RAVE was supported by the Immune Tolerance Network (ITN), which is funded in turn by the National Institute of Allergy and Infectious Disease (NIAID). RTX, matching placebo, and partial funding were provided by Genentech, Inc. (South San Francisco, CA) and Biogen IDEC, Inc. (Cambridge, MA).

The RAVE Research Group comprised eight clinical centers^*, a Coordinating Center [Pharmaceutical Products Development, Inc. (PPDI) until April 2009, then Rho,Inc.], a drug distribution center (Eminent Services, Inc., Frederick, MD), and the Tolerance Assay and Data Analysis Group of the ITN (See Mechanistic Studies, below). The list of personnel at the trial sites and all branches of the RAVE Research Group are shown in the Appendix.

Each RAVE clinical site included a primary patient assessor (termed the “Investigator”), a Safety Officer(s), a Trial Coordinator(s), and a laboratory technician. An individual assigned to be the Investigator or the Safety Officer for one patient could fulfill the other role for other patients.

RAVE compared a new treatment (RTX) to a conventional standard of care (CYC), which was first employed in AAV in 1954 [27]. The hypothesis of the trial was that the combination of RTX plus glucocorticoids was not inferior to conventional therapy in its ability to induce disease remission and permit glucocorticoid discontinuation by 6 months. Critical RAVE Trial definitions are shown in Table 1.

Table 1

Critical RAVE Trial Definitions

Two of the required entry criteria for RAVE were: 1) a clinical diagnosis of GPA or MPA that fulfilled the Chapel Hill Consensus Conference definitions of GPA or MPA [1]; and, 2) presence of PR3-ANCA or MPO-ANCA at trial entry.

The eligibility criteria for the trial are summarized in Table 2. All patients enrolled had active disease within 28 days before enrollment, defined by a Birmingham Vasculitis Activity Score for WG (BVAS/WG) of > 3 [28] (see Appendix). Patients who were starting their first courses of treatment for AAV or those suffering a flare of previously quiescent disease were eligible. All patients had severe disease at screening, meaning that outside the trial they would have been treated with CYC and high-dose glucocorticoids.

Table 2

Major Inclusion and Exclusion Criteria for Enrollment in RAVE

Patients treated with certain immunosuppressive or biologic agents within specific periods of time before trial entry were ineligible. The reasons for this were two-fold: to prevent confounding of the primary outcome by pre-trial therapy; and to permit certain mechanistic studies on baseline samples (Table 2).

Treatment assignments were generated in permuted blocks. The randomization schedule, generated, written, and controlled by the Coordinating Center, was yielded an assignment ratio of 1:1 between the two treatment groups and within each stratum. The randomization was stratified by both ANCA subtype (PR3- or MPO-ANCA) and clinical site.

Data collected at the baseline visit are shown in Table 3. At each trial visit, serum, plasma, and peripheral blood mononuclear cell samples were collected. DNA samples were collected at the 2 month visit only (Table 3).

Table 3

Schedule of Assessments for Original Treatment Assignment

There were three treatment phases (Fig.1of appendix):

Disease response, assessed formally at month 1, was defined as: 1) no new disease manifestation; 2) no worsening of existing disease; and, 3) reduction of at least 1 in the BVAS/WG compared to the score at screening (See Assessment of DiseaseActivity, below). Early treatment failures were defined as failure to achieve a disease response by month 1. Because this was a blinded trial of a new, unproven therapy compared to an entrenched standard of care regimen that is widely regarded as being effective, the investigators believed it important to ensure that patients were responding to their assigned treatment one month into therapy.

Clinical events that lead to categorization of subjects having experienced a treatment failure (early or otherwise) are shown in Table 4.

Table 4

Clinical Events Categorized as Treatment Failures

Subjects who experienced severe disease flares between the month 1 and month 6 study visits were crossed over to the opposite treatment arm. This feature was deemed essential to providing equipoise and maintenance of the double-blind design. Crossover patients were considered treatment failures.

The treatment of disease recurrences was dictated by flare severity and the study phase in which it occurred. An algorithm for the treatment of disease flares is shown in Fig. (2) (Appendix).

The types and frequency of data collected for the purpose of analyzing trial outcomes are shown in Table 3.

We designated primary, secondary, and tertiary outcome measures (Table 5).

Table 5

Rave Outcome Measures

The narrow mode of action of RTX provided unique opportunities for mechanistic studies that address the central hypothesis related to tolerance; namely, that tolerance to the self-antigens PR3 or MPO can be restored by anti-CD20 therapy because of the elimination of PR3- or MPO-specific memory B cells.

2.19.1. Sample Collection

Whole blood flow cytometry was performed centrally at the time of each visit to evaluate the development, frequencies, and activation status of B and T cells, natural killer cells, and myeloid and dendritic cells. The flow cytometry panel is shown in Table 6.

Table 6

Flow-Cytometry Antibody Panels

Peripheral blood mononuclear cells (PBMC) were prepared at each visit. We plan to perform PBMC-Elispot assays to determine the proliferative responses as well as antibody and cytokine production of B and T lymphocytes upon antigen stimulation.

Serum and plasma samples were also collected at each visit for batch measurements of ANCA, B cell lymphocyte stimulator (BLyS/BAFF), other cytokines, and future ancillary studies. Both high and low molecular weight DNA was harvested from peripheral blood for studies designed to understand genetic predictors of clinical outcomes. Whole-blood RNA was stored for investigations of gene expression profiling and the identification of associations with treatment response, specific drug resistance and toxicities, and disease relapses.

The initial concept proposal for RAVE was submitted to the ITN in November, 2002. Funding was granted in May, 2003. Protocol development was completed in the fall of 2004. The first patient was enrolled on December 28, 2004. The 197th patient was enrolled at the end of June, 2008. The decision was made to stop enrollment at that time because the observed drop-out rate was lower than anticipated.

The effects of both CYC and RTX posed potential for unblinding. To conceal the diffuse alopecia that is often associated with CYC use from the investigators who assessed clinical outcomes, all patients wore hats to their study visits for 6 months following the discontinuation of CYC or CYC placebo. Laboratory results such as leukopenia or macrocytosis could also have led to unblinding. Thus, each subject was evaluated by two clinicians, a Safety Officer and an Investigator. The Investigator, who performed the BVAS/WG assessment, viewed only redacted reports of laboratory results, with white blood cell count, lymphocyte count, and mean corpuscular volume values blacked out by the site Trial Coordinator. Mild infusion reactions attributable to RTX occur in approximately 10% of patients [Rituximab Investigator’s Brochure] and infusion reactions can also occur among placebo-treated patients. Thus, the occurrence of an infusion reaction did not necessarily unblind the patient’s treatment assignment. To prevent unblinding of the Investigator, the Safety Officer evaluated and treated all patients with possible infusion reactions. Moreover, subjects were asked to report potentially CYC- or AZA-related adverse events such gastrointestinal symptoms only to the Safety Officer.

AZA was used in lieu of CYC in the remission maintenance phase of the trial. Subjects’ ability to metabolize AZA is determined by the presence or absence of alleles for thiopurine methyltransferase (TPMT) [36]. Because the time required to receive the results of TPMT testing would have constituted an unacceptable delay before initiating trial therapy, TPMT alleles were tested at entry but randomization proceeded before the results became available. Patients heterozygous for TPMT continued in the protocol but at a reduced dose of AZA/AZA placebo. Patients who were homozygous negative for the TPMT allele were unblinded at 6 months and treated thereafter according to best medical judgment.

The six-month primary outcome data have been published [37]. The two treatment groups were equivalent at baseline with regard to baseline disease activity, organ involvement, and the proportion of patients with new disease at entry. Sixty-three (64%) of patients assigned to RTX achieved the primary outcome as compared with 52 (53%) in the CYC arm, meeting the criterion for non-inferiority (p<0.001). The superiority comparison for RTX versus CYC fell short of statistical significance (p = 0.089), but RTX was more efficacious than CYC for remission induction among the subset of patients with relapsing disease at trial entry: 34/51 (66.7%) patients allocated to RTX achieved this measure, compared to 21/50 (42.0%) randomized to CYC (p=0.01). This finding persisted even after controlling for differences in ANCA type and clinic site (odds ratio 1.40; 95% confidence interval [RCI] 1.03, 1.91; p=0.03). RTX was also as effective as CYC for treating subsets of patients regarded as having the most severe disease; namely, patients with either major renal disease or alveolar hemorrhage. There were no differences between treatment groups in adverse event (AE) rates, serious AE rates, or non-disease related AEs at 6 months [37].

Analyses of data pertaining to treatment efficacy and adverse effects beyond six months, extending to 18 months of follow-up and beyond, are ongoing. Mechanistic studies on samples up to 18 months and beyond are also under way.

Despite the fact that CYC has been established in the late 1960s as the standard of care for remission induction in AAV, strong equipoise existed for RAVE. The conventional regimen of CYC and high-dose glucocorticoids has well-recognized toxicities and is ultimately unsuccessful in maintaining remission for the majority of patients [10]. Preliminary data on the use of RTX in AAV were promising [22-24], and the early studies of RTX used strict definitions of remission with a validated instrument [28]. Further uncontrolled studies were felt unlikely to augment the knowledge base related to the safety of rituximab, and such studies are not reliable tests of efficacy. A randomized, double-blind trial was the only way to achieve the dual goals of testing RTX rigorously and determining the impact of this therapy on tolerance induction.

Because of concern among the Research Group related to treating severe AAV without CYC, we built into the protocol several provisions designed to protect patients in the event that the hypothesized efficacy of RTX proved unfounded.

• Frequent follow-up after randomization. This feature was inherent in the design of RAVE, because patients had to be evaluated clinically for at least four consecutive weeks to receive their treatment infusions. In addition, patients were evaluated for a strictly-defined disease response by the 1 month visit in order to continue on their assigned therapy (See Disease Response, Section 2.11).
• Careful definition of early treatment failures (Table 1). The DSMB was instructed to recommend stopping the trial if an unexpected number of such events occurred among the first patients enrolled.
• Treatment with glucocorticoids. Patients in both arms were treated with pulse methylprednisolone. High doses of glucocorticoids were expected to temper the manifestations of severe AAV. Thus, the glucocorticoid regimen provided some effective therapy during the period when the CYC and RTX are only beginning to exert their effects.
• Availability of crossover. Blinded crossover was available for patients who experienced severe disease flares during the remission induction phase.
• Physician judgment. Trial physicians were allowed to discontinue patients from the trial at any time if they believed that treatment on the basis of best medical judgment was in the patients’ best interest.
• Exclusion of patients with AAV with the most severe involvement of the kidneys or lungs. Patients with serum creatinine concentrations > 4.0 mg/dL or on mechanical ventilation were not eligible for RAVE.

The decision to treat severe disease flares in the remission maintenance phase with open-label RTX deserves comment. If a patient (randomized to either RTX or CYC) had achieved disease control and completed the first 6 months of the trial, then the therapy to which the patient had been randomized was successful in inducing remission.

Assuming then that the patient had been randomized originally to CYC, the readministration of CYC to induce remission would have subjected the patient to a significantly increased risk of treatment-associated morbidity. The side effects of CYC relate strongly to the dose and duration of therapy [11,16,38]. Given the equipoise of this trial, an attempt to induce remission with RTX in that setting was appropriate. On the other hand, if the patient had been randomized originally to RTX, a second administration of RTX was reasonable because of the indications from clinical studies in other diseases that the side effect profile of RTX might be superior. Moreover, information to date from the use of RTX in non-Hodgkin’s lymphoma and rheumatoid arthritis indicates that the administration of a second course of RTX is as safe as the first course and not associated with loss of efficacy [39-42].

The funding source for the trial had an important impact on the design of RAVE. The overarching aim of the ITN is to develop therapies for immunologically-mediated disorders, inflammatory diseases, and organ transplantation that restore tolerance. This ITN focus had two implications for the design of RAVE.

First, although remission induction and successful glucocorticoid taper by 6 months were designated as the primary outcome, secondary outcome measures related to immune tolerance necessitated follow-up for a period significantly longer than 6 months. The minimum follow-up of 18 months in this trial offered the opportunity to understand the effects of RTX administration in a variety of clinical scenarios, e.g., after a previous course of RTX or following the first induction of remission with a regimen involving CYC.

Second, the desire to understand the impact of RTX on immune tolerance meant that second courses of this medication could not be built routinely into the protocol. Indeed, with regard to AAV, the optimal timing of RTX readministration, if warranted, was not clear at the time the trial was designed and remains unclear at the time of this writing.

Genentech staff had a consultative role in study design and participated in a pre-trial meeting with the FDA related to the potential labeling of RTX for remission induction in AAV. Genentech representatives also approved the trial protocol, participated in regular conference calls of the Study Management Team, and provided safety updates on RTX studies in other immunologic indications such as rheumatoid arthritis. However, Genentech/Biogen IDEC staff had no direct input into the procedures for data collection or the analysis of trial data, and no decision-making rights regarding what data to publish or when to publish it.

The RAVE trial marked a major challenge to CYC as the cornerstone of remission induction therapy in severe AAV. No head-to-head comparison of any agent against CYC for patients severe AAV had ever been attempted before the RAVE trial. The primary outcome results of the trial demonstrated that treatment with RTX and glucocorticoids is not inferior to the standard regimen in patients with new-onset severe AAV. The trial also showed that RTX and glucocorticoids are superior to the standard regimen of CYC and glucocorticoids for remission induction in severe relapsing AAV.

The RAVE results reported to date focus exclusively on remission induction but do not address the question of retreatment. RTX was not re-administered following the return of peripheral blood B-cells (anticipated by 9-12 months). Longer follow-up in the present trial is important to understand the benefits and risks of RTX therapy for AAV. Analyses of patients followed for a minimum of 18 months will explore issues such as the need for re-treatment, particularly in the context of peripheral B-cell reconstitution.

The acceptance of CYC as the standard of care for remission induction in severe AAV for several decades is based on results from large uncontrolled cohort studies and expert opinion. However, no medication (including CYC) has ever been approved by the FDA for the treatment of AAV. This fact posed a hurdle in the design of this non-inferiority trial and is a problem encountered commonly by investigators who intend to design trials that challenge the “standard of care” in any condition when no such standard is recognized in the form of an FDA-approved regimen.

Because of the lack of data from randomized, controlled trials on the effect of CYC in AAV, the FDA required us to demonstrate not only the relevance of proving non-inferiority to CYC, but also superiority to the expected rate of primary outcome achievement in untreated patients. This point was more difficult to demonstrate, because of the paucity of data on the natural history of untreated disease in the modern era. However, case series published in 1958 [5] and 1967 [43] were instrumental in showing that the lower limit of the confidence interval proposed for our primary outcome measure – set at 50% of the patients in the RTX group achieving a BVAS/WG of zero at 6 months (off glucocorticoids) – would far exceed the anticipated remission rate in patients with severe AAV treated with placebo.

Key elements of our discussions with FDA about the selection of the appropriate non-inferiority margin, primary outcome selection, and demonstration of efficacy of a novel therapy in this rare disease have been published in a Draft Non-inferiority Guidance Document for Industry. (http:// www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM202140.pdfhttp://www.fda. gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM202140.pdf).

Important discussion points in the development phase of RAVE were the orphan disease status of AAV and the practical implications of this status for sample size. The FDA Guidance Document indicates that flexibility in the design of non-inferiority trials may be given by the FDA when: 1) the difference between the active control response and the response of untreated patients is large; 2) the primary endpoint does not involve an irreversible outcome; 3) the experimental treatment is associated with fewer serious adverse events than currently available therapies; or, 4) when the experimental medication is in a new pharmacologic class and is shown to be better tolerated than other available treatments. The design of the RAVE trial aligned with all of those elements and thus represents a paradigm for designing a pivotal trial in an orphan disease indication.