associated with increasesin serious infection incidence,9,10,33,34 although in a compre-hensive analysis of available trials and their extension phases,intergroup differences were NS.35 The most important safetysignal was the occurrence of diverticulitis in two patients withpre-existing diverticulosis, one event resulting in colon perfo-ration. The risk of gastrointestinal perforation is known to besignificant under IL-6 blockade.11,36 In a large US real-worlddataset 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, andpaired 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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Figure 3. Serum Ig concentrations. Shown are the percent levels (in relation to values at baseline) of (A) IgG, IgM, and (B) IgG sub-classes 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 pairedWilcoxon test to evaluate changes under clazakizumab (overall cohort) in the open-label extension (part B).
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associated with a lower gastrointestinal perforation rate of 1.26per 1000 patient-years.37 A study by Choi et al.12 showed zeroof 36 patients receiving tocilizumab for ABMR experiencingdiverticular disease complications; one case of colon perfora-tion, however, was recorded in a small trial of tocilizumab forrecipient desensitization.38 Given that none of the trials evalu-ating clazakizumab in rheumatologic disease (.500 treatedpatients) reported any patients with gastrointestinal perfora-tion (as in our study, patients with a history of diverticulitis orgastrointestinal perforation were not included),10,15 one mayassume that additional immunosuppression, in particularlong-term steroid use, and the background of certain predis-posing conditions, including diverticulosis and polycystic kid-ney disease, have increased the risk significantly.39 Our datasuggest the need for careful evaluation of transplant patientsconsidered for IL-6 signaling blockade, for example, in thecontext of future trials. This should include a thorough
evaluation of the patient’s history, including documented colondiverticulosis and, at least for patients deemed to be at partic-ular risk, such as those with polycystic kidney disease, use ofdiagnostic tests (e.g., colonoscopy or computer tomography)to rule out significant colon diverticulosis. Of note, the resultsof our safety analysis have significantly influenced the design ofan ongoing pivotal phase 3 trial (IL 6 Blockade ModifyingAntibody-Mediated Graft Injury and eGFR Decline;ClinicalTrials.gov identifier: NCT03744910), including a re-duction in monthly clazakizumab doses (from 25 to 12.5 mgper month) and a strict definition of exclusion criteria (historyof gastrointestinal perforation; diverticular disease or divertic-ulitis, except if disease has been fully excised; or inflammatorybowel disease).
A major limitation of our trial is its small sample size. Astrength, however, may be the granularity of analyzed end-points, including a detailed work-up of three sequential
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Figure 4. Kidney allograft function. The individual course of eGFR is shown for patients allocated to receive clazakizumab (A) versusplacebo (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; shadedareas represent 95% CIs) in relation to treatment in part A (clazakizumab: red lines; placebo: black lines).
JASN 32: 708–722, 2021 Clazakizumab in Late Rejection 719
biopsies. Although the trial was not primarily powered to de-tect efficacy outcome differences, the double-blind phase ofthe trial revealed significant differences in DSA levels andprogression of graft dysfunction, and observed changes in sec-ondary outcomes were pronounced upon treatment in theopen-label extension. One may argue that intergroup differ-ences in the course of renal function in part A could partlyrelate to a bias by chance due to imbalances in baselinevariables associated with the severity of rejection or immuno-suppressive therapy (despite stratified randomization), inparticular, higher numerical levels of DSAMFI, CRP, and pro-teinuria and a lower dose of enteric-coated mycophenolic acidin the placebo arm. In contrast, however, baseline eGFR andmorphologic features of rejection and/or chronic injury weresimilar between groups, and molecular rejection-relatedscores were even higher in the clazakizumab arm.
In conclusion, this phase 2 trial suggests the efficacy ofclazakizumab in late ABMR after kidney transplantation.Recorded safety outcomes—the primary trial endpoint—warrant careful patient selection and monitoring to minimizethe risk of diverticular disease complications and serious in-fections. The results of secondary endpoint analysis, includingfirst systematic data on the size of expected outcome effects,may provide a valuable basis for larger phase 3 trials withlonger duration of follow-up to systematically evaluate theconcept of IL-6/IL-6R blockade in transplantation (in com-parison to placebo and/or other treatment concepts).
DISCLOSURES
B. Jilma reports having consultancy agreements with Guardian Therapeu-tics; and Speakers Bureau from Sanofi. E. Chong is employed by and hasownership interest in Vitaeris Inc., Vancouver, Canada. G. Böhmig andK. Budde are members of the steering committee for an ongoing pivotal phase3 trial evaluating clazakizumab in chronic active ABMR (ClinicalTrials.govnumber, NCT03744910; sponsored by Vitaeris Inc.). G. Böhmig reports con-sultancy agreements from Vitaeris Inc., Vancouver, Canada; research fundingfrom Vitaeris Inc., Vancouver, Canada; honoraria from Astellas, FreseniusMedical Care, OneLambda, Sandoz; and being a scientific advisor or member-ship of Vitaeris Inc, Vancouver, Canada. G. Bond reports receiving researchfunding from Chiesi. H. Regele reports receiving honoraria from Roche,Menarini, and other interests/relationships with Gerson Lehrman Group.K. Budde reports consultancy agreements with Abbvie, Alexion, Astellas,Bristol-Myers Squibb, Chiesi, CSL-Behring, Fresenius, Hansa, Hexal, Novar-tis, MSD, Otsuka, Pfizer, Quark, Roche, Sandoz, Shire, Veloxis, Vifor, andVitaeris; reports receiving research funding from Abbvie, Alexion, Astellas,Bristol-Myers Squibb, Chiesi, CSL-Behring, Fresenius, Hansa, Hexal, Novar-tis, MSD, Otsuka, Pfizer, Quark, Roche, Sandoz, Shire, Veloxis, Vifor, andVitaeris; receiving honoraria from Abbvie, Alexion, Astellas, Bristol-MyersSquibb, Chiesi, Fresenius, Hexal, Novartis, Otsuka, Pfizer, Quark, Roche, San-doz, Shire, and Veloxis Pharma; being a scientific advisor or membership ofAstellas, Bristol-Myers Squibb, Chiesi, Hansa, Hexal, MSD, Novartis, Pfizer,Roche, and Veloxis. M. Duerr reports receiving research funding from Bristol-Myers Squibb; honoraria from Shire, Novartis; being a scientific advisor ormember of Novartis. N. Kozakowski reports receiving research funding fromthe Austrian Science Fund—Erwin Schrödinger Fellowship J-4377; being ascientific advisor or member as Associate Editor—BMC Nephrology; otherinterests/relationships with 2012–2020 Austrian, German, French, and
European Societies for Pathology, 2018–2020 French Speaking Club of RenalPathology, 2012–2020 Austrian, French, and European Societies for Trans-plantation, 2018–2020 Member then Co-chair of the Banff Working groupfor Thrombotic Microangiopathy, and 2020 Co-chair of the Banff Workinggroup for Peritubular Capillaritis. N. Lachmann reports consultancy agree-ments with BmT GmbH. P. Halloran reports consultancy agreements withAstellas, CSL Behring; ownership interest in Transcriptome Sciences Inc., aUniversity of Alberta research company with an interest in molecular diag-nostics, has given lectures for Thermo Fisher, and is a consultant for CSLBehring; reports receiving research funding from Transcriptome SciencesInc.; reports receiving honoraria from Astellas, One Lambda; is a scientificadvisor or member of CEO, Transcriptome Sciences Inc., Editor-in-Chiefemeritus, American Journal of Transplantation; Speakers Bureau for Astellasand One Lambda. All remaining authors have nothing to disclose.
FUNDING
The trial was funded by an investigator-initiated unrestricted grant fromVitaeris Inc., Vancouver, Canada (to G. Böhmig and B. Jilma).
ACKNOWLEDGMENTS
The authors wish to thank Prof. Josef Smolen for his scientific advice, andSusanne Haindl, Sarah Ely, Dr. Christa Drucker, Rene Nadolny, and SilkeKasbohm for excellent assistance.
SUPPLEMENTAL MATERIAL
This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2020071106/-/DCSupplemental.
Supplemental Table 1. Single lesions and molecular classifiers at baseline.Supplemental Table 2. Baseline immunosuppression.Supplemental Table 3. Adverse events by system organ class.Supplemental Table 4. Diverticular disease and complications.Supplemental Table 5. Resolution of ABMR activity in four recipients: serial
biopsy results.Supplemental Figure 1. Summary of trial protocol.Supplemental Figure 2. Immunosuppression levels and dosage.Supplemental Figure 3. Safety lab.Supplemental Figure 4. CRP and Torque Teno virus (TTV) levels.Supplemental Figure 5. Morphologic and molecular features of chronic
injury.Supplemental Figure 6. Analysis of renal function including two patients
withdrawn from the trial.Supplemental Figure 7. Spot urine protein/creatinine ratio.Supplemental Figure 8. CRP and renal function after the end of the trial.
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AFFILIATIONS
1Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria2Department of Nephrology, Charité Universitätsmedizin Berlin, Berlin, Germany3Alberta Transplant Applied Genomics Centre, University of Alberta, Edmonton, Alberta, Canada4Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria5Centre for Tumor Medicine, Histocompatibility & Immunogenetics Laboratory, Charité Universitätsmedizin Berlin, Berlin, Germany6Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria7Department of Surgery, Medical University of Vienna, Vienna, Austria8Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria9Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Vienna, Austria10Vitaeris Inc., Vancouver, Canada
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CLINICAL RESEARCH www.jasn.org
1
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
2
Supplementary Table 1. Single lesions and molecular classifiers at baseline.
