American Journal of Transplantation 2008; 8: 1480–1485 Blackwell Munksgaard

 C 2008 The Authors C 2008 The American Society of Journal compilation  Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2008.02273.x

Alemtuzumab (Campath-1H) and Tacrolimus Monotherapy After Renal Transplantation: Results of a Prospective Randomized Trial R. Margreitera, ∗ , J. Klempnauerb , P. Neuhausc , F. Muehlbacherd , C. Boesmuellera and R. Y. Calnee a

Department of General and Transplant Surgery, University Hospital Innsbruck, Anichstrasse Innsbruck, Austria b Klinik fur ¨ Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany c Klinik fur ¨ Allgemein-, Viszeral- und Transplantationsmedizin, Universitatsklinikum ¨ Charite´ Berlin, Augustenburger Berlin, Germany d Abteilung fur ¨ Transplantation, Universitatsklinik ¨ fur ¨ Chirurgie, Wahringer ¨ Gurtel ¨ Vienna, Austria e University of Cambridge, Cambridge, UK ∗ Corresponding author: Raimund Margreiter, [email protected] Clinical trials registry: Clinical Trials.gov Trial identifier number: NCT00147381 The lymphocyte-depleting antibody alemtuzumab was evaluated in a prospective randomized multicenter trial in deceased donor kidney transplantation. The 65 patients in the study group received induction with alemtuzumab followed by delayed tacrolimus monotherapy, while the 66 patients in the control group were started on tacrolimus in combination with mycophenolate mofetil and steroids. Tacrolimus levels of 8–12 ng/mL for the first 6 months and 5–8 ng/mL thereafter were aimed for in both groups. At 12 months the biopsy-proven rejection rate was 20% in the study group and 32% in the control group (p = 0.09). Patient survival at 1 year was 98% for both groups. Graft survival was 96% for the study group versus 90% for the control group (p = 0.18). Graft function was identical in both groups. Adverse events were similar in both groups apart for more CMV infections in the study group. At the end of the first year 82% of the patients in the study group were steroid-free and 71% continued on tacrolimus monotherapy. These results suggest that alemtuzumab induction together with tacrolimus monotherapy is at least as efficient in renal transplantation as is a tacrolimus-based triple-drug regimen with a similar safety profile but more CMV infections. 1480

Key words: Alemtuzumab, kidney transplantation, tacrolimus monotherapy Received 31 October 2007, revised 12 March 2008 and accepted for publication 01 April 2008

Introduction The many adverse events associated with the administration of immunosuppressive drugs prompted a search for new protocols for posttransplant immunosuppression that employ no steroids and lower dosages of or no calcineurin inhibitors (CNI), but that would be equally effective with regard to graft survival and possibly encourage the development of unresponsiveness to the graft. The combination of mycophenolate mofetil plus sirolimus, however, has been reported to produce significantly worse results as compared with CNI-containing regimens (1). Several experimental studies have shown that lymphocyte depletion at the time of transplantation is associated with a limited T-cell response with subsequent development of tolerance in some models but not routinely in nonhuman primates (2,3). Although peripheral depletion is a widely accepted induction strategy in renal transplantation, it was found to be insufficient to prevent rejection if induction was not followed by maintenance immunosuppression (4,5). It was first reported by Calne et al. that alemtuzumab (Campath-1H), a humanized monoclonal antibody to CD52 that causes profound depletion of most immunocompetent cells including T- and B-lymphocytes, monocytes and NK cells, was effective in the prophylaxis of rejection when given together with cyclosporine monotherapy starting 3 days after transplantation (6,7). The rationale behind this strategy of leaving a time window with no immunosuppression was to provide an opportunity for immunological engagement (6,7). It was shown that T cells with a memory-like function were the most resistant to alemtuzumab depletion, while at least in vitro being more sensitive to tacrolimus than to cyclosporine (8). This was the reason why we decided to design a protocol that would be similar to that for the initial Campath trial but would replace cyclosporine with tacrolimus. A prospective randomized, controlled, multicenter trial was initiated to assess the efficacy and safety of alemtuzumab induction with tacrolimus

Alemtuzumab in Renal Transplantation

monotherapy as compared to a tacrolimus, mycophenolate mofetil and steroid triple-drug regimen.

Material and Methods Patients This investigator-initiated multicenter, randomized controlled trial was conducted at four major European transplant centers (Berlin, Hannover, Innsbruck and Vienna). The protocol was approved by the institutional review board at each center and the pertinent national health authorities. Patients aged 18 to 65 years with end-stage renal failure awaiting a first renal transplant from a deceased donor and having given written consent were eligible to participate. Patients with a positive cross match against donor cells, who had more than 25% panel-reactive antibodies, who had prior renal transplants or who were multiorgan recipients were excluded. Other exclusion criteria were previous treatment with alemtuzumab, the use of other investigational agents within 6 weeks, active systemic infection, HIV-positive patients or donors, autoimmune hemolytic anemia and a history of anaphylaxis following exposure to humanized monoclonal antibodies. Pregnant or breast-feeding women were also excluded, as were recipients of a live donor transplant.

Immunosuppressive protocol Between January 8, 2004 and June 27, 2005 a total of 131 recipients of a first deceased donor renal transplant were randomized in a 1:1 manner to either group A (study group) or group B (control group) at each center. Group A patients were given 250 mg of methylprednisolone i.v. immediately after completion of surgery, followed 1 h later by 20 mg alemtuzumab infusion over 3 to 6 h. On day 1 patients received the same treatment as on day 0. After a day without immunosuppressive therapy patients received the initial tacrolimus dose of 0.05 mg/kg twice daily. Trough blood levels of 8–12 ng/mL were aimed for during the first 6 months and 5–8 ng/mL thereafter. Centers were asked to prevent trough levels from falling below 10 ng/mL in the first 3 months. Group B was given tacrolimus preoperatively or immediately posttransplant at a dosage of 0.05 mg/kg twice daily orally with target whole blood trough levels of 8–12 ng/mL for the first 6 months and of 5–8 ng/mL between months 7 and 12. Again, centers were asked to avoid trough levels below 10 ng/mL during the first 3 months. In addition, 1–1.5 g mycophenolate mofetil was given and adjusted on the basis of clinical evidence of toxicity. Steroids were prescribed according to the center’s standard regimen. In brief: at three of the four centers steroids were given at an identical dosage, namely 500 mg on day 2. On day 3, patients were switched to oral prednisolone that was rapidly tapered to 25 mg on day 10 and further reduced to 5 mg at 1 year. At the fourth center steroids were started with 200 mg prednisolone at the day of transplantation and stepwise reduced to 20 mg on day 10 and further to 5 mg at 1 year. Histologically confirmed rejections were treated with 500 mg of methylprednisolone on three consecutive days, steroid-resistant rejections with antilymphocyte preparations. Infection prophylaxis consisted of trimethoprim-sulphamethoxazole twice daily three times a week for 2 months and oral gancyclovir or valgancyclovir for 90 days in patients with EBV+ and/or CMV+ donors. The primary endpoint of the study was the proportion of patients with a first biopsy-proven acute rejection within 6 months of transplantation. Rejection was defined as any episode with relevant clinical and laboratory

American Journal of Transplantation 2008; 8: 1480–1485

signs and symptoms. According to the study protocol, all clinically apparent episodes of rejection had to be confirmed by core biopsy. Biopsies were assessed locally by a histopathologist and later confirmed by a single expert. Rejection was classified according to the Banff 97 grading system (9). Biopsy-proven acute rejection was defined as any histologically confirmed episode for which a Banff score of borderline, I (mild), II (moderate) or III (severe) was recorded. For patients who had several biopsies during a single rejection episode the highest grade was used. Secondary efficacy endpoints included biopsy-proven acute rejection episodes for 12 months after transplantation, time to first biopsy-proven rejection, patient and graft survival, incidence of corticosteroid-resistant rejection, serum creatinine as well as clearance at 1 year and adverse events. Graft loss was defined as the need to resume chronic hemodialysis, for retransplantation, transplant nephrectomy or death. A change in the immunosuppressive treatment because of corticosteroid-resistant rejection was considered treatment failure. The glomerular filtration rate was calculated with the Cockcroft-Gault formula (10). For safety and tolerability assessment the overall rate of adverse events, the rate of those leading to withdrawal from the study, laboratory tests (hematology, biochemistry, urine analysis) and vital signs were recorded on days 0, 7, 14, 28 and at months 3, 6 and 12. New-onset diabetes mellitus (NODM) was defined as taking any oral hypoglycemic medication or insulin for more than 2 weeks between day 15 and the end of the first year. Patients with normal total cholesterol and/or triglyceride levels at baseline, but who had abnormal readings (more than 200 mg/dL total cholesterol, more than 150 mg/dL triglycerides) at month 6 and/or 12 were considered hyperlipidemic.

Statistical analysis Intention-to-treat analysis was performed on all included randomized patients who underwent transplantation and received at least one dose of study medication. Statistical testing was done by means of appropriate techniques depending on data distribution (Mann-Whitney U-test, t-test, Pearson’s chi-square test, Fisher’s exact test). The rate of acute rejection at month 6 and month 12 was analyzed with a one-sided chi-square test at a level of 5%. Freedom from rejection and graft survival were analyzed with Kaplan-Meier survival procedures, and the hazard ratio was estimated with Cox-proportional hazard regression. Relative risks were calculated for the adverse events. Relative risks and hazard ratios were calculated with 95% confidence intervals. The analyses were done with SPSS 15 (Chicago, IL).

Results A total of 131 patients were randomly assigned to receive tacrolimus monotherapy after alemtuzumab induction (n = 65) or tacrolimus-based triple-drug therapy (n = 66). Donor and recipient characteristics are depicted in Table 1. With regard to these baseline data the study group contained more female patients (p = 0.04). Furthermore, the donors for recipients of the study group were somewhat older than in the control group (p = 0.06) but on the other hand somewhat better matched with the recipients for HLA A and B (p = 0.06). Differences in the latter two characteristics, however, were not statistically significant. 1481

Margreiter et al. Table 1: Baseline characteristics of donor and recipients Campath group n = 65

Control group n = 66

Donors Age (years), mean (SD) 50 (13.1) 45 (14.9) Male, n(%) 37 (57%) 34 (52%) Cold ischemia time 15.7 (4.9) 16.4 (6.1) (h), mean (SD) CMV neg n (%) 22 (34%) 28 (42%) Recipients Age (years), mean (SD) 50 (10.6) 49 (12.7) Male, n (%) 38 (58%) 50 (76%) Primary disease Glomerulonephritis 14 13 Polycystic disease 13 10 Nephrosclerosis 4 4 Interstitial nephritis 6 2 Diabetic nephropathy 2 4 Other 25 30 Unknown 1 3 Dialysis (months), 55 (27.0) 55 (32.7) mean (SD) CMV mismatch 24 (36.9%) 25 (39.1%) HLA mismatch, mean A+B 1.48 1.89 DR 0.74 0.70

p = 0.06 p = 0.54 p = 0.67 p = 0.31 p = 0.86 p = 0.04

p = 0.91 p = 0.94 p = 0.06 p = 0.75

Rate of rejection, time to rejection and histological severity are summarized in Table 2. Frequency of biopsy-proven rejection at 6 months was 15% (10/65) in the study group and 29% (19/66) in the control group (p = 0.05). At 12 months a total of 13 (20%) acute biopsy-proven rejections were reported for the study group and 21 (32%) for Table 2: Frequency and severity of acute rejection episodes and time to first biopsy-proven rejection

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Most of the rejections were grade I and were evenly distributed between the two groups (Table 2). Grade II rejections, however, were mainly seen in patients in the control group. Only one grade III rejection was observed in each group. There were no steroid-resistant rejections requiring antilymphocyte preparations in the study group, but three in the control group. Overall, two (3%) grafts were lost in the study group, and 6/66 (9%) in the control group. The reasons for graft loss are listed in Table 3. Graft survival at 1 year was calculated to be 96% in the study group and 90% in the control group (HR = 0.33; 95% CI 0.07–1.66).

Otherwise there were no apparent differences between the two treatment groups in any demographic or baseline characteristics.

Biopsy proven rejection up to month 6 Median time to rejection (months) Biopsy proven rejection up to month 12 Median time to rejection (months) Histological severity Borderline Mild (Banff I) Moderate (Banff II) Severe (Banff III)

the control group (p = 0.09). Time to first biopsy-proven rejection at 1 year was 4.9 months in the study group versus 0.4 month in the control group (HR = 0.55, 95% CI 0.27–1.09) (Figure 1).

Campath group n = 65

Control group n = 66

10

19

p = 0.05

4.2

0.3

HR = 0.46 (0.22–1.00)

13

21

p = 0.09

4.9

0.4

HR = 0.55 (0.27–1.09)

0 11 1 1

3 10 7 1

Mean serum creatinine concentrations at various times were almost identical, namely 1.58 mg/dL in the study group and 1.56 mg/dL in the control group at 1 year. Mean creatinine clearance was 61.7 mL/min in the study group and 64.2 mL/min for patients in the control group (p = 0.38). One patient died in each group: in the study group from intracerebral hemorrhage with a functioning graft and in the control group from septicemia after graft loss. Patient survival at 1 year was thus 98% in both groups. Tacrolimus whole blood trough concentrations were within the desired range during the course of the study (Figure 2). At 1 year 46 (71%) of the 65 patients in the study group were on tacrolimus monotherapy, and 49 (74%) of the 66 patients in the control arm were on their initial immunosuppressive regimen. At the end of the study 53 (82%) patients in the study group were steroid-free. The reasons for changing immunosuppression in the study group were adverse events in 14 patients and rejection in five, whereas in the control group 14 patients changed immunosuppression for drug side effects and three for rejection (Table 4). Overall, adverse events observed in the study group and in the control group were similar to those previously reported for tacrolimus trials except for CMV infections: Eighteen CMV infections were seen in the study group versus only eight in the control group. (RR = 2.28, 95% CI 1.07–4.88). Three of these eight infections in the control group, however, were tissue invasive in contrast to the alemtuzumab group where all infections were nontissue invasive. Two patients in the study group and one in the control group developed a polyoma virus infection; all three were subsequently switched from tacrolimus to cyclosporine. American Journal of Transplantation 2008; 8: 1480–1485

Alemtuzumab in Renal Transplantation

Figure 1: Freedom from rejection.

In particular, alemtuzumab was well tolerated and no cytokine storm was observed after premedication with methylprednisolone. Study-relevant adverse events are summarized in Table 5.

Discussion To date, alemtuzumab induction followed by calcineurin inhibitor monotherapy was previously tested in merely one prospectively randomized trial including a total of only 30 patients (11). While that trial used cyclosporine as calcineurin inhibitor, our study opted for tacrolimus. Our decision was based on the findings that effector memory T cells, which are most resistant to depletion and are known to be associated with rejection, proved to be in vitro resistant to steroids, sirolimus and deoxyspergualin but responded to calcineurin inhibitors, particularly to tacrolimus (91% vs. only 55% to cyclosporine) (8).

Table 3: Causes of graft loss

Surgical Rejection (Banff III) Death with functioning graft Recurrent glomerulonephritis Hemolytic uremic syndrome Total

Campath group n = 65

Control group n = 66

0 1 1 0 0 2

2 1 0 2 1 6

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The 6 months results of the alemtuzumab-arm in our study appear to indicate that tacrolimus might be at least as efficacious as cyclosporine after alemtuzumab induction (15% rejection in this study vs. 25% in the alemtuzumab-arm of the cyclosporine study). Also our 96% graft survival rate at 1 year compares favorably with their 85% results at 6 months. According to the protocol there were, however, lower doses and corresponding levels of cyclosporine in the alemtuzumab-arm versus the control arm, in contrast to our study where no difference in tacrolimus levels was observed between both arms (11). Although the rate of biopsy-proven rejection at 6 months was significantly lower (p = 0.05) in the alemtuzumab group of our study, this difference was no longer significant by the end of the first year (p = 0.09). Accordingly, time to first rejection was substantially longer in patients in the study group (HR = 0.55; 95% CI 0.27–1.09). The majority of rejections in patients in the control arm occurred in the first postoperative months, whereas in the study group they were mainly observed from month 4 onward. By that time lymphocytes in the alemtuzumab group had increased from 1% to 15% and reached 34% at the end of the first year as compared to 24% in the control group (data not shown). It has to be mentioned, however, that lower rejection rates have been achieved with tacrolimus based triple-drug immunosuppression after induction with an IL-2 receptor antibody (12). A similar observation concerning rejections mainly occurring during months 6 to 12 was made in the largest series to date of 205 living donor kidney transplants in Pittsburgh

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Margreiter et al.

Figure levels.

using alemtuzumab induction with tacrolimus maintenance therapy, reporting 2.9% acute rejection by month 6 but 22% rejection by month 12 (13). With regard to severity of rejection, the number of mild and severe rejections in our study was similar in both groups (only one case of severe rejection in each group). There were, however, more Banff 2 rejections in the control arm (n = 7) than in the study group (n = 1). This finding together with the fact that three steroid-resistant rejection episodes in the control group, but none in the study group, required antilymphocyte preparations can be interpreted as a higher efficacy of tacrolimus monotherapy following

2: Tacrolimus

trough

alemtuzumab induction compared with a tacrolimus-based triple-drug regimen. This higher grade of efficacy has been achieved without compromising tolerability. Indeed, the number of adverse events was similar in both our trial groups, except for CMV infections, which were more frequent in the study group. It needs to be mentioned, however, that none of the 18 CMV infections in the study group were tissueinvasive, while three of the eight in the control group were. Other viral infections were not seen more frequently in the study group. For this reason, it appears advisable to routinely administer CMV prophylaxis to patients receiving alemtuzumab.

Table 4: Reasons for change in immunosuppression

Rejection Polyoma virus infection CMV infection Tacrolimus toxicity ATN Diarrhea Diabetes mellitus Leucopenia Hemolytic uremic syndrome Focal segmental glomerulonephritis Proteinuria Vomiting Tremor Total

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Campath group n = 65

Control group n = 66

5 2 1 4 2 1 0 0 1 1 1 1 0 19

3 1 1 2 1 2 3 3 0 0 0 0 1 17

Table 5: Adverse events Campath Control group group n = 65 n = 66 Infections Viral: non-CMV CMV Bacterial Fungal Cardiovascular Gastrointestinal Hematologic Metabolic Hyperlipidemia New onset diabetes Malignancies

16 18 17 7 13 30 49

15 8 29 9 14 30 48

RR = 1.08 (0.59–2.00) RR = 2.28 (1.07–4.88) RR = 0.60 (0.36–0.97) RR = 0.79 (0.31–1.99) RR = 0.94 (0.48–1.85) RR = 1.02 (0.70–1.47) RR = 1.04 (0.85–1.27)

19 2 0

18 2 0

RR = 1.07 (0.62–1.85) RR = 1.02 (0.15–6.99)

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Alemtuzumab in Renal Transplantation

Interestingly, only two patients in each group developed diabetes posttransplant. Similarly, the Pittsburgh group had reported a remarkably low incidence of NODM of merely 0.5% (12). In our study, neither malignancy nor autoimmune disease has been observed to date. The latter was reported to occur in a significant number of patients treated with alemtuzumab for multiple sclerosis (14). Although no malignancy and particularly no posttransplant lymphoma was observed in the short term and although the Cambridge group, using a similar protocol, reported no increased incidence of malignancies with a longer follow-up of 5 years, nevertheless, caution is warranted when using this type of immunosuppression in patients with a high risk of developing malignancy (15). Its simplicity and its higher cost-effectiveness must be considered as another advantage of this new immunosuppressive regimen. In conclusion, antibody pre-conditioning with alemtuzumab together with tacrolimus monotherapy is at least as efficient as a tacrolimus based triple-drug regimen with a similar safety profile except for CMV infections. To identify the optimum alemtuzumab regimen long follow-up of a larger cohort of patients is needed.

Acknowledgment This work was supported by Astellas Pharma GmbH, Munich–Germany.

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2. Monaco AP, Morris PJ. Clinical tolerance: The end of the beginning. Transplantation 2004; 77: 921–925. 3. Kean LS, Gangappa S, Pearson TC, Larsen CP. Transplant tolerance in non-human primates: Progress, current challenges and unmet needs. Am J Transplant 2006; 6: 884–893. 4. Kirk AD, Mannon RB, Kleiner DE et al. Results from a human renal allograft tolerance trial evaluating T-cell depletion with alemtuzumab combined with deoxyspergualin. Transplantation 2005; 80: 1051–1059. 5. Kirk AD, Hale DA, Mannon RB, Kleinert DE, Hoffmann SC, Kampen RL. Results from a Human Renal Allograft Tolerance Trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (Campath-1H). Transplantation 2003; 76(1): 120–129. 6. Calne R, Friend P, Moffatt S et al. Prope tolerance, perioperative Campath 1H, and low-dose cyclosporine monotherapy in renal allograft recipients. Lancet 1998; 351: 1701–1702. 7. Calne R, Moffatt SD, Friend PJ et al. Campath 1H allows low-dose cyclosporine monotherapy in 31 cadaveric renal allograft recipients. Transplantation 1999; 88: 1613–1616. 8. Pearl JP, Parris J, Hale DA et al. Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody mediated T-cell depletion. Am J Transplant 2005; 5: 465–474. 9. Racusen LC, Solez K, Colvin RB et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55: 713– 723. 10. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41. 11. Vathsala A, Ona ET, Tan S-Y et al. Randomized trial of alemtuzumab for prevention of graft rejection and preservation of renal function after kidney transplantation. Transplantation 2005; 80: 765–774. 12. Ekberg H, Tedesco-Silva H, Demirbas A et al. Reduced exposure to calcineurin inhibitors in renal transplantation. N Engl J Med 2007; 357: 2562–2575. 13. Tan HP, Kaczorowski DJ, Basu A et al. Living donor renal transplantation using alemtuzumab induction and tacrolimus monotherapy. Am J Transplant 2006; 6: 2409–2417. 14. Coles AJ, Wing M, Smith S et al. Pulsed monoclonal antibody treatment and autoimmune thyroid disease in multiple sclerosis. Lancet 1999; 354: 1691. 15. Watson CJE, Bradley JA, Friend PJ et al. Alemtuzumab (Campath 1H) induction therapy in cadaveric kidney transplantation–efficacy and safety at five years. Am J Transplant 2005; 5: 1347–1353.

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