Despite major advances in immunosuppression in the last three decades, a combination of agents, each with their pleomorphic toxicities, is usually necessary to prevent rejection in solid-organ transplant recipients. Because these agents have to be given indefinitely for the life of the allograft, transplant recipients experience the consequences of immunosuppressive toxicities such as infection and malignancy, and nonimmunosuppressive toxicities of the drugs themselves, including renal damage, hypertension, diabetes, hyperlipidemia, hirsutism, cushingoid features, and avascular necrosis of bone. These toxicities contribute significantly to long-term morbidity and mortality in allograft recipients. The search for treatment regimens that allow reprogramming of the recipient immune system thereby permitting the development of allograft tolerance is a long awaited goal in solid-organ transplantation.
Although there are a number of possible mechanisms for the development of allograft tolerance, several experimental studies have suggested that in the absence of an aggressive T-cell response, an engagement of the immune system with donor antigen can lead to tolerance (1). Lymphocyte depletion at the time of transplantation has been associated with a limited T-cell response with the subsequent development of tolerance (2). In 1998, Calne et al first reported that Campath-1H, or Alemtuzumab, a humanized monoclonal antibody to CD52 that induces profound T- and B- lymphocyte depletion, was effective in the prophylaxis of rejection in renal transplant (RTx) recipients, thereby permitting patients to be maintained on low-dose Cyclosporine (CsA) monotherapy without corticosteroids(3). They suggested that Campath-1H may afford a “window of opportunity for immunological engagement” whereby low-dose immunosuppressive therapy may prevent rejection. They speculated that limited engagement of the recipient immune system with donor antigen promoted the development of almost or “prope tolerance.” (3, 4).
A randomized, controlled trial was initiated to assess the safety and efficacy of Alemtuzumab with low-dose CsA monotherapy, in comparison with a standard CsA-corticosteroid-azathioprine (AZA)-based regimen, for the prevention of rejection in RTx.
MATERIALS AND METHODS
This multicenter, randomized controlled trial was performed at three transplant centers in Asia: the Singapore General Hospital, Singapore; the National Kidney and Transplant Institute, Quezon City, Philippines; and the University of Malaya Medical Center, Kuala Lumpur, Malaysia. The protocol was approved by the institutional review boards of each center and relevant regulatory authorities. The trial was conducted according to Good Clinical Practice Guidelines. Data management was performed by the Clinical Trials and Epidemiology Research Unit, Singapore. The planned follow-up of study patients was for 3 years post-RTx. The results reported here summarize the 6-month analysis.
Patients aged 18 to 65 years with renal failure who had given written informed consent before RTx were eligible to participate. Patients with a positive lymphocyte cytotoxicity cross-match against donor cells, who had panel reactive antibodies greater than 85%, who had prior RTxs, or who were multiorgan transplant recipients were not eligible. Patients deemed to require mycophenolate mofetil (MMF) as primary immunosuppression (criteria as per institutional practice) were excluded. Other exclusion criteria were prior treatment with Alemtuzumab, use of other investigational agents within 6 weeks, history of anaphylaxis after exposure to humanized monoclonal antibodies, pregnant or nursing women, unwillingness or inability to practice an acceptable form of birth control, and presence of major systemic or other illness likely to interfere with the patient's compliance with the protocol or compromise patient safety. In addition, patients with active infection, who were human immunodeficiency virus antibody positive, who were hepatitis B surface antigen or anti-hepatitis C antibody positive, who had autoimmune hemolytic anemia, or who were unable to undergo transplant biopsy, including patients who would require anticoagulation, were excluded. Within 5 hr posttransplant surgery, the presence of graft function was confirmed by documentation of urine output greater than 50 mL/hr; alternatively, the presence of graft perfusion was confirmed by perfusion scan or Doppler examination. Transplant recipients in whom graft function or perfusion could be demonstrated were then randomized to a CAMPATH or Standard group in a 2:1 ratio.
Patients assigned to CAMPATH received two 20-mg doses of intravenous (IV) Alemtuzumab (ILEX Pharmaceuticals, LP) over 2 hr, 60 min after premedication with 500 mg IV methylprednisolone (MP); the first dose was administered within 6 hr post-RTx, and the second dose was administered 24 hr after the first dose. Forty-eight hours after the second dose of Alemtuzumab, CsA was initiated at 5 mg/kg orally twice daily for two doses. Subsequently, maintenance CsA doses were reduced to 4 mg/kg per day as twice daily dosing for patients with immediate graft function (IGF). Patients assigned to CAMPATH who demonstrated delayed graft function (DGF), as defined by the requirement for dialysis within the first week post-RTx, received maintenance CsA doses of 3 mg/kg per day as twice daily dosing. Thereafter, CsA doses were adjusted to maintain CsA whole-blood trough levels of 90 to 110 ng/mL, and patients were maintained on low-dose CsA monotherapy.
Patients assigned to the Standard group received CsA twice daily, initially at 6 mg/kg per day if they demonstrated DGF and 8 mg/kg per day if they demonstrated IGF, with subsequent doses adjusted to achieve CsA whole-blood trough levels of 180 to 225 ng/mL. Azathioprine (AZA) was started at 1 mg/kg per day (to the nearest 25 mg and titrated to maintain total white count >4,000/μL and platelet counts >100,000/μL), and corticosteroids were administered according to institutional practice.
Concomitant Drug Therapy
Administration of corticosteroids, at a dose not exceeding 500 mg IV MP or its equivalent, was permitted at the time of surgery in both groups. Patients randomized to CAMPATH received an additional dose of 500 mg IV MP, as premedication for Alemtuzumab infusion, if the time between the dose given at surgery and the first dose of Alemtuzumab exceeded 1 hr. The total amount of corticosteroid given to patients in either group on the day of RTx was not to exceed 1 g IV MP or its equivalent. Premedication with chlorpheniramine, pethidine, or paracetamol was allowed 30 min before each infusion of Alemtuzumab. Treatment with maintenance corticosteroids was not allowed for patients in the CAMPATH group except after treatment of steroid resistant or recurrent rejection. Induction therapy with OKT3 monoclonal antibody, other antilymphocyte preparations, or anti-interleukin-2 receptor antibodies was not permitted in either group. Likewise, medications that could potentially alter CsA blood levels were to be avoided.
All patients received trimethoprim-sulfamethoxazole (TMP-SMX) for prophylaxis of Pneumocystis carinii pneumonia (PCP) or alternative for 6 months. Prophylaxis with ganciclovir for 3 months was given to patients at high risk for cytomegalovirus (CMV) infection, namely, donor+, recipient− patients; the remainder received acyclovir, valacyclovir, or ganciclovir as prophylaxis for 3 months.
Randomization, which was performed after the functionality or perfusion status of the transplanted kidney was confirmed, was effected by sealed envelopes balanced in blocks of three, placed with the principal investigator of each center; the envelopes were opened in serial order within 5 hr post-RTx. Completed registration forms (contained in each envelope) were faxed to the Clinical Trials and Epidemiology Research Unit. All unused envelopes were collected once patient recruitment was completed.
Posttransplant, patients were monitored for graft function by measuring serum creatinine (SCr) and urea, creatinine clearance, 24-hr urinary protein, and urinalysis at varying intervals post-RTx. Glomerular filtration rate was estimated using the Nankivell formula (5) as:
In addition, full blood counts, liver function tests, fasting lipids, serum calcium, phosphate, and intact parathyroid hormone levels were measured at various intervals post-RTx. Renal biopsies were performed for graft dysfunction, as defined by a greater than 25% increase in SCr, other stable graft dysfunction, or other clinical indications, and interpreted by the institutional histopathologist on the basis of Banff 1997 criteria. Acute rejection was defined as graft dysfunction confirmed by histologic findings on allograft biopsy. Steroid responsiveness was defined by return of SCr to within 25% of baseline within 1 week after treatment for acute rejection was started. Contrariwise, steroid resistance was defined by failure of SCr to return to within 25% of baseline or by occurrence of a second episode of acute rejection within up to 1 month after the first episode. A second episode of acute rejection occurring more than 1 month after the first episode was defined as recurrent rejection.
First-line therapy for acute rejection was IV MP up to 3 g per episode of rejection. In both groups, patients with steroid-responsive acute rejection continued their baseline therapies, namely, low-dose CsA monotherapy for the CAMPATH group and CsA, AZA, and corticosteroids for the Standard group. Steroid-resistant acute rejection and recurrent acute rejection were, by definition, treatment failures, and management was per institutional practice.
According to protocol, patients with DGF received lower doses of CsA in comparison with those with IGF. Regardless of the presence of DGF, CsA was adjusted to target levels of 90 to 110 ng/mL and 180 to 225 ng/mL in the CAMPATH and Standard groups, respectively. Allograft biopsies were performed at days 5 to 7 post-RTx for persistent DGF; acute rejection was treated, if present on biopsy. Patients with DGF persisting beyond 12 to 14 days post-RTx were converted to other therapies per institutional practice and were also considered to have failed treatment.
Safety and Efficacy
Safety parameters included CMV infections, other moderate and severe infections, lymphoproliferative disease, and other malignancies and adverse reactions to Alemtuzumab. CMV infection was defined by CMV antigenemia or a fourfold increase in CMV antibody titer with or without clinical or laboratory manifestations. CMV infections were further classified as CMV syndrome in the presence of fever, pneumonia, hepatitis, gastrointestinal ulceration, chorioretinitis, leukopenia or thrombocytopenia, asymptomatic CMV infection when there was CMV antigenemia in the absence of these manifestations, or asymptomatic increase in CMV titers in the presence of an isolated increase in CMV antibody titer.
At 6 months, the primary efficacy parameters were SCr levels, the incidence of biopsy-proven acute rejection and graft survival. For the purposes of this trial, graft loss was defined as return to continuous dialysis, and graft survival was defined as a functioning graft at 6 months; patient death with a functioning graft was also assessed as graft loss. The secondary efficacy measures at 6 months were the incidence of steroid resistant/recurrent acute rejection, incidence of DGF, treatment failure from all causes, patient survival, cumulative and actual steroid dose, CsA dose in mg/kg per day, and lymphocyte counts. Treatment failure was defined as graft loss from any cause, patient death, steroid resistant, or recurrent rejection.
Practical constraints rather than formal statistical methods determined sample size for this feasibility study, as did the decision to randomize 2:1 in favor of Alemtuzumab. As a consequence, no formal statistical testing was envisaged, but estimates of differences between treatment groups or the hazard ratios are expressed with the corresponding 95% confidence interval (CI) (6). In view of the multiplicity of the endpoints considered, due caution is needed in the interpretation of the CIs. Graft and patient survivals were summarized by Kaplan-Meier analysis.
All analyses were by intention to treat unless otherwise indicated as “per protocol.” In the box and whisker plots, the horizontal bars within the box represent the 25%, 50% (median), and 75% percentiles of the data, whereas the whiskers represent the remainder with circles indicating extreme values.
Between October 26, 2001, and September 20, 2003, of a total of 50 patients screened, 30 eligible patients were recruited into the study; 20 were randomized to CAMPATH and 10 to Standard. The flow diagram depicts the progress of patients through the trial (Fig. 1). Baseline characteristics of patients and donors and transplant-related variables were similar between CAMPATH and Standard groups (Table 1). Of note is the high proportion of sensitized patients (panel reactive antibody > 0), those with a 3, 4 human leukocyte antigen mismatch, and those receiving non-heart-beating cadaver donor kidneys in the CAMPATH group.
Alemtuzumab was administered at a median interval of 4.3 hr (range 0.5–6 hr) after anastomosis. All patients receiving Alemtuzumab were administered corticosteroids per protocol and an antihistamine (chlorpheniramine in 55%, promethazine in 45%), whereas all but one received paracetamol as premedication. After the loading dose, the median starting CsA dose on day 4 was 3.9 mg/kg per day for the CAMPATH group (4 mg/kg per day IGF, 3 mg/kg per day DGF) and 7.6 mg/kg per day for the Standard group (7.6 mg/kg per day IGF, 5.9 mg/kg per day DGF). Among patients who completed the treatment protocol, CsA levels were maintained within the target ranges throughout the study period (Fig. 2). Although the starting AZA dose for the Standard group was higher than per protocol (median: 1.5 mg/kg per day; range: 0.8–2.2 mg/kg per day), this was considered clinically insignificant. Antiviral prophylaxis was with valacyclovir or ganciclovir (n=13 each), acyclovir (n=1), or a combination (n=2) for at least 3 months. In all but four patients assigned to CAMPATH, prophylaxis for PCP was with TMP-SMX for at least 6 months. In the CAMPATH group, one patient received TMP-SMX for only 46 days, another for only 18 days, and the third received monthly pentamidine inhalation for 2 months followed by oral dapsone for 4 months. One patient in CAMPATH who had graft loss on day 1 received neither PCP nor antiviral prophylaxis.
Efficacy at Six Months
Outcome measures at 6 months demonstrated comparable SCr, incidence of acute rejection, and graft and patient survivals between the CAMPATH and Standard groups (Table 2). SCr over time post-RTx showed no differences between the groups (Fig. 3A); likewise, there were no differences in the measured creatinine clearance or glomerular filtration rate as estimated with the Nankivell formula (Fig. 3B). By 6 months, there were three graft losses in the CAMPATH group: one on day 1 because of renal arterial thrombosis attributed to surgical causes; a second on day 10 because of CsA-induced hemolytic uremic who syndrome; and a third patient who died of bleeding from the gastrointestinal tract on day 139 with a functioning graft was also deemed a graft loss. There were no graft losses or patient deaths in the Standard group.
Per protocol, at 6 months post-RTx, trough CsA levels were markedly lower in the CAMPATH group in comparison with the Standard group (Table 2). On per protocol analysis, a significant proportion of patients in the CAMPATH (60%–71%) and Standard groups (67%–80%) had trough CsA levels greater than 110 ng/mL and 225 ng/mL, respectively, in the first 2 weeks posttransplant. Nevertheless, the 75th percentiles for trough CsA levels at 2 weeks for CAMPATH and Standard groups were 145 ng/mL and 384 ng/mL, respectively. Despite the low levels, cumulative first acute rejection rates were comparable between the groups. In the CAMPATH group, five patients experienced acute rejection between 2 and 30 days post-RTx; two patients (40%) had steroid-responsive acute rejection. Of the three patients assigned to CAMPATH who had steroid-resistant acute rejection, the intervals between rejection treatments were 2, 6, and 25 days; these three patients were treated by initiation of maintenance steroids and either by resumption of full-dose CsA together with initiation of MMF (n=2) or conversion from CsA to tacrolimus (n=1). No grafts were lost as the result of acute rejection in the CAMPATH group. In the Standard group, acute rejection occurred in two patients at 10 and 15 days (one was steroid responsive). The other patient with steroid-resistant acute rejection had two episodes of acute rejection in an interval of 9 days, and this patient underwent conversion from AZA to MMF. Three of four patients in the CAMPATH group with DGF had received kidneys from non-heart-beating donors.
At 6 months, 15 of 17 patients (88%) assigned to CAMPATH who had functioning grafts were on steroid-free low-dose CsA monotherapy; the remaining 12% had been administered maintenance corticosteroids after steroid-resistant acute rejection. Per protocol, all patients in the Standard group were on maintenance corticosteroids (Table 2). For patients assigned to CAMPATH, cumulative corticosteroid dosage was significantly lower than for those on Standard therapy.
Among the other endpoints, although total white blood cell counts were similar between the groups, lymphocyte counts were profoundly depleted after the second dose of Alemtuzumab (median lymphocyte count at day 6, 53/μL vs. 1,306/μL in CAMPATH and Standard groups, respectively; 95% CI −1,679 to −728) and remained lower until 6 months post-RTx (Fig. 4). Indeed, among the 19 patients assigned to CAMPATH who were alive at 6 months, only 12 had recovery of lymphocyte counts to greater than 1,000/μL (median time to recovery 125 days), while 7 patients remained depleted throughout this period.
Among patients assigned to CAMPATH, tachycardia with a pulse rate 100/min or greater, temperature greater than 37.5°C, and a decrease in diastolic blood pressure to less than 50 mm Hg occurred in 50%, 35%, and 30%, respectively, within 2 days post-RTx; the occurrence of these parameters for patients in the Standard group was 30%, 0%, and 10%, respectively. Thrombocytopenia with a platelet count 140,000/μL or less occurred in 40% and 30% of patients in the CAMPATH and Standard groups, respectively, within 2 weeks post-RTx. The incidence of these adverse events, although possibly attributable to Alemtuzumab, was not significantly different from that in patients in the Standard group. The overall incidence and spectrum of infections were comparable between the CAMPATH and Standard groups. Eight of 20 patients in the CAMPATH group (40%) and 3 of 10 patients in the Standard group (30%) experienced moderate or severe infections. None of the patients in the CAMPATH group with an asymptomatic increase in CMV antibody titers were treated for CMV infection (Table 3). There was no posttransplant lymphoproliferative disorder or other malignancies recorded for either group in the 6-month period.
Alemtuzumab or Campath-1H, a humanized monoclonal antibody directed against the CD52 antigen, is a powerful lytic agent for both T and B lymphocytes but not bone marrow stem cells (7). Alemtuzumab administration effectively depletes lymphocytes without causing permanent bone marrow depression. Alemtuzumab is known to be effective in the treatment of a number of conditions mediated by lymphocytes; it was used initially in patients with hematologic malignancies or autoimmune diseases, such as rheumatoid arthritis, vasculitis, and multiple sclerosis (8, 9), and was first introduced into clinical RTx by Calne et al. in 1995 (10). Although initially used in the treatment of rejection, subsequent studies by Calne and colleagues were for the prophylaxis of rejection in clinical RTx. In the initial study on 31 cadaveric RTx recipients treated with Alemtuzumab on days 0 and 1 after transplant surgery together with low-dose CsA monotherapy, Calne et al. demonstrated a 19.4% incidence of acute rejection on follow-up to 21 months after transplantation (3, 4).
Since then, Alemtuzumab has been used at several other centers in different regimens. The results reported here are those of the first randomized controlled trial of Alemtuzumab, together with low-dose CsA monotherapy in RTx, with a protocol similar to that of the original study by Calne (4). Although not powered to demonstrate differences in rejection incidence and graft and patient survivals, this pilot study demonstrated a comparable incidence of these endpoints and stability in renal function at 6 months between CAMPATH and Standard therapies. Alemtuzumab administration was not associated with an increase in any adverse reactions; moreover, although lymphocytes were depleted for a prolonged period in this group, there was no increased incidence of immune toxicities such as infections or malignancies, attesting to its relative safety in clinical RTx.
In the past five decades of clinical solid-organ transplantation, efforts have been directed to reducing acute rejection, improving long-term graft and patient survivals and reducing immunosuppressive and nonimmunosuppressive toxicities from the drugs required to avoid rejection. Induction of tolerance, namely, successful engraftment without recourse to any maintenance immunosuppressive therapy, has remained a long elusive goal in solid-organ transplantation. Experimental studies have suggested that lymphocyte depletion, induced primarily by antibodies at the time of transplantation, establishes a milieu in which potentially alloreactive cells are destroyed, and such a lymphocyte-depleted state is permissive to the subsequent development of a nondestructive alloimmune response (2). Although transplantation tolerance has been achieved using such immunologic interventions in laboratory animals, few tolerance induction protocols that permit complete maintenance immunosuppressive drug avoidance or withdrawal have been successful in humans to date.
Indeed, Calne has suggested that given the heterogeneity of donor-recipient combinations and immune status, and the susceptibility to disturbances in the tolerant state by infections or other immune reactions, full tolerance may not be clinically feasible in the majority of human solid-organ transplantation (11). In fact, he suggests that the preferred approach is “operational tolerance,” a state in which, after a period of induction immunosuppression, nontoxic doses of a maintenance immunosuppressive agent should be used to keep these low-grade immune reactions at abeyance. This concept of “prope” or almost tolerance serves as the basis for the immunosuppressive regimen used in the original studies of Alemtuzumab by Calne et al. (4) and in the randomized controlled trial reported here. As in the original series reported by Calne et al., the 6-month results of this study support the premise that lymphocyte depletion by Alemtuzumab followed by low-dose CsA monotherapy is effective in preventing rejection in the majority of patients and is as safe as standard therapy despite the profound lymphocyte depletion. Although immunologic studies documenting the state of donor-specific alloreactivity are lacking in this study, one can speculate that the 17 patients with functioning grafts at 6 months who were treated with Alemtuzumab while on minimal maintenance immunosuppression in this series have some degree of functional tolerance to their allograft.
Two other remarkable findings from our study were the high proportion of patients (88%) who were on corticosteroid-free immunosuppression with low-dose CsA monotherapy at 6 months post-RTx and the characteristics of the acute rejection episodes. Although a large proportion of the patients in our study were sensitized, were mismatched at 3 to 4 human leukocyte antigen loci, were recipients of cadaveric transplants, and experienced DGF, the overall incidence of acute rejection in the CAMPATH group was 25%. Notwithstanding the difficulties in diagnosing and classifying acute rejection in lymphocyte-depleted patients, apart from one patient in the CAMPATH group with biopsy diagnosis of Banff IIA rejection, the remainder had borderline or Banff I rejection. All acute rejection episodes in the CAMPATH group responded to corticosteroids and/or institution of full-dose CsA together with MMF or switch from CsA to tacrolimus. These findings are similar to that of the original study by Calne et al., in which 27 of 31 patients were on steroid-free immunosuppression at follow-up (3, 4). Five of the 31 patients reported by Calne et al. experienced an acute rejection episode by the sixth month; all acute rejections were steroid responsive, and of these, only one had a vascular component, attesting to the relatively mild nature of rejection under Alemtuzumab with CsA monotherapy.
There have been several other recent reports of outcomes after Alemtuzumab therapy in RTx. In a study by Kirk et al. in which several doses of Alemtuzumab were administered without any other immunosuppressive therapy in seven live donor RTxs, a monocyte-mediated rejection was detected in all patients (12). All of these rejection episodes were steroid responsive, and at 6 months, all patients were receiving sirolimus monotherapy. In another study in which Alemtuzumab was administered with sirolimus monotherapy, Knechtle et al. demonstrated a 27.6% incidence of acute rejection, all requiring institution of corticosteroids (13). A large proportion of acute rejection episodes in this study had a humoral component, many of which required aggressive therapy with Thymoglobulin, rituximab, steroids, and plasma exchange for reversal.
In contrast with the studies without calcineurin inhibitors as maintenance immunosuppression, among patients receiving a calcineurin inhibitor, MMF, and steroids, Knechtle et al. reported a significantly lower incidence of acute rejection after induction with two doses of Alemtuzumab, in comparison with that after Thymoglobulin or an anti-CD25 monoclonal antibody (14). Likewise, the Miami group reported a remarkably low incidence of acute rejection of 9.1% when Alemtuzumab administration was followed by “half” doses of tacrolimus and MMF without concomitant corticosteroids (15). The lower incidence of acute rejection in the Miami study in comparison with the study reported here may be because of the use of higher levels of maintenance immunosuppression together with Alemtuzumab in the former. Nevertheless, taken together with the results from our study, Alemtuzumab permits steroid-free immunosuppression in the majority when administered with low-dose calcineurin inhibitor therapy.
Another finding of note from this study was the issue of safety. Adverse reactions to Alemtuzumab were clinically insignificant and easily avoided with the use of prophylactic steroids, paracetamol, and antihistamine as premedications. No malignancy or posttransplant lymphoproliferative disorder was documented up to 6 months post-RTx, and the spectrum of infections was not dissimilar between the groups. Finally, although all patients receiving Alemtuzumab were CMV seropositive before transplantation, 44% manifested an asymptomatic increase in CMV antibody titers without CMV antigenemia, suggesting that humoral immunity in these patients is largely retained despite lymphocyte depletion.
This pilot study demonstrates the safety and efficacy of Alemtuzumab together with low-dose CsA monotherapy in the prophylaxis of rejection in RTx at 6 months. An analysis of long-term outcomes for graft and patient survival, and renal function and drug-related toxicities, as planned in this clinical trial, will further elucidate the potential applicability of Alemtuzumab in clinical transplantation.
The authors thank Dr. Stuart Knechtle (University of Wisconsin) for serving as the safety monitor for this clinical trial. In addition, the authors thank Dr. Florecita Padua and Dr. Januario Veloso Jr. (National Kidney Transplant Institute, Quezon City, Philippines) for immunologic work and flow cytometric studies, respectively, Dr. Sufi M. Suhail and Dr. Stephen Chew (Singapore General Hospital, Singapore) for their clinical contributions, and Dr. Jorgen Seldrup (Clinical Trials and Epidemiology Research Unit, Singapore) for statistical support.