New Biopsy Version
Eculizumab Prevents Recurrent Antiphospholipid Antibody Syndrome and Enables Successful Renal Transplantation
Article first published online: 8 JAN 2014
© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons
American Journal of Transplantation
Volume 14, Issue 2, pages 459–465, February 2014
How to Cite
Lonze, B. E., Zachary, A. A., Magro, C. M., Desai, N. M., Orandi, B. J., Dagher, N. N., Singer, A. L., Carter-Monroe, N., Nazarian, S. M., Segev, D. L., Streiff, M. B. and Montgomery, R. A. (2014), Eculizumab Prevents Recurrent Antiphospholipid Antibody Syndrome and Enables Successful Renal Transplantation. American Journal of Transplantation, 14: 459–465. doi: 10.1111/ajt.12540
- Issue published online: 28 JAN 2014
- Article first published online: 8 JAN 2014
- Manuscript Accepted: 30 SEP 2013
- Manuscript Revised: 16 SEP 2013
- Manuscript Received: 19 JUL 2013
- complement regulation;
- kidney transplantation
Renal transplantation in patients with antiphospholipid antibodies has historically proven challenging due to increased risk for thrombosis and allograft failure. This is especially true for patients with antiphospholipid antibody syndrome (APS) and its rare subtype, the catastrophic antiphospholipid antibody syndrome (CAPS). Since a critical mechanism of thrombosis in APS/CAPS is one mediated by complement activation, we hypothesized that preemptive treatment with the terminal complement inhibitor, eculizumab, would reduce the extent of vascular injury and thrombosis, enabling renal transplantation for patients in whom it would otherwise be contraindicated. Three patients with APS, two with a history of CAPS, were treated with continuous systemic anticoagulation together with eculizumab prior to and following live donor renal transplantation. Two patients were also sensitized to human leukocyte antigens (HLA) and required plasmapheresis for reduction of donor-specific antibodies. After follow-up ranging from 4 months to 4 years, all patients have functioning renal allografts. No systemic thrombotic events or early graft losses were observed. While the appropriate duration of treatment remains to be determined, this case series suggests that complement inhibitors such as eculizumab may prove to be effective in preventing the recurrence of APS after renal transplantation.
atypical hemolytic uremic syndrome
antiphospholipid antibody syndrome
catastrophic antiphospholipid antibody syndrome
complement-dependent cytotoxicity crossmatch
cytomegalovirus immune globulin
calculated panel reactive antibody
end-stage renal disease
flow cytometric crossmatch
Food and Drug Administration
human leukocyte antigen
investigational new drug
membrane attack complex
mean fluorescence intensity
paroxysmal nocturnal hemoglobinuria
thrombotic thrombocytopenic purpura
Antiphospholipid antibody syndrome (APS) is an autoimmune hypercoagulable disorder characterized by large or small vessel thrombosis often in the presence of circulating antibodies directed against phospholipid binding proteins . Renal impairment is associated with primary APS, and for those who progress to end-stage renal disease (ESRD), long-term survival is poor. Dialysis dependence alone is associated with increased mortality, and for APS patients, the risk of exhaustion of vascular access is considerable .
The catastrophic antiphospholipid antibody syndrome (CAPS) is a rare but particularly devastating manifestation of APS that affects approximately 1% of APS patients. A diagnosis of CAPS requires that three specific criteria are fulfilled: vascular occlusions involving three or more organ systems either simultaneously or within less than 1 week, histopathological confirmation of small vessel occlusion in at least one organ or tissue and serologic demonstration of circulating antiphospholipid antibodies (APLA) [3-5]. CAPS frequently culminates in multiorgan system failure and is fatal in 50% of patients in whom it develops . Unlike APS in which both small and large vessel thrombosis is typically seen, CAPS results in diffuse multisystem microvascular thromboses. CAPS is also episodic, and crises are often preceded by a triggering event such as infection, trauma or surgery. Studies of the small number of patients who have survived an episode of CAPS report that up to 40% of those who experienced a recurrent thrombotic event did so in the setting of surgery. The perioperative death rate among these patients is 33% . For this reason there are no published reports of successful renal transplantation among patients with CAPS, although renal failure is common in survivors of this disease .
Mechanistically, at a molecular level, the tissue injury that occurs in APS and CAPS as a result of APLA binding to phospholipid proteins is a complement-dependent process. Complement activation results in the subsequent cleavage of C5 to its effector molecules C5a and C5b. C5a promotes recruitment and activation of neutrophils and monocytes and mediates endothelial cell activation. Binding of C5b to the target initiates the nonenzymatic assembly of the C5b-9 membrane attack complex (MAC) which inserts into cell membranes resulting in either cell lysis and/or the activation of other proinflammatory pathways when at sublytic levels. The resultant deposition of C5b-9 on endothelium has been implicated as the primary biologic process in the pathogenesis of APS-associated thrombosis. If activation of complement with the subsequent deposition of C5b-9 drives large vessel and microvascular thrombosis, then pharmacologic blockade of C5 may prove effective in preventing APS- and CAPS-associated posttransplant complications.
Eculizumab (Soliris®; Alexion Inc., Cheshire, CT) is a humanized mAb that binds the complement protein C5, preventing cleavage into C5a and C5b, thereby preventing generation of the MAC . This biologic has proven to be highly effective in other catastrophic C5b-9-mediated pauci-inflammatory thrombogenic vasculopathy syndromes including atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP) and Degos disease. Here, we report the successful transplantation of three APS patients, two with CAPS, by using prophylactic continuous systemic anticoagulation together with the terminal complement inhibitor eculizumab.
Informed consent and regulatory oversight
For the administration of eculizumab, all three patients were enrolled in a prospective study protocol approved by the Institutional Review Board (IRB) at the Johns Hopkins Hospital. Specific informed consent for participation in the study protocol was obtained from all patients. An investigational new drug (IND) application for off-label use of eculizumab was approved by the Food and Drug Administration (FDA) and the IND was held by the investigators.
One thousand two hundred milligrams of eculizumab was administered on the day prior to transplantation followed by 900 mg on postoperative day (POD) 1, and weekly thereafter for the first month. Beginning on week 5, patients received 1200 mg every 2 weeks and continue on this regimen. For the two patients requiring plasmapheresis for donor-specific antibody (DSA), each plasmapheresis treatment was followed by a dose of 600 mg of eculizumab. This dosing was based on the pharmacokinetic studies performed in these patients (Figure 1) as well as an index case that we treated under a separate IND for severe oliguric antibody-mediated rejection in 2004 .
All three patients were converted from Coumadin to either heparin or bivalirudin infusion. Patients remained therapeutic on heparin or bivalirudin throughout the surgery and perioperative period until plasmapheresis was discontinued, at which time the patients were transitioned back to oral warfarin. Anticoagulation goals were set at the high end of the therapeutic range and correlated with unfractionated heparin anti-Xa assays.
Desensitization and immunosuppression
Patients 2 and 3 were sensitized from previous transplants and required plasmapheresis for removal of DSA. Immunosuppression, plasmapheresis, cytomegalovirus immune globulin (CMVIg; 100 mg/kg) and monitoring of DSA were performed as previously described . Patient 2 received 4 pre- and 19 posttransplant plasmapheresis treatments, and patient 3 received 2 pre- and 7 posttransplant. All three patients received anti-CD20 antibody (rituximab, 375 mg/m2) before transplantation. Induction included daclizumab for patient 1 who was not sensitized and antithymocyte globulin for patients 2 and 3.
All patients were immunized with the meningococcal vaccine (Menactra®; Sanofi-Pasteur, Swiftwater, PA) at least 4 months prior to transplantation. In addition, prophylaxis with either ciprofloxacin or amoxicillin was continued throughout the course of eculizumab administration.
APLA screening and skin biopsy pathology
APLA surveillance was performed using tests for cardiolipin and β2-glycoprotein I (β2-GPI) antibodies. Tests were performed pretransplant, weekly during the inpatient hospitalization and with bi-monthly eculizumab infusions thereafter. Immunofluorescent studies to assess for the deposition of IgG, IgM, IgA, C3, C3d, C4d and C5b-9 were conducted on fresh frozen tissue of the skin according to previously published protocols .
Patient 1 was 51 years old at the time of transplantation. He was diagnosed with CAPS 2 years prior and following a mitral valve replacement for multiple valvular thrombi. The operation precipitated an episode of CAPS with thrombosis and infarction of the liver, spleen and kidneys. The patient was acutely managed with anticoagulation, corticosteroids and plasmapheresis. Although he received massive blood product transfusions at the time of his CAPS crisis, he did not become sensitized. He emerged with renal failure and a chronic autoimmune thrombocytopenia. At the time of referral, he was on low-dose prednisone with a baseline platelet count of 40–50 000/µL. CMVIg and rituximab were administered 6 weeks preoperatively for correction of thrombocytopenia prior to transplantation. With this therapy, the patient's platelet count increased to 170 000/µL at the time of transplant operation. He underwent a live donor renal transplant under the cover of eculizumab in July 2009.
The renal allograft had immediate function and no evidence of thrombosis was noted intraoperatively. Within 2 days of transplantation, serum creatinine (Cr) fell to normal levels (Figure 2). An open renal biopsy performed at the time of re-operation for hematoma evacuation on POD 7 demonstrated only mild inflammation and no evidence of thrombosis or rejection. Immunostaining for the complement MAC revealed granular deposition of C5b-9 within glomerular capillaries but weaker deposition within interstitial peritubular capillaries.
Preoperatively, serum titers of APLA were within normal limits. Anticardiolipin antibody titers remained within normal limits in the postoperative period. However, serum titers of anti-β2-GPI antibodies tripled by POD 4. Anti-β2-GPI IgG, and to a lesser degree IgA, remained elevated throughout the patient's hospitalization as has been previously reported . At nearly 4 years postoperatively, on bi-monthly eculizumab infusions, the patient has serum Cr of 1.2 mg/dL, no evidence of thrombotic events and persistently elevated anti-β2-GPI IgG.
Patient 2 was 39 years old at the time of transplantation. He had developed ESRD due to focal segmental glomerulosclerosis and had previously undergone a deceased donor kidney transplant in 2003. His diagnosis of CAPS was made the following year when he suffered a stroke, a myocardial infarction and lost the renal allograft due to thrombosis. He also underwent a mitral valve replacement for mitral insufficiency related to multiple valvular thrombi. He had become highly sensitized as a result of his previous transplant and multiple blood transfusions. He was both broadly sensitized with flow cytometric crossmatch (FCXM) calculated panel reactive antibodies (cPRAs) of 100% and complement-dependent cytotoxicity crossmatch (CDCXM) of 84%. We found a donor in our paired kidney exchange pool with whom he had positive CDCXM but only at an undiluted titer. In February 2010 after four plasmapheresis treatments and a single dose of anti-CD20, his CDCXM was negative, and he underwent a living donor kidney transplant. The operation was uneventful and he had prompt graft function. Eculizumab was begun the night before transplant and continues presently.
Currently his baseline serum Cr is 2.4–2.6 mg/dL. He is 188 cm tall and received a kidney from a small woman in a paired kidney exchange. His lowest Cr was 2.0 mg/dL. He has had transiently elevated Cr (to 3.6 mg/dL) in the setting of biopsy proven cellular rejection and has been successfully treated with pulse corticosteroids. His early biopsies had glomerulitis and capillaritis but were C4d negative. His DSAs were to HLA-A3, -B65 and -DQA5. The mean fluorescence intensities remained between 10 000 and 20 000 using a single antigen bead assay (OneLambda®; One Lambda, Inc., Canoga Park, CA) throughout his postoperative course. Plasmapheresis was continued for a prolonged period postoperatively due to the persistently elevated DSAs.
Immediately prior to the administration of the eculizumab, the patient had a skin biopsy of normal skin, demonstrating extensive vascular deposits of C5b-9 throughout the dermis (Figure 3A). A few weeks following the administration of the drug, he continued to have prominent deposits of C5b-9 within the cutaneous microvaculature, although he had no clinical signs of CAPS and his graft function was excellent, indicative of effective clinical blockade of C5 despite its persistence in tissue (Figure 3B).
He had two retroperitoneal re-explorations for hematoma evacuation, on POD 6 and on POD 55 following a percutaneous renal biopsy. His postoperative course was also complicated by the development of several large full-thickness necrotic lesions on his legs. Biopsies of the lesions revealed a striking subcutaneous calcific thrombogenic vasculopathy with attendant ischemic necrosis of the skin and subcutis diagnostic of calciphylaxis (Figure 4A); the microvasculature changes involved capillaries, venules and small arteries of the subcutaneous fat comprising vascular thrombosis and endoluminal calcification (Figure 4B).
Seven renal biopsies have been performed over the 3 years since transplantation. Cd4 staining has been negative in all biopsies, though he has had persistent capillaritis. His 1-year biopsy had a cg score of 0, although a “for cause” biopsy at 2 years had a cg score of 1. Titers of anti-β2-GPI and anticardiolipin antibodies had been elevated at the time of his initial CAPS presentation but have not been elevated during the course of his renal transplant.
Patient 3 was 38 years old at the time of renal transplantation. He had developed ESRD 10 years prior when he suffered bilateral renal artery thrombosis from APS in the setting of an appendectomy. He had previously had a deep venous thrombosis and pulmonary embolus and was systemically anticoagulated, but his anticoagulation was discontinued at the time of the appendectomy. He was initially referred to our institution in 2005 and underwent a living-unrelated renal transplant at that time. The transplant was performed with continuous anticoagulation, but despite this, he developed an early acute rise in Cr and biopsy demonstrated diffuse thrombotic microangiopathy consistent with recurrent APS. Anticardiolipin IgG and anti-β2-GPI IgG acutely increased to 51 and 21 U, respectively. He was treated with anticoagulation and plasmapheresis, and ultimately was discharged from the hospital with a marginally functioning renal allograft and a Cr of 3.5 mg/dL. He returned to dialysis after 7 years. He presented for re-transplantation but was sensitized as a consequence of his previous failed transplant and blood transfusions, with cPRA 62% at the level of multianalyte bead assay (Luminex®; Luminex Corporation, Austin, TX). He had a willing living donor for whom he had multiple DSAs but his FCXM was negative. His strongest antibody was specific for HLA-DR53. He underwent desensitization with plasmapheresis and, in February 2013, received a live donor kidney transplant. Eculizumab and rituximab were administered the night before surgery. His renal allograft functioned immediately. His renal function has remained stable with a baseline Cr of 1.6 mg/dL and he continues to receive twice monthly eculizumab infusions. His DSA was present but below the level of FCXM. His APLA titers had been elevated at the time of his appendectomy 10 years prior and during his recurrence after his first transplant, but did not become elevated with this transplant. A biopsy performed for transiently elevated Cr at 6 months posttransplant demonstrated no evidence of rejection, and no thrombotic microangiopathy to suggest any evidence of recurrent APS.
To date, systemic anticoagulation remains the only effective treatment for prevention of APS-associated thrombosis, including APS-associated renal allograft loss. Historically renal transplants in APS patients treated with anticoagulation have had only limited success [14, 15] but survival on dialysis is also poor. The risk of triggering diffuse systemic activation of coagulation heretofore has made CAPS a contraindication to transplantation and any elective surgery. New approaches to treatment of these challenging patients are needed. Here we report the use of the terminal complement inhibitor eculizumab to enable renal transplantation in a patient with APS and two patients with CAPS. We also demonstrate that it is feasible to desensitize patients with APS and recommend an eculizumab dosing regimen that allows for the maintenance of therapeutic serum levels of the drug during plasmapheresis.
The pathophysiology of thrombosis in APS and CAPS remains incompletely understood. Whether the binding of APLA is primarily a cause or a consequence of endothelial cell activation is unclear, but complement cascade activation with subsequent C5b-9 deposition leads to the generation of a potently pro-thrombotic environment [16, 17]. In APS, large vessel thrombosis can be seen, but in CAPS, most of the vascular thrombosis involves the microvasculature, analogous pathologically to prototypic pauci-inflammatory microthrombotic syndromes such as TTP and aHUS. Inhibition of the MAC assembly has been shown to protect against thrombosis in mouse models of immune-mediated thrombotic microangiopathy, and additionally, mice lacking functional C5 or C5a receptors are protected from APLA-induced microvascular thrombosis . Mechanistically eculizumab inhibits the formation of C5a and the MAC component C5b, thereby interrupting complement-mediated pro-inflammatory events.
In addition, MAC at sublytic concentrations stimulates endothelial cells to express adhesion molecules and enhances tissue factor procoagulant activity primarily via the inactive terminal C complex . Furthermore, sublytic C5b-9 deposition induces apoptosis by up-regulating the interferon regulatory factor-1 caspase 8/caspase 3 pathways, an event that leads ultimately to endothelial cell detachment and subsequent activation of the indirect clotting pathway via exposure of type IV collagen . Eculizumab would effectively block these MAC-mediated procoagulant events.
Eculizumab is FDA approved for the treatment of paroxysmal nocturnal hemoglobinuria [21, 22] and for aHUS  and has been used off-label in the treatment of TTP refractory to plasmapharesis . In renal transplant recipients, it has been used for the treatment of a variety of complement/antibody-mediated microangiopathy syndromes including aHUS [24, 25] and antibody-mediated rejection [10-26] and in single case studies of patients with CAPS  and APS .
APS patients often have received large volumes of blood products during their primary episode, had miscarriages and/or had previous transplants that failed due to disease recurrence. As a result, they can be highly sensitized. Eculizumab has previously been shown to provide additional protection against antibody-mediated rejection after desensitization . Since the pathogenesis of APS- and HLA-antibody-mediated rejection overlap and control of humoral responses is paramount to effective therapy for both diseases, it has been intriguing to consider prevention of both conditions with a single therapeutic cocktail. Two patients in the study had lost previous transplants due to recurrent APS and became sensitized. These two patients required extensive desensitization for their second transplants in addition to eculizumab and continuous anticoagulation.
Kidney biopsies can be treacherous in the setting of APS and anticoagulation, so alternative indicators of therapeutic efficacy are needed. We have also used a bioassay as a functional measure of complement blockade and investigated the utility of MAC staining of kidney tissue  and skin biopsies. A previous study demonstrated that skin is an extremely sensitive site for the detection of C5b-9 deposition . In this current study, a biopsy of normal skin prior to the initiation of eculizumab demonstrated prominent deposits of C5b-9 within the microvasculature of patient 2, providing a clear rationale for the administration of the drug despite the absence of elevated APLA at the time of surgery. It should be emphasized, however, that employing the skin and kidney biopsy to monitor treatment response to eculizumab by measuring a progressive decrement in C5b-9 is not useful. In particular, it characteristically takes several months to almost a year for the tissue samples to no longer demonstrate C5b-9 deposition despite the effective blockade of complement almost immediately after the initial administration of the drug. While complement activation is critical to thrombosis in the setting of CAPS/APS, the fact that one patient in our series developed calciphylaxis while on eculizumab shows its inhibition does not preclude the contribution of other pathways that may be independent of complement and serves as a cautionary note.
The limitations of this study include the fact that we were unable to randomize patients to treatment and placebo arms due to the unacceptably high risk of transplantation in APS patients with anticoagulation alone. Although APS patients are at increased risk of thromboembolism, there is no correlation between the degree of antibody elevation among APS patients and thrombosis risk. Thus, negative assays for APLA in APS patients cannot be used as justification to discontinue eculizumab .
We believe that eculizumab shows promise in the prevention of APS-mediated thrombosis, and report here a series of successful kidney transplants in APS and CAPS patients. A recently published study by Canaud et al  demonstrates the efficacy of eculizumab in rescuing renal allografts with plasmapheresis-resistant APS; however, the kidneys were not protected against chronic APS-associated vascular changes appearing after eculizumab was discontinued. We have maintained our CAPS transplant patients on eculizumab therapy indefinitely given their lifelong risk for thrombotic events and the poor outcome associated with recurrent CAPS episodes. For the APS patient, the cost/benefit ratio is less clear. Larger trials are warranted and will be needed to prove its efficacy, and to determine the necessary duration of treatment.
The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Alexion, Inc., which markets eculizumab (Soliris®), provided a research grant and no-cost drug for the study. The company had no direct involvement in the design or performance of the study or preparation of the manuscript.