Objectives: This prospective study was designed to develop a steroid and calcineurin inhibitor-free regimen for kidney transplants using alemtuzumab.

Materials and Methods: A single dose of alemtuzumab (30 mg) was given preoperatively. Phase 1: Twenty-one patients were randomized into 2 groups; the tacrolimus (n=11) and the sirolimus groups (n=10). Steroids were given for 5 days. Azathioprine (1 mg/kg) was added when white blood cells ≥ 4000 cells/cm³. Mean follow-up was 48 ± 2.8 and 48.2 ± 1.6 months for the tacrolimus and sirolimus groups. Phase 2: Twenty patients were included and the study design was modified. Tacrolimus was given for 2 months, and was replaced by sirolimus thereafter. The mean follow-up was 28.3 ± 2.1 months.

Results: Phase 1: Acute rejection episodes were encountered in 5 patients of the tacrolimus versus 2 cases in the sirolimus group (P = .44). Antibody-mediated rejection was diagnosed in 2 recipients in each group. Four patients were switched from sirolimus to tacrolimus owing to resistant rejection, significant proteinuria, persistent thrombocytopenia, lymphocele, and urinary leakage. One patient was shifted from tacrolimus to sirolimus owing to Kaposi sarcoma. Glomerular filtration rate was significantly higher in the sirolimus group. Currently, 14 patients (8 tacrolimus, and 6 sirolimus) are steroid-free. One patient died from the tacrolimus group owing to fulminant hepatitis. Two grafts were lost in the sirolimus group versus 1 graft in the tacrolimus group. Phase 2: Five patients developed successfully treated borderline changes with no antibody-mediated rejection. Mean serum creatinine was 114.9 ± 17.7 µmol/L. Currently, 17 patients are steroid-free and 15 of them are calcineurin inhibitor-free as well. In this phase, only 1 patient died with a functioning graft.

Conclusions: This clinical trial provides a good insight into a potentially effective steroid and calcineurin inhibitor-free protocol with the use of alemtuzumab induction in combination with sirolimus.

Key words : Kidney, Transplant, Campath-1H, Sirolimus

Introduction

Campath-1H, a humanized anti-CD 52 monoclonal antibody, is a powerful lytic agent for T lymphocytes and B lymphocytes. It has been used in renal transplants to facilitate a steroid-free protocol together with significantly reduced calcineurin inhibitor immunosuppressions, thereby potentially minimizing their adverse effects.¹ Recent reports indicated that Campath-1H has been used as an induction therapy in kidney,² pancreas,³ liver,⁴ and lung transplant.⁵ In kidney transplant, Campath-1H was used as an induction agent followed by maintenance therapy in the form of cyclosporine,⁵ tacrolimus,⁶ sirolimus,⁷ or mycophenolate mofetil.⁸

In this prospective trial, our aim was to develop a steroid and calcineurin inhibitor-free regimen in live-donor renal transplant recipients using Campath-1H as an induction therapy.

Patients and Methods

Inclusion criteria
Candidates for a first kidney transplant at Mansoura Urology and Nephrology Center who are legally able to give informed consent. Patients must not have any condition that precludes serial follow-up. The white blood count should be > 4000 cells/cm³ and a platelet count > 100 000 platelets/cm³.

Immunosuppression
Campath-1H infusion
A single dose of 30 mg of alemtuzumab (MabCampath- ILEX Pharmaceuticals Ltd, Guilford, UK) was diluted in 100 mL of normal saline or glucose 5% and given by IV infusion over 2 hours before transplant. Patients were premedicated with acetaminophen, diphenhydramine, and 500 mg of IV methylprednisolone.

Phase 1
A total of 21 patients were randomized into 2 groups: tacrolimus group (11 patients) and sirolimus group (10 patients). Tacrolimus was given in a dose of 0.1 mg/kg aiming for a trough level of 4 to 8 ng/mL, while sirolimus was given in a dosage of 5 mg/d aiming for a level of 10 to 15 ng/mL. Both groups received a short course of steroids for 5 days posttransplant. Azathioprine (1 mg/kg) was added when the white blood cells > 4000 cells/cm³ which was replaced by mycophenolate mofetil 500 mg twice daily owing to high liver enzymes or the onset of acute rejection.

Phase 2
A total of 20 patients were assigned to receive tacrolimus aiming for a trough level of 4-8 ng/mL. Azathioprine in a dose of 1-2 mg/kg was added when the total leukocytic count ≥ 4000 cells/cm³. The steroid course was extended to 14 days and tacrolimus was maintained for 2 months in all patients. Provided that the patient was rejection-free and urinary protein excretion < 0.5 g/d, sirolimus was started aiming for a target level 10-15 ng/mL and tacrolimus was withdrawn at this point.

Follow-up and evaluation
Postoperatively, patients of both phases received prophylaxis for Pneumocystis Carinii with trimethoprim-sulfamethoxazole for 6 months posttransplant. Complete blood count, electrolytes, creatinine, and glucose were monitored at least twice weekly for the first 3 months, weekly for the next 3 months, then twice monthly thereafter. Liver functions and lipid profiles were monitored monthly for the first year. Posttransplant viral, fungal, and bacterial infections were recorded. Cytomegalovirus infections were diagnosed with cytomegalovirus PCR testing and clinical correlation.

Kidney biopsies were done for all episodes of elevated serum creatinine not clearly attributable to dehydration or a high calcineurin inhibitor levels. All rejections were graded as per the Banff Criteria.⁹ Antibody-mediated rejections were diagnosed based on histomorphologic features, C4d staining, and the presence of donor specific antibodies against human leukocyte antigens. Acute cellular rejections were treated with 5 pulses of IV methyl prednisolone (500 mg/d). While for antibody-mediated rejection episodes, a single dose of rituximab (375 mg/1.73m²) and/or intravenous immuno­globulin (500 mg/kg/d) for 5 days were given in addition to 5 sessions of plasma exchange.

The protocol was approved by the ethics committee of our institution before the study began, and conformed to the ethical guidelines of the 1975 Helsinki Declaration. Written, informed consent was obtained from all patients.

Statistical Analyses
Intention-to-treat analysis was performed in all included randomized recipients who underwent transplant and received at least 1 dose of the study medication. Differences between continuous variables were tested by the t test. Differences among categoric variables were evaluated by the chi-square test. Values for P less than 0.5 were considered significant in all statistical tests.

Results

Phase 1
The patient characteristics were essentially similar among the 2 studied groups (Table 1). The mean follow up period was 48 ± 2.8 and 48.2 ± 1.6 months for the tacrolimus and sirolimus groups. Campath-1H was generally well tolerated. Dose elimination and/or reduction were not required. Six patients experienced mild self-limited symptoms indicative of cytokine release. Peripheral lymphocyte depletion occurred within 1 hour of administration. Repopulation was gradually observed 1 month posttransplant. Normal lymphocyte counts were reached approximately 6 months after transplant.

Rejection episodes
Table 2 is a summary of patients who developed acute rejection episodes. In the tacrolimus group, 5 cases experienced acute rejection episodes; borderline in 2, grade IA in 1, and antibody-mediated rejection with positive C4d staining in 2 cases. All these episodes occurred within the first month posttransplant. They were successfully reversed with either steroid pulses alone (borderline and grade IA rejection) or with additional 5 sessions of plasma exchange and 1 dose of rituximab for cases with antibody-mediated rejection. Recipients who developed antibody-mediated rejection or grade IA rejections were maintained on steroid, tacrolimus, and mycophenolate mofetil. Patients who were rejection-free or who developed border line changes remained steroid-free.

In the sirolimus group, 2 recipients developed antibody-mediated rejection within the first month posttransplant. In spite of steroid pulses, plasma exchange, and rituximab, the first patient lost his graft while the other one was salvaged; this patient is currently enjoying normal graft function on triple tacrolimus-based immunosuppression. The remaining 8 patients were maintained on sirolimus monotherapy.

Posttransplant complications
Immediate surgical complications
One week posttransplant, urinary leakage and lymphocele were encountered in a patient of the sirolimus group. Surgical intervention was necessary and maintenance immunosuppression was switched over to tacrolimus.

Infections
In the tacrolimus group, 1 patient caught hepatitis B viral infection 1 year posttransplant. Initially, viral replication was controlled with lamivudine therapy, later; the virus became lamivudine-resistant with development of chronic active hepatitis, in spite of adding entecavir, with progressive hepatic failure. Cessation of immunosuppression was necessary with subsequent deterioration of graft function. In the sirolimus group, 1 recipient developed pulmonary tuberculosis 9 months posttransplant, which responded to a full course of antituberculous therapy, and currently she is enjoying normal graft function on sirolimus monotherapy (3 mg/d) and isoniazid prophylaxis.

Malignancy
Two years posttransplant, a 54-year old man from the tacrolimus group developed extensive cutaneous Kaposi sarcoma of the abdominal wall. The diagnosis was confirmed by skin biopsy. The patient received 8 cycles of IV paclitaxel and intralesional vincristine together with switching to sirolimus and cessation of azathioprine. The lesion significantly regressed and currently the patient is enjoying a normal graft function (SCr 114.9 µmol/L, CrCl 1.63 mL/s) on sirolimus monotherapy (4 mg/d).

Posttransplant diabetes
After transplant, 2 recipients in the tacrolimus group and 3 in the sirolimus group developed new onset diabetes.

Current immunosuppression
A total of 14 patients (8 from tacrolimus group and 6 from sirolimus group) are enjoying a steroid-free regimen (Table 3). Four patients in the sirolimus group were switched to tacrolimus in view of resistant rejection, urinary leakage, massive proteinuria, and severe thrombocytopenia. On the other hand, 1 patient was switched from tacrolimus to sirolimus because of extensive cutaneous Kaposi sarcoma.

Laboratory assessment
A summary of the laboratory findings at the last follow up are shown in Table 4. All the data were comparable except for the mean creatinine clearance, which was significantly higher in the sirolimus group.

Graft and patient survival
In the tacrolimus group, only 1 recipient in whom immunosuppression was stopped after 3 years posttransplant in view of fulminant hepatitis B viral infection, lost her graft and died shortly afterwards with liver cell failure.

In the sirolimus group, no patients were lost. Only 2 patients in this group lost their graft. The first lost his graft owing to early irreversible antibody-mediated rejection. While the other one, who was switched early to tacrolimus because of severe thrombocytopenia, developed severe hemolytic uremic syndrome 1 week postconversion. The lesion partially recovered, and the patient was maintained on steroids and mycophenolate mofetil with gradual decline of graft function over 9 months. Both patients are currently maintained on regular hemodialysis.

Phase 2
The patient characteristics are shown in Table 5. The mean follow-up was 28.3 ± 2.1 months.

Rejection episodes
Out of the 20 patients included in this phase, 5 recipients experienced acute rejection episodes. Three occurred preconversion to sirolimus while the other 2 episodes developed after conversion to sirolimus. Graft biopsies revealed borderline changes, and all of them were successfully treated with steroid pulses. Patients who experienced preconversion rejections were maintained on steroid and azathioprine was switched to mycophenolate mofetil, while in postconversion rejections, patients were switched back to a tacrolimus-based regimen. All rejection episodes were successfully reversed and serum creatinine went back to the prerejection values. The characteristics of the recipients who developed acute rejection episodes are detailed in Table 6.

Posttransplant complications

Tacrolimus nephrotoxicity
This was histologically proven in 3 cases. Two of them had high tacrolimus levels at the time of graft biopsy while the third one had an acceptable tacrolimus trough level. All showed good response to dose reduction.

Infections
Viral infections were encountered in 4 recipients. In 3 patients, cytomegalovirus disease was confirmed clinically and serologically. They received IV ganciclovir for 14 days followed by 3 months of oral treatment to eradicate the infection. One patient developed Herpes zoster and was treated with IV acyclovir for 14 days and maintenance oral therapy for 3 months. One patient developed Pseudomonas bronchopneumonia, which necessitated aggressive culture-based antibiotic therapy.

Proteinuria
Five of the 15 sirolimus-based group developed proteinuria less than 0.5 g/d (0.31 ± 0.08 g/d). All showed significant reduction of urinary proteins with a small dose of angiotensin-converting enzyme inhibitors.

Current immunosuppressions
A total of 17 recipients are enjoying steroid-free regimen. Of them, 15 patients are also enjoying calcineurin inhibitor-free regimen. The remaining recipients included in this phase (3 patients) are maintained on tacrolimus, mycophenolate mofetil, and steroids, owing to occurrence of acute rejection episodes in the first 60 days before conversion to sirolimus (Table 7).

Laboratory assessment
At last follow-up, all recipients had good functioning grafts, acceptable hematologic profile, and acceptable tacrolimus and sirolimus levels. Hyper­cholesterolemia was encountered in only 6 sirolimus-treated patients and was easily controlled with 10 mg of simvastatin daily (Table 8).

Graft and patient survival
A young female, who was maintained on sirolimus monotherapy, developed severe depression and refused to receive her medications. Perfect graft function was maintained for 5 months, and she died thereafter from an assumed suicide.

Discussion

To improve transplant outcomes, new strategies that allow minimization of chronic immunosuppression, while achieving long-term graft function, must be explored. Developed by Waldmann and Hale, Campath-1H is currently the most potent lymphoid-depleting antibody.^{10 11} It was first used as induction therapy for kidney transplant by Calne and associates¹² in combination with minimal cyclosporine-based maintenance immuno­suppression.

In this prospective trial, we aimed for a steroid-calcineurin inhibitor-free regimen using Campath-1H as induction therapy for our live-donor renal transplant recipients. In the first phase of this study, 7 patients (5 tacrolimus-based and 2 sirolimus-based) developed acute rejection episodes within the first month posttransplant. Out of these, antibody-mediated rejection was encountered in 2 recipients in each group. Although the incidence of rejection was high in the tacrolimus group, there was no graft loss secondary to rejection. Currently, 14 recipients are maintained on steroid-free regimen, 6 of them are calcineurin inhibitor-free as well.

Histopathology of their grafts showed scarce lymphocytic infiltration with plasma cells and monocytic predominance. Similar findings were reported by Knechtle and associates¹⁷ who performed a pilot study at the University of Wisconsin with alemtuzumab induction and sirolimus monotherapy in 29 patients. These authors had to modify their protocol for the last 5 cases to include thymoglobulin and a 2-week course of steroids. Nevertheless, of these 5 patients, 3 experienced acute rejection. Similarly, Flechner and associates,¹³ in another pilot trial on 22 patients, used alemtuzumab induction with mycophenolate mofetil and sirolimus maintenance to avoid the use of calcineurin inhibitor and steroids. Acute rejection occurred in 8 patients of which 2 were humoral. The early occurrence of antibody-mediated rejection in the first phase of our study could be explained by an escape of plasma cells and/or monocytes from the powerful depleting effect of alemtuzumab.^{14, 15}

In terms of posttransplant malignancy, only 1 case in phase 1 (2.4%), from the tacrolimus group, developed extensive cutaneous Kaposi sarcoma, which was successfully managed with chemotherapy and conversion to sirolimus monotherapy. Also a low incidence of serious infectious complications was encountered among the recipients of this phase; pulmonary tuberculosis in 1 case of the sirolimus group and acute hepatitis B viral infection in another case of the tacrolimus group. Similar low incidence of malignancy and infections after Campath-1H induction was previously reported by Ortiz and associates¹⁶

In phase 2, the immunosuppression protocol was modified in view of the high incidence of antibody-mediated rejection among recipients in the previous phase. The steroid dosage was extended for 2 weeks together and tacrolimus for 2 months posttransplant. This modification was based on a retrospective analysis reported in the OPTN/UNOS database. Evidence was provided that Campath-1H induction in recipients receiving calcineurin inhibitor and steroids had improved graft function and rejection-free survival, especially those receiving tacrolimus.¹⁷

The incidence and severity of rejection in this phase improved significantly after the former modification. No antibody-mediated rejection was encountered and only easily-reversed borderline changes developed in 5 recipients. Three of them occurred in the first 2 months before conversion to sirolimus while the other 2 developed postconversion. For these 5 patients, tacrolimus was maintained as it was judged that they are in need for more immunosuppression.

Three cases with cytomegalovirus disease and 1 case of Herpes zoster were encountered in the second phase. This low incidence of infectious complications is similar to that of Malek and associates¹⁸ who reported a low incidence of infection with campath-1H induction in comparison to conventional immunosuppression. Non­significant proteinuria (< 0.5 g/d) was encountered in 5/15 recipients who were converted to sirolimus, who were then easily stabilized with a small dose angiotensin-converting enzyme inhibitors.

In phase 2, conversion to sirolimus was feasible in 17/20 recipients 60 days posttransplant. We were not able to convert the other 3 patients owing to the occurrence of early border line changes. Furthermore, 2 of the 17 converted recipients were reswitched to tacrolimus in view of border line changes that occurred after conversion. Currently, of 20 recipients in phase two, 15 (75%) are enjoying steroid-calcineurin inhibitor–free regimen.

In conclusion, antibody preconditioning with alemtuzumab in kidney transplant could allow use of a steroid-calcineurin inhibitor-free regimen. The important limitation of this report is the small number of patients included. However, while the data are encouraging, additional larger trials are required to confirm these relatively favorable outcomes. Meanwhile, this regimen appears to be a reasonably effective and attractive one, with good patient acceptance, excellent patient and graft survival rates. An antiplasma cell agent, such as bortezomib, which has been recently reported to successfully treat antibody-mediated rejection19 could be used preemptively in combination with alemtuzumab and/or therapeutically to overcome the plasma cell escape phenomena.

References:

1. Calne R, Moffatt SD, Friend PJ, et al. Campath IH allows low-dose cyclosporine monotherapy in 31 cadaveric renal allograft recipients. Transplantation. 1999;68(10):1613-1616.
2. Watson CJ, 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(6):1347-1353.
3. Sundberg AK, Roskopf JA, Hartmann EL, Farney AC, Rohr MS, Stratta RJ. Pilot study of rapid steroid elimination with alemtuzumab induction therapy in kidney and pancreas transplantation. Transplant Proc. 2005;37(2):1294-1296.
4. Dhesi S, Boland B, Colquhoun S. Alemtuzumab and liver transplantation: a review. Curr Opin Organ Transplant. 2009;14(3):245-249.
5. Reams BD, Musselwhite LW, Zaas DW, et al. Alemtuzumab in the treatment of refractory acute rejection and bronchiolitis obliterans syndrome after human lung transplantation. Am J Transplant. 2007;7:2802-2808.
6. Shapiro R, Ellis D, Tan HP, et al. Alemtuzumab pre-conditioning with tacrolimus monotherapy in pediatric renal transplantation. Am J Transplant. 2007;7(12):2736-2738.
7. Knechtle SJ, Pirsch JD, H Fechner J Jr, et al. Campath-1H induction plus rapamycin monotherapy for renal transplantation: results of a pilot study. Am J Transplant. 2003;3(6):722-730.
8. Giessing M, Fuller TF, Tuellmann M, et al. Steroid- and calcineurin inhibitor free immunosuppression in kidney transplantation: state of the art and future developments. World J Urol. 2007;25:325-332.
9. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant. 2008;8:753-760.
10. Hale G, Dyer MJ, Clark MR, et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet. 1988;2(8625):1394-1399.
11. Waldmann H. A personal history of the CAMPATH-1H antibody. Med Oncol. 2002;19(suppl):S3-S9.
12. Calne R, Friend P, Moffatt S, et al. Prope tolerance, perioperative campath 1H, and low-dose cyclosporin monotherapy in renal allograft recipients. Lancet. 1998;351(9117):1701-1702. Erratum in: Lancet. 1998;352(9125):408.
13. Flechner SM, Friend PJ, Brockmann J, et al. Alemtuzumab induction and sirolimus plus mycophenolate mofetil maintenance for CNI and steroid-free kidney transplant immunosuppression. Am J Transplant. 2005;5:3009-3014.
14. Kirk AD, Hale DA, Mannon RB, et al. Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (CAMPATH-1H). Transplantation. 2003;76(1):120-129.
15. Buggins AG, Mufti GJ, Salisbury J, et al. Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab. Blood. 2002;100(5):1715-1720.
16. Ortiz J, Palma-Vargas J, Wright F, et al. Campath induction for kidney transplantation: report of 297 cases. Transplantation. 2008;85(11):1550-1556.
17. Huang E, Cho YW, Hayashi R, Bunnapradist S. Alemtuzumab induction in deceased donor kidney transplantation. Transplantation. 2007;84:821-828.
18. Malek SK, Obmann MA, Gotoff RA, et al. Campath-1H induction and the incidence of infectious complications in adult renal transplantation. Transplantation. 2006;81:17-20.
19. Everly MJ, Everly JJ, Susskind B, et al. Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection. Transplantation. 2008;86(12):1754-1761.