The advent of new immunosuppressive agents over the past 15 years has allowed the development of a wide range of different regimens for kidney transplantation, breaking the wide consensus around cyclosporine A (CsA)-based triple therapy (CsA/azathioprine/steroids) that had prevailed from the late 1980s. The combination of induction therapy and the use of tacrolimus-based regimens offer the promise of steroid-free maintenance regimens (1, 2), without the established cost (in terms not only of rejection but also of graft survival) associated with steroid withdrawal from CsA-based regimens (3–5), or the heavy price in terms of calcineurin-inhibitor toxicity associated with aggressively dosed CsA-based regimens (6, 7). The low rejection rates seen in the most successful of the modern regimens suggest that a substantial proportion of recipients are receiving more long-term immunosuppression than they require to maintain good, rejection-free, renal transplant function (7) leading to consideration of regimens combining potent induction therapies with decreased intensity of long-term maintenance therapy.
One of the induction agents considered in this way has been the anti-CD52 monoclonal antibody (MoAb) Campath, first developed in the late 1970s with the specific intention of finding agents with high lytic efficiency against human lymphocytes (8). Campath 1-H (alemtuzumab) is a potent agent, which produces profound depletion in circulating mononuclear cells lasting from 1 to 2 months (for natural killer cells and monocytes) to 6 to 12 months (for circulating B cells and T cells), with some T-cell subsets such as CD4+ cells showing only partial recovery of circulating numbers over greater than 1 year (9). Its widespread adoption as an immunosuppressant agent has been inhibited by the poor patient survival associated with its use in patients with multiply relapsed vasculitis exposed to Campath 1-H during the 1990s (10), and with reports of fatal fungal infections in kidney/pancreas transplant recipients exposed to multiple doses (11). These data strongly suggest that Campath 1-H should not be used in repeated, multiple dosing regimens or in individuals already carrying a heavy burden of immunosuppression. However, an extensive body of reports, both cohort studies, and randomized prospective trials are now available to show that when used at total cumulative doses of between 30 and 60 mg, delivered intravenously (IV) or subcutaneously as induction therapy at the time of renal transplantation, Campath 1-H is a safe and effective therapy (12). A series of ambitious studies demonstrated that Campath 1-H (alemtuzumab), the humanized form of this monoclonal, does not allow allotransplantation without maintenance therapy (13, 14), nor provide sufficient immunosuppressive cover to allow calcineurin inhibitors-free maintenance without high rejection rates (15). Less audacious regimens using Campath however have been reported to produce good medium term results with low rates of rejection, and of opportunistic infection (16, 17). Recent retrospective United Network for Organ Sharing registry analysis suggests that, corrected for known risk factors, induction with alemtuzumab produces significant benefit in terms of graft survival compared with non-lymphocyte-depleting induction, or no induction (18).
Prospective randomized trials have demonstrated equivalent efficacy and safety of regimens using Campath induction without long-term steroids compared with classical CsA-based triple therapy (19), to tacrolimus/mycophenolate mofetil (MMF)/steroid triple therapy without induction (20), to induction with interleukin-2 receptor (IL-2R) MoAb or anti-thymocyte globulin with tacrolimus and MMF used in all three limbs (21), and in a small cohort comparing CsA with sirolimus monotherapy after Campath (22) Two large trials to have compared induction with Campath with anti-thymocyte globulin or basiliximab followed by tacrolimus and MMF in both groups, with or without early steroid withdrawal, and both showed equivalent patient and graft survival with less rejection in the Campath arm (23, 24). The Pittsburgh group has taken the use of Campath further, reporting that the simple combination of alemtuzumab with low-dose tacrolimus maintenance monotherapy without steroids or MMF produces good outcomes particularly in live donor transplantation (25), and this regimen has been shown to be as efficient as tacrolimus-based triple therapy without induction (20).
We believed that this trial was needed to compare the attractive and simple alemtuzumab/tacrolimus monotherapy regimen to a more conventional regimen which combined the anticipated long-term benefits (and attractiveness to patients) of long-term steroid-free maintenance with the low rejection rates associated with the use of induction therapy. Having developed and validated just such a regimen between 2001 and 2004 (following on from regimens developed in Chicago) (26, 27), we undertook this prospective, randomized study to compare Campath and tacrolimus monotherapy with a conventional regimen consisting of daclizumab induction, tacrolimus, and MMF. Neither group received maintenance steroids.
From the initiation of trial recruitment in October 2005, until the completion of recruitment in April 2008, 123 patients underwent randomization (Fig. 1). The two arms were well balanced with no major differences in terms of live versus deceased donor transplant, recipient gender, age, primary renal diagnosis, ethnicity, and first versus subsequent transplant (Table 1). More patients in the daclizumab arm were sensitized against human leukocyte antigens, with sensitization in the context of a previous transplant in two of four patients in the Campath arm and four of eight patients in the daclizumab arm with calculated PRA more than 50%. On single-antigen bead analysis, three of the sensitized patients in each arm had detectable donor-specific antibodies. This did not have a detectable effect on rejection or graft survival because none of the sensitized patients (calculated PRA >50%) in either arm suffered acute rejection during the first 2 years, and only one sensitized patient (in the Campath arm) suffered graft failure (due to progressive fibrosis in a scarred graft which functioned poorly from implantation).
The two arms showed no significant difference at the primary endpoint (survival with a functioning graft, not censored for death with a functioning graft, at 1 year) or at 2 years (Table 2, Fig. 2a). Survival with a functioning graft at 1 year in the alemtuzumab arm was 97.6% and 95.1% in the daclizumab arm (P=0.467 by log-rank test, 95% confidence interval of difference 6.9% to −1.7%). Patient and graft outcomes were excellent in both arms, with overall 97.5% patient survival at 2 years and 93.4% survival with a functioning graft.
Analysis of the primary outcome measure on a per-protocol (which is heavily influenced by rejection-free survival, because rejection episodes are the most important reason for failure of per-protocol persistence) showed no significant difference between the two arms (patient survival with functioning graft at 1 year in alemtuzumab arm patients remaining steroid-free on tacrolimus monotherapy at 1 year was 75.6%, and in the daclizumab arm the comparable proportion of patients remaining steroid-free on tacrolimus/MMF therapy was 74.6%, P=0.937 by log-rank test).
Eight patients in the Campath arm suffered 13 rejection episodes in the first 2 years, and seven patients in the daclizumab arm suffered 11 episodes (Fig. 2b). Although rejection was more frequent in the daclizumab arm, the difference did not reach statistical significance. Banff 1997 classifications of rejection grade are summarized in Table 2. One patient in each arm suffered acute antibody mediated rejection (both in the presence of concomitant acute cellular rejection). Both of these responded to treatment with plasma exchange with 2 g/kg human pooled intravenous immunoglobulin. Two patients in the alemtuzumab arm suffered repeated rejection episodes, responsive to steroids initially, but treated as steroid resistant with intravenous immunoglobulin after the second recurrence, and eventually leading to graft loss. Two patients died in the alemtuzumab arm (sudden death at home at 12.7 months, and sepsis with respiratory failure at 22 months after severe pancreatitis). One patient in the daclizumab arm died of hemophagocytosis at 4.4 months associated with disseminated cytomegalovirus (CMV) disease after the cessation of CMV prophylaxis after a D+/R− live donor transplant.
At 2 years, six grafts were lost in the alemtuzumab group, and two in the daclizumab group (Table 3) with one of the alemtuzumab group graft losses being related to donor organ quality with significant interstitial fibrosis and tubular atrophy on early biopsies for poor initial graft function progressing to graft failure without any evidence of rejection on multiple biopsies, despite the donor being sensitized. (The sister organ was randomized to the daclizumab group and provided significantly impaired function in a preemptively transplanted recipient with significant residual native renal function.)
Graft function did not differ significantly between the two groups (Fig. 2c), despite lower tacrolimus trough levels as per protocol in the alemtuzumab than daclizumab groups (Fig. 2d). Proteinuria (measured as spot urine protein/creatinine ratio P/Cr) did not differ at 6, 12, or 24 months between the two groups (alemtuzumab arm 34.2/28.9/33.5 mg/mmol, daclizumab arm 31.4/59.7/49.9 mg/mmol).
Preliminary analysis of surveillance biopsies taken between 6 and 12 months in a subset of patients with stable graft function did not show any significant difference in the degree of interstitial fibrosis.
Adverse events were similar between the two groups (Table 2), with no significant difference in infection rates at 1 year. There was one episode of autoimmune thrombocytopenia in the alemtuzumab group, which responded to treatment with Rituximab. There were no episodes of BK virus transplant nephropathy during the first year, although one patient in the daclizumab arm developed BK nephropathy at 13 months, which responded successfully to withdrawal of MMF therapy.
Our approach (24) (in line with the regimen developed by Shapiro and coworkers in Pittsburgh) has been to take advantage of the profound initial suppression of the adaptive immune system provided by alemtuzumab induction to allow a long-term maintenance regimen, which avoids both steroids and anti-proliferative agents, leaving innate and nonspecific immune defenses substantially intact and providing a regimen which is simple to administer (and for our patients to take), and substantially cheaper than regimens based on induction with IL-2R blockade or modern polyclonal anti-T cell antisera combined with a calcineurin inhibitors and mycophenolate-based immunosuppression.
This alemtuzumab/tacrolimus monotherapy regimen has been shown to have good outcomes compared with an induction-free regimen using tacrolimus, MMF, and steroids (20), with similar graft and patient survival, and lower rejection rate at 6 months (although not significantly so at 1 year). We have now shown that this regimen produces equivalent, excellent graft, and patient survival at 1 and 2 years compared with a more directly comparable regimen without long-term steroid exposure but with monoclonal IL-2R induction therapy.
In common with most trials of alemtuzumab induction, we have demonstrated a low incidence of acute rejection in the first 6 months associated with the use of alemtuzumab. Unlike some of the reported studies (20, 21), our cohort did not suffer a significant level of “catch-up” acute rejection episodes beyond 6 months (although the difference in rejection rate between the two arms did not reach statistical significance at 6 or 12 months). We did not observe the increased risk of CMV disease (20, 28) or antibody mediated rejection that has been speculated to be associated with the use of alemtuzumab induction therapy (29).
Our trial was conservatively powered to detect a large difference in survival with a functioning graft at 1 year, so we are not able to exclude the possibility of smaller differences, and the single-center nature of the study may limit its applicability (although the similarity of our outcomes to those reported with similar regimens in other centers suggest that these good short-term results do not simply reflect local practice) (24, 25). We did not attempt to undertake prospective surveillance for subclinical CMV or polyoma virus infection, and cannot therefore comment on their incidence in the two regimens. In the absence of stratification for, or exclusion of, sensitized or regrafted recipients, our study populations were not balanced for sensitization against human leukocyte antigen antigens, but this did not impact the outcome measures reported here.
The combination of low-dose alemtuzumab induction, rapid steroid withdrawal, and tacrolimus monotherapy provides excellent outcomes with 1- and 2-year patient and graft survival equivalent to more conventional immunosuppression with IL-2R blocking MoAb and tacrolimus/mycophenolate combination maintenance.
MATERIALS AND METHODS
Study Design and Patients
We undertook a 12-month, prospective, randomized, open-label single center study, with NHS research ethics committee approval (Research Ethics Committee reference 05/Q0403/119) and with a Clinical Trial Agreement from the UK Medicines and Healthcare products Regulatory Agency (European Union Drug Regulating Authorities Clinical Trials [EudraCT] Number 2005-002856-17) under the sponsorship of The Hammersmith Hospital NHS Trust (now Imperial College Healthcare NHS Trust). The trial is registered at ClinicalTrials.gov (NCT00246129).The trial was undertaken in accordance with the principles of the Declaration of Helsinki and was funded by the Imperial College Kidney and Transplant Centre.
Patients due to undergo single-organ live or deceased donor kidney transplantation were eligible for recruitment. Exclusion criteria were as follows: potential recipients of simultaneous kidney/pancreas or donation-after-circulatory-death kidney transplants, patients with HIV, Hepatitis B, or Hepatitis C infection, and patients previously treated with myelosuppressive doses of immunosuppressive therapy. All patients provided written informed consent. The local reference for the trial was “CamTac” (Campath/Tacrolimus).
Randomization and Masking
Patients were randomized by computer-generated random permuted blocks (with concealment of block size from the clinical team). Allocation and masking was through computer-generated sheets in opaque, tamper-evident envelopes, opened after patient consent to take part in the study. Randomization was stratified in both arms by live versus deceased donor transplant origin and was undertaken in a 2:1 ratio for alemtuzumab/tacrolimus versus daclizumab/tacrolimus/MMF, this ratio having been chosen as providing an optimum balance of power versus cost in this internally funded study. The study was open-label, with no blinding of patients or staff to treatment group.
Patients received alemtuzumab induction as a single IV infusion of 30 mg alemtuzumab (MabCampath, Genzyme) on return from theaters with tacrolimus (Prograf, Astellas) monotherapy long-term maintenance (alemtuzumab group) or daclizumab (Zenapax, Roche) induction given as 2×2 mg/kg infusions on return from theaters and on day 14, with combined tacrolimus/mycophenolate mofetil (CellCept, Roche) long-term maintenance (daclizumab group). Both groups received a rapid steroid withdrawal regimen (0.5 g IV methyl-prednisolone intra-operatively at release of vascular clamps with oral prednisolone 1 mg/kg up to max 60 mg on postoperative days 1 to 3 then prednisolone 0.5 mg/kg up to max 30 mg on days 4 to 7 followed by steroid cessation, unless rejection had occurred during the first week).
All patients received 3 months of CMV prophylaxis with 450 mg once daily Valganciclovir initially, adjusted for estimated glomerular filtration rate, and 6 months Pneumocystis prophylaxis with Co-Trimoxazole 480 mg three times per week.
Drug Target Levels and Treatment of Rejection
Patients in the alemtuzumab group received tacrolimus initially 0.1 mg/kg in two equal divided doses, adjusted to achieve target 12 hr trough levels of 5 to 8 ng/mL by liquid chromatography/tandem mass spectrometry (equivalent to 6.5–10 ng/mL measured by immunoassay). Patients in the daclizumab group received tacrolimus, initially 0.15 mg/kg in two divided doses, adjusted to target trough levels of 8 to 12 ng/mL (equivalent to 10–15 ng/mL). Patients in the daclizumab group received MMF initially 500 mg BD adjusted to achieve target 12 hr trough mycophenolic acid levels of 1.5 to 3.0 mg/L.
Biopsy-proven rejection episodes were conventionally treated with 3×500 mg pulsed IV methylprednisolone followed by oral prednisolone 30 mg OD tapered to 10 mg and maintained for at least 1 year, with upwards adjustment of target tacrolimus levels to 8 to 12 ng/mL and addition of MMF in Campath-arm patients on tacrolimus monotherapy at the time of rejection. Preimplantation biopsies were not taken.
Efficacy, Safety, and Adverse Effects
The primary endpoint of the study was survival with a functioning graft at 1 year posttransplant. Secondary endpoints were as follows: the incidence and histological type of biopsy-proven acute rejection episodes and episodes of infection, graft function, maintenance treatment survival (steroid-free tacrolimus monotherapy and tacrolimus/MMF dual therapy, respectively), the incidence of new-onset diabetes, and patient and graft outcomes at 2 years.
Secondary outcome measures not reported in this article include 5-year graft and patient outcomes, length of initial hospital admission, and the number and duration of subsequent admissions during the first year, overall cost of treatment during the first year, and the prevalence of interstitial scarring on surveillance biopsies taken between 6 and 12 months and at 3 years posttransplant.
Adverse events (including death, episodes of rejection, infection, or graft dysfunction due to drug toxicity) were reviewed monthly at an internal research and clinical practice group, and by an external data monitor at quartiles of target recruitment and follow-up. Serious adverse events (death, life-threatening infection, or graft failure) were referred to the external data monitor at the time of occurrence.
Stata version 11.0 (StataCorp, Texas) was used for all statistical analysis. The primary outcome (and other event-free survival measures) was measured using Kaplan-Meier survival with the Log-rank method used to compare and assess the significance of difference between the two arms. All analysis was on an intention-to-treat basis, determined by the induction therapy given at transplantation. Graft function was estimated using the Modification of Diet in Renal Disease four-variable formula and comparison of graft function between arms undertaken with Student's t test. The trial was designed with a noninferiority endpoint, with 2:1 randomization to Campath and daclizumab arms, producing a required sample size of 120 (80:40) to give a 92% power to demonstrate a 10% inferiority in the primary outcome measure at a confidence level of 90% (based on estimated 95% survival with functioning graft in the control group).
The authors thank the NIHR Biomedical Research Centre funding scheme and Dr. Mark Harber of the Centre for Nephrology, Royal Free Hospital, University College London, for providing external data review.
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