Sensitized renal transplant recipients with high levels of donor-specific alloantibody (DSA) commonly develop antibody-mediated rejection (AMR), which may cause acute graft loss or shorten allograft survival. We examined the efficacy of terminal complement inhibition with the humanized anti-C5 antibody, eculizumab, in the prevention AMR in renal transplant recipients with a positive crossmatch against their living donor. The incidence of biopsy-proven AMR in the first 3 months posttransplant in 26 highly sensitized recipients of living donor renal transplants who received eculizumab posttransplant was compared to a historical control group of 51 sensitized patients treated with a similar plasma exchange (PE)-based protocol without eculizumab. The incidence of AMR was 7.7% (2/26) in the eculizumab group compared to 41.2% (21/51) in the control group (p = 0.0031). Eculizumab also decreased AMR in patients who developed high levels of DSA early after transplantation that caused proximal complement activation. With eculizumab, AMR episodes were easily treated with PE reducing the need for splenectomy. On 1-year protocol biopsy, transplant glomerulopathy was found to be present in 6.7% (1/15) eculizumab-treated recipients and in 35.7% (15/42) of control patients (p = 0.044). Inhibition of terminal complement activation with eculizumab decreases the incidence of early AMR in sensitized renal transplant recipients (ClincalTrials.gov number NCT006707).
Donor-specific alloantibody (DSA) against class I and class II human leukocyte antigens (HLA) remains a major barrier to successful renal transplantation. Using desensitization protocols involving multiple plasma exchange (PE) treatments and/or high dose intravenous immunoglobulin (1–3), hyperacute rejection generally has been avoided. However, early antibody-mediated rejection (AMR) primarily occurring with the first month after transplantation has emerged as the next major complication with an incidence of up to 40% using current protocols (2,4). AMR may require aggressive treatment involving multiple PEs and splenectomy to prevent acute graft loss. Even successfully treated AMR is associated with shortened allograft survival (4).
Since the mechanism of AMR appears to involve the activation of complement in the setting of high levels of DSA (5–7), we hypothesized that blocking terminal complement activation might prevent AMR in highly sensitized renal transplant recipients. The current study examined the efficacy of terminal complement blockade in the prevention of AMR using eculizumab, a humanized IgG2/4k monoclonal antibody with high affinity for C5 (8).
The study used an open-label design. Patients were enrolled with informed consent using protocols approved by the Institutional Review Board of the Mayo Foundation and Clinic (ClincalTrials.gov number, NCT006707). The goal was to examine whether eculizumab could reduce the incidence of AMR in the first 3 months after kidney transplant in sensitized recipients with DSA against their living donor.
To assess the presence and amount of DSA, we primarily used cell-based T- and B-flow cytometric crossmatch channel shift (2,9). T-cell antihuman globulin cytotoxicity assays were also performed. The B flow cytometric crossmatch (BFXM) is used primarily by our group because it estimates total DSA against both class I and class II HLA. BFXM data are presented here as channel shifts using a 1024 linear scale. The channel shift is the observed number of channels (raw fluorescence) above the negative control. We previously have published the correlation between the channel shift in our FXM assays to an independent standard (molecules of equivalent soluble fluorochrome units) (2). A solid-phase assay (LABscreen, One Lambda, Canoga Park, CA, USA) was also used to verify alloantibody specificities and amount (reported as the highest mean fluorescence index (MFI) against donor HLA). DSA levels were measured at baseline (prior to any therapy), the day prior to transplant (after multiple PE in patients undergoing pretransplant PE) and posttransplant on days 4, 7, 14, 21 and 28.
Patients and treatment protocols
Inclusion criteria primarily were age greater than 18 years and a baseline BFXM against their living donor with a channel shift <450 and ≥200. The eculizumab group consisted of consecutive recipients transplanted between January 6, 2008 and January 8, 2010 and a historical control group consisted of consecutive transplants between January 1, 2005 and January 10, 2007 that met the inclusion criteria. A history of a splenectomy was an exclusion criterion. In both eras, we excluded patients from these protocols who, in the investigators opinion, had severe cardiovascular or pulmonary disease, or otherwise were too sick to undergo positive crossmatch kidney transplantation. Note, in both time periods, patients with a baseline BFXM >450 were considered to have DSA levels too high for transplantation in our program and thus were excluded a priori.
Eculizumab group patients were immunized against meningococcus and pneumococcus 1 month prior to transplantation. Otherwise, pretransplant management was the same in the two groups. Recipients with a channel shift ≥300 underwent pretransplant PE to achieve both T- and B-flow crossmatch channel shifts <300 on the day of transplant (2). Recipients with a T- and B-flow crossmatch channel shift <300 at baseline received no pretransplant PE. Patients in the control group who received pretransplant PE also underwent 4–14 posttransplant PE treatments. Three patients in the eculizumab group received posttransplant PE early in the study, but this approach was discontinued as it was deemed unnecessary for the prevention of AMR. All patients received induction with antithymocyte globulin (Thymoglobulin®; 1.5 mg/kg/d × 5–7 doses) and maintenance immunosuppression with tacrolimus, mycophenolate mofetil and prednisone as previously described (9).
Renal allograft biopsies were obtained percutaneously using ultrasound guidance, processed for light microscopy and immunofluorescence for peritubular capillary staining for C4d. All biopsies were reviewed by a pathologist (LDC) in a blinded fashion. AMR was diagnosed using standard Banff criteria (10) in combination with graft dysfunction (increase in serum creatinine ≥0.3 mg/dL over nadir). In the control group, all patients had at least one protocol biopsy in the first month after transplantation (between posttransplant day 7 and 14) and most also had a 1-year protocol biopsy. Eculizumab-treated patients underwent biopsies on posttransplant days 7, 14, 28, 90 and 365. Biopsy findings were correlated with DSA levels performed on serum collected within 24 h of the biopsy.
Serum creatinine values were determined almost daily for the first 28 days in all recipients and at least monthly thereafter.
The eculizumab dosing regimen was modified from that used in the treatment of paroxysmal nocturnal hemoglobinuria and consisted of 1200 mg immediately prior to transplantation, 600 mg on postoperative day 1, and 600 mg weekly thereafter for 4 weeks (Figure 1). At week 4, assessment of DSA levels was performed. Eculizumab was discontinued in patients whose DSA had significantly decreased (B flow crossmatch channel shift <200). In patients with persistently high DSA and thus believed to have continued high risk for AMR, eculizumab treatment continued (1200 mg week 5, and then every 2 weeks). Another DSA assessment was performed at week 9 and eculizumab was discontinued if the B flow crossmatch channel shift was <200. In three patients who received PE posttransplant, an additional dose of eculizumab was administered (600 mg) immediately after PE to ensure eculizumab levels were sufficient to completely block terminal complement activity.
The pharmacokinetics of eculizumab were determined in two ways: (1) serum drug levels were determined using a validated enzyme-linked immunosorbent assay that detects both free and C5-bound eculizumab (11) and; (2) activity was determined as the ability of the eculizumab-treated patient's serum to lyse chicken erythrocytes in a validated total human serum–complement hemolytic assay (12).
The primary endpoint of the study was the incidence of AMR in the first 3 months after living donor kidney transplantation. Secondary endpoints were (1) incidence of AMR at 30 min, 4 days, 7 days, 14 days, 1 month, 3 months, 6 months and 12 months posttransplant; (2) the incidence of AMR in patients with B-cell FXM >350 at any time point in the first 12 months after transplant; (3) in patients with B-cell FXM >350 at any time point in the first 12 months after transplant, the percentage of patients demonstrating an increase in serum creatinine ≥0.3 mg/dL from nadir determined within the first week posttransplant throughout the duration of the 3-month dosing period; (4) incidence of delayed graft function posttransplant (defined as the requirement of dialysis within the first week posttransplant; (5) percentage of patients requiring splenectomy; (6) creatinine clearance (calculated) at 6 and 12 months; (7) patient and graft survival at 3, 6, 9 and 12 months; (8) incidence of transplant glomerulopathy at 12 months after transplant; (9) incidence of acute cellular rejection (ACR) at 1, 2, 3, 6 and 12 months; (10) number of patients whose FXM remained elevated (>200) to warrant 12 months of treatment; (11) number of patients whose FXM decreased sufficiently (<200) to warrant discontinuation of the drug prior to the 12-month mandatory drug discontinuation and (13) wound infections and other infectious complications. Since the use of PE posttransplant was discontinued after the first transplant in the eculizumab group, this should be considered a poststudy initiation protocol change.
Numerical data were expressed as mean ± standard deviation and nominal data by counts and percents. The proportions of nominal data were tested using Fisher's exact test, two-tailed; numerical data, means were compared using pooled t-test (two sample). A p value of < 0.05 was considered statistically significant.
One patient in each era met the inclusion criteria by baseline crossmatch data, but did not achieve a crossmatch <300 despite multiple PE and thus did not proceed to transplantation. All other patients during these time periods who had a BFXM <450 and ≥200 were included in this study. There were no differences in the baseline demographics in the eculizumab group (n = 26) and the control group (n = 51) (Table 1) with respect to sex, age, ethnicity, cause of renal failure and pretransplant PE management. Baseline DSA levels were similar in the two groups (B flow cytometric crossmatch channel shift = 330 ± 84 in eculizumab vs. 327 ± 73 in controls, p = 0.85) and solid-phase assay (MFI 7188 ± 3503 in eculizumab vs. 6473 ± 4946 controls, p = 0.51, see Table S1 for detailed DSA data). More patients in the eculizumab group had a previous transplant compared to controls (50% (13/26) vs. 25%, (13/51); p = 0.043). No patient was lost to follow-up or removed from the study for any reason.
Table 1. Baseline demographic characteristics and alloantibody data in all patients
Eculizumab group (n = 26)
Control group (n = 51)
1Race or ethnic group was self-reported; patients receiving pretransplant plasmapheresis were those with a baseline B- or T-cell flow cytometric crossmatch channel shift >300.
Age in years at time of transplantation (mean ± SD)
48.6 ± 12.5
48.4 ± 11.5
Cause of renal failure
Prior kidney transplant
Pretransplant donor-specific alloantibody levels
B flow crossmatch
Baseline channel shift (mean ± SD)
330 ± 84
327 ± 73
Single antigen bead assay Baseline mean fluorescence index (mean ± SD)
7188 ± 3503
6473 ± 4946
Anti-class 1 only
Anti-class 2 only
Both anti-class 1 and 2
Pretransplant plasma exchange
Number of patients receiving
Number of PEs (mean ± SD)
4.0 ± 3.6
3.7 ± 3.4
Patient and graft survival
All patients had at least 3 months follow-up (eculizumab group, mean = of 11.9 ± 6.1 months, range 3.0–27.5; control group, mean = 48.8 ± 14.1 months, range 7.8–69.8) (Table 2). In the historical control group, one patient lost their graft at 1 month due to early AMR and another with a functioning graft died due to a bowel perforation at 7.8 months. Sixteen patients in the eculizumab group had 1-year follow-up and 50 in the control group had 1-year follow-up. No patient in the eculizumab group lost their graft in the first year. Graft survival at 1 year was 100% in the eculizumab group and 96% in the control group. One-year protocol biopsy was obtained in 15 eculizumab-treated patients (1 refused) and 42 control patients (4 refused and 3 were on anticoagulation, one had died and another had lost their graft early).
Table 2. Posttransplant outcomes in the eculizumab-treated and control groups
Eculizumab group (n = 26)
Control group (n = 51)
1B flow crossmatch channel shift >350 at any time point in the first 3 months.
11.8 ± 6.3
48.8 ± 14.1
(mean months ± SD, range)
Graft survival at 1 year (n, %)
Antibody-mediated rejection ≤ 3months (n, %)
Patients developing high DSA levels ≤ 3 months1
High DSA biopsies C4d+ (n, %)
High DSA and C4d+ biopsies showing AMR (n, %)
Cellular rejection ≤3 months (n, %)
Plasma exchange posttransplant
Patients receiving PE (n, %)
Number of PE treatments (mean ± SD)
0.35 ± 1.1
7.9 ± 7.5
Splenectomy (n, %)
Graft dysfunction in first month (mg/dL) (maximum serum creatinine – nadir serum creatinine)
0.45 ± 0.37
0.93 ± 1.15
Histology at 1 year
Transplant glomerulopathy incidence (n, %)
Cg score (mean ± SD)
0.20 ± 0.78
0.74 ± 1.13
Ci score (mean ± SD)
1.00 ± 0.76
0.79 ± 0.80
Ct score (mean ± SD)
1.13 ± 0.74
0.91 ± 0.80
Cv score (mean ± SD)
0.80 ± 0.68
0.59 ± 0.74
The incidence of AMR in the first 3 months was 7.7% (2/26) in the eculizumab group and 41.2% (21/51) in the historical control group (Table 2, p = 0.0031) with all episodes occurring within 1 month of transplant. The percentage of patients who developed high levels of DSA (B FXM >350 and/or MFI >10 000) in the first 3 months after transplantation was similar in both groups (43% in controls and 50% in the eculizumab group, p = 0.63). Evidence of proximal complement activation on biopsy (C4d+ immunostaining) was observed in 91% of control and in 100% of eculizumab-treated patients who developed high levels of DSA early after transplantation. In the control group, all patients with high levels of DSA and C4d+ staining (20/20) showed AMR while only 15% of patients (2/13) in the eculizumab group with this combination showed AMR (p < 0.001). All cases of AMR in both groups were accompanied by an increase in serum creatinine from baseline of >0.3 mg/dL and thus would be considered clinical AMR. One patient in the control group with an increase of 0.2 mg/dL showed clear evidence of AMR on biopsy and thus was designated as AMR.
The two cases of AMR in the eculizumab group occurred on posttransplant day 7 and 14, respectively, in the setting of an increasing serum creatinine, increased DSA (BFXM >400 and MFI>14 000), and a biopsy that showed both C4d+ staining and glomerular microthrombi (13,14). PE was instituted resulting in the resolution of the histologic features of AMR within 1 week, decreased serum DSA and a decrease in serum creatinine without the need for dialysis.
Histology in the setting of high serum DSA levels
Figure 2A shows typical changes of AMR in a control group patient with high levels of DSA. In addition to the light microscopic changes of AMR, the biopsy showed C4d+ immunostaining of peritubular capillaries and ultrastructural endothelial cell changes typical of AMR (loss of fenestrations, cell enlargement, or microvillous changes in glomerular and peritubular capillaries). Figure 2B shows typical microscopic findings in an eculizumab-treated patient with similar levels of DSA 5 days posttransplant. While the biopsy was C4d+, the light and electron microscopy showed normal features.
Other early outcomes
Graft dysfunction and the need for PE and splenectomy: The eculizumab group generally demonstrated stable renal function during the first month. For example, the mean increase in serum creatinine from the posttransplant nadir in the first month in the eculizumab group was less than that of the control group (0.45 ± 0.37 mg/dL vs. 0.93 ± 1.15, p = 0.05, Table 2).
Posttransplantation, 76% of patients in the control group underwent posttransplant PE compared to only 12% in the eculizumab group (Table 2, p < 0.0001). The first patient transplanted in the eculizumab group developed high levels of DSA posttransplant despite daily PE therapy, yet the serum creatinine remained stable and biopsies did not demonstrate AMR. Based on this case, the protocol was modified and the practice of routine, preemptive PE was discontinued. Indeed, 77% (10/13) of patients with a high BFXM posttransplant did not undergo PE. The development of severe AMR required aggressive intervention. Splenectomy was performed for severe AMR in the setting of a rising serum creatinine (usually >2.0) and a rising serum DSA levels despite daily PE treatments. Splenectomy was performed in nine (17.7%) patients in the control group, including 43% (9/21) of the patients that developed AMR, but was not required in any eculizumab-treated patients (p = 0.025) including the patients that developed AMR.
Duration of eculizumab treatment and DSA levels over time
By protocol, eculizumab was discontinued if the DSA level was low (i.e. B flow crossmatch channel shift <200) at either 4 or 8 weeks. In the 16 patients followed for 1 year, eight discontinued eculizumab at 4 weeks, six discontinued drug at 8 weeks, including one patient who developed AMR. Two patients continued eculizumab for 1 year. No patient developed acute AMR after discontinuation of eculizumab.
At 3, 6 and 12 months, the mean serum creatinine was similar in the historical control group and the eculizumab group. Three eculizumab patients had serum creatinine levels >2.0 mg/dL at 1-year follow-up. One patient had evidence of transplant glomerulopathy and a creatinine of 3.9 mg/dL. The other two patients did not have transplant glomerulopathy and had only mild chronic changes with serum creatinines of 2.4 and 2.3 mg/dL. Detailed posttransplant serum creatinine and DSA data are presented in Table S2. Serum creatinine levels were similar at all time points. DSA levels generally were lower in both groups at 6 months compared to baseline.
On the 1-year protocol biopsy, the incidence of transplant glomerulopathy, the histologic lesion most closely associated with chronic antibody-mediated damage was decreased in the eculizumab group (6.7%, 1/15) compared to the control groups (36%, 15/42), (p = 0.044, Table 2). Detailed biopsy data are presented in Table S3. The mean chronicity scores (including the mean cg score) were similar in the two groups at all time points. The patient in the eculizumab group that had TG at 1 year (#10) received eculizumab for a full year. This patient had persistent decline in renal function and returned to dialysis by 2 years after transplantation.
In the eculizumab group, there was one episode of subclinical acute cellular rejection (Banff grade Ia) identified on a 3-month protocol biopsy that was successfully treated with bolus methylprednisolone. There was one wound infection in the eculizumab group. One patient in the eculizumab group developed Burkitt's lymphoma 2.5 years after transplantation and died with a functioning graft after deciding against chemotherapy treatment.
The dosing regimen generally was sufficient to completely block terminal complement throughout the treatment period (Figure 3). However, in one patient, terminal complement activity was not blocked at the end of the initial dose period correlating with a low eculizumab level.
AMR has emerged as one of the most significant complications of kidney transplantation in highly sensitized patients. Most series report an incidence of 30–40% in the first few months after transplant with the incidence of early AMR increasing with increasing DSA levels at baseline (1–3). In the current study, using a BFXM between 200 and 450 as the inclusion criterion, the incidence of AMR in our historical control group was 41.2%. Similarly, a recent study using solid-phase assays to estimate the amount of DSA found that the incidence of AMR was 36.4% in patients with an MFI between 3001 and 6000 at baseline and 51.3% for MFI >6000 (13). All except two eculizumab-treated patients had MFI >3000 at baseline. Thus, an AMR rate of 7.7% in the current study using eculizumab represents a significant advance in the management of this common and often difficult-to-treat posttransplant complication. This study also extends previous reports suggesting efficacy in a rat cardiac allograft model (14) and one case report (15).
As expected, eculizumab did not appear to have an impact on DSA levels after transplantation. The percentage of patients who developed high levels of DSA (B FXM >350 and/or MFI >10 000) in the first 3 months after transplantation was similar in both groups. However, all patients with high levels of DSA after transplantation in the control group showed AMR while only 15% of patients in the eculizumab group with high DSA after transplantation showed AMR. Eculizumab also did not appear to prevent the activation of proximal complement components as all patients with high posttransplant DSA showed C4d deposition in the allograft. Thus, eculizumab prevented the development of AMR in the vast majority of patients that we would have expected to develop AMR, i.e. those with high levels of DSA in the first month after transplantation.
The mechanism of the two cases of AMR is unclear, but could be due to the lack of terminal complement blockade at the level of the allograft or via a pathway that is either complement independent or only involves proximal complement components.
Eculizumab treatment also simplified the posttransplant management by decreasing the need posttransplant PE. In addition, fewer patients developed graft dysfunction as a result of the decreased AMR rate. Both episodes of AMR were easily treated with PE and did not require splenectomy. Eculizumab was well tolerated and did not result in treatment-related infections despite concomitant use of immunosuppressive therapy. The dosing regimen resulted in the complete blockade of terminal complement similar to that observed in patients with paroxysmal nocturnal hemoglobinuria where the drug has been shown to be almost universally effective in preventing the clinical expression of disease (8).
The primary endpoint of this study was the incidence of AMR in the first 3 months after transplantation and any long-term data should be interpreted with caution at this juncture. Early AMR has been associated with chronic histologic changes such as transplant glomerulopathy (4,16), and the decreased incidence of TG at 1 year is certainly encouraging. However, the patient with TG at 1 year went on to develop graft loss during the second year. Thus, longer follow-up will be needed to determine the impact of eculizumab on the incidence and severity of chronic changes. Since persistently high DSA levels have been associated with decreased renal allograft survival (17), developing therapies aimed at reducing antibody in this subset of patients might be a useful addition to the protocol used in this study. The need to continue eculizumab therapy for months after transplantation in patients who have persistently high DSA also requires further study.
This study has several limitations. First, the use of a historical control group raises the question of whether or not unrecognized biases affected the outcome of two groups. We contend that the two groups are comparable and were treated similarly except that the treatment group received eculizumab and underwent posttransplant PE less frequently. However, a prospective, randomized trial will be needed to validate these initial findings. Second, given the variability of crossmatch assays between different centers, these data should be generalized with caution. Specifically, the current study does not show efficacy in patients who have high-titer T-cell cytotoxicity crossmatches. However, we suggest that our BFXM data can be compared to other center's data if MESF standardization is used.
We conclude that eculizumab significantly decreases the incidence of AMR in highly sensitized renal allograft recipients avoiding significant morbidity. Mechanistically these data suggest that in most cases, terminal complement activation is necessary for the development of the histologic and clinical manifestations of AMR. Further study is needed to determine if overcoming this early barrier will lead to improved long-term outcomes.
MDS has research contracts with Alexion Pharmaceuticals (sponsor for this study) and Millennium Pharmaceuticals.