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Durability of Antibody Removal Following Proteasome Inhibitor-Based Therapy

Everly, Matthew J.1; Terasaki, Paul I.2; Trivedi, Hargovind L.3

doi: 10.1097/TP.0b013e31824612df
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Background. Evidence of the short-term effect of bortezomib on donor-specific human leukocyte antigen (HLA) antibody (DSA) removal capacity has emerged. However, no published data characterize the durability of DSA response. Here, we report the long-term DSA response results on renal transplant patients treated with bortezomib.

Methods. In this single-center study, 26 living-donor renal transplant patients with a positive level of de novo DSA were preemptively treated with bortezomib (1.3 mg/m2×4 doses). A total of 15 patients received bortezomib as part of a combination regimen; 11 received bortezomib alone. Weekly serial measurements of HLA antibody were noted before, during, and after treatment using single-antigen beads.

Results. At a median follow-up of 25.8 months posttreatment, allograft function remained good in each of the patients. Following treatment, 96% of the patients achieved at least a partial response. Eighteen patients (69%) experienced a complete response followed by a period of DSA remission. Ten patients had DSA relapse after remission, at a median of 3.8 months. The remaining eight patients are still in remission at 14 months posttreatment (median). Patients with remission enjoyed better allograft functional stability than those who relapsed (P=0.023). After bortezomib therapy, the addition of a calcineurin inhibitor or mycophenolate mofetil was predictive for maintaining a DSA remission (hazard ratio 0.09, 95% confidence interval 0.01–0.76).

Conclusions. Bortezomib therapy consistently provides reduction in DSA and in many a DSA remission may occur. However, sustaining remission is likely necessary to improve allograft stability.

1One Lambda Inc., Research 2 Division, Los Angeles, CA.

2Terasaki Foundation, Los Angeles, CA.

3Department of Nephrology and Transplantation Medicine, Institute of Kidney Diseases and Research Centre (IKDRC)-Institute of Transplantation Sciences (ITS), Ahmedabad, India.

The authors declare no funding. P.T. is the chairman and a major shareholder in One Lambda Inc., a company that produces HLA antibody testing kits used in this study.

M.E. is an employee of One Lambda Inc., a company that produces HLA antibody testing kits used in this study.

Address correspondence to: Matthew J. Everly, Pharm.D., One Lambda Inc., Research 2 Division, 11570 W. Olympic Blvd, Los Angeles, CA 90064. E-mail: meverly@onelambda.com

H.T. treated the patients; P.T. and M.E. supervised the HLA antibody testing and analyzed the data; and M.E. and P.T. wrote the manuscript.

Received 29 August 2011. Revision requested 8 November 2011.

Accepted 9 December 2011.

In the past 10 years, epidemiologic studies have demonstrated that approximately 5% of primary renal transplant recipients will develop de novo donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) within the first year and continually thereafter (1). Because the rate of DSA positivity accumulates, the majority of patients will become positive posttransplant (26). This is concerning given that a major cause of allograft loss in transplant is DSA (7, 8). Fortunately with the advent of solid-phase (single-antigen bead) monitoring tests, early detection of the onset of DSA production is possible and has been recommended as a way to improve long-term transplant outcomes (9). However, monitoring alone will only identify patients at highest risk, and immunosuppression modifications for those at risk patients will be necessary. One such modification that has improved long-term outcomes in lung transplant recipients is preemptive monitoring and treating DSA with rituximab followed by monthly treatments with intravenous immune globulin (IVIg) and plasmapheresis (10). However, IVIg-based therapy is costly and the durability of antibody reduction is unknown.

Beginning in 2008, transplant practitioners began using bortezomib, a proteasome inhibitor, as a new option for treatment of antibodies and antibody-mediated injury (11, 12). Bortezomib has been shown to have significant apoptotic effect on alloantibody-specific plasma cells (1214), while leaving vaccine-related protective immunity intact (15). Based on published case reports, it is estimated that more than 150 transplant patients have been treated with bortezomib to date. Although a small number of patients have failed to show a decrease in DSA levels, the majority of patients experience both allograft stabilization and antibody reduction following treatment with bortezomib (16).

On the basis of these early case reports, proteasome inhibition and specifically the use of bortezomib has become an increasingly accepted treatment strategy to combat HLA antibodies and antibody-mediated rejection. However, to date, long-term data describing relapse rates and overall durability of bortezomib-based therapy have not been documented. This study presents the short- and long-term DSA response in patients treated with a bortezomib-based combination regimen or bortezomib-alone.

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RESULTS

Patients and Treatment

In 26 patients, bortezomib-based regimens were used preemptively (before serum creatinine increase) to remove donor-specific anti-HLA antibodies. Table 1 shows baseline characteristics of all 26 patients. Most (88%) patients developed DSA in the first year posttransplant. The majority (54%) of these patients had class II DSAmax antibody, most of which (71%) were DQ-loc specificities. DSA was considered de novo in all patients; however, all patients were exposed to donor antigen within the weeks immediately before transplant through the use of donor-specific transfusion(s). The median patient age was 28 years; 93% were men. The median follow-up of the 26 patients is 25.8 months posttreatment. To date, no treated patient has lost his or her allograft.

TABLE 1

TABLE 1

The median duration between DSA appearance and initiation of bortezomib therapy was 30 days. All patients received corticosteroids at the time of bortezomib therapy. Bortezomib-based combination therapy was used in 15 patients (bortezomib+plasmapheresis+rituximab, n=9; bortezomib+plasmapheresis, n=5; bortezomib+IVIg, n=1). One patient in the bortezomib-based combination therapy group received two bortezomib cycles consecutively. The nine remaining patients received one cycle of bortezomib alone (bortezomib-alone group).

Of the 26 patients treated, no patients discontinued therapy because of adverse events. Additionally, no grade 3 or 4 hematologic or nonhematologic toxicities occurred. Patients were not routinely administered surveys to analyze lower grade toxicities. Therefore, no analysis can be done with respect to lower grade toxicities.

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DSA Response Following Bortezomib

Analyzing all 26 patients together, a bortezomib-based therapeutic regimen caused a statistically significant reduction in DSA intensity (P=0.002) by 1 year posttreatment (Fig. 1). The types of response for all patients combined and divided into the bortezomib-based combination and bortezomib-alone patient groups are shown in Table 2. Complete response is defined as obtaining DSA removal. Partial response is defined as a 50% reduction in DSAmax but not DSA removal.

FIGURE 1

FIGURE 1

TABLE 2

TABLE 2

Twenty-five out of 26 patients achieved complete or partial response to a bortezomib regimen. The median time to response was 1.5 months. Eighteen patients had a complete response. An analysis of the degree and time of DSAmax response for the patients revealed a partial response was achieved by 1 month in 11 patients, between 1 and 2 months in 3 patients, and between 2 and 3 months in 10 patients. One additional patient achieved a partial response beyond 3 months. One patient remained refractory at last follow-up.

When comparing bortezomib-alone to bortezomib-based combination therapy, rates of partial and complete response were similar. Time to any response (partial or complete) was slightly faster in the bortezomib-based combination group, but this was not statistically different from the bortezomib-alone group (median 29 vs. 88 days, respectively). Bortezomib-alone-treated patients achieved a partial response by 1 month in 36% of patients, between 1 and 2 months in 9% of patients, and between 2 and 3 months in 45% of patients. Bortezomib-based combination patients achieved a partial response by 1 month in 46% of patients, between 1 and 2 months in 13% of patients, and between 2 and 3 months in 33% of patients. The one refractory patient was treated with bortezomib-based combination therapy.

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Durability of DSA Remission Following Bortezomib

The goal of treatment in the 18 patients who achieved complete response was remission. At last follow-up, eight patients remain in remission. The median time of remission in these patients is 14 months. The remaining 10 patients (56%) who achieved a complete response relapsed. The median time to relapse was 3.8 months. The rate and median time to relapse did not differ based on the use of bortezomib-alone or bortezomib-based combination therapy (Fig. 2).

FIGURE 2

FIGURE 2

Achievement of DSA remission was correlated with better allograft function at last follow-up. For those patients in remission, the median time of DSA freedom (after achievement of complete remission) is 14.4 months (2.3–33.2 months). In those who relapsed, the median time of DSA freedom was 3.8 months (0–12.5 months; Fig. 3). The baseline mean serum creatinine in the remission, relapsed, and noncomplete response groups was 1.32±0.20, 1.20±0.22, and 1.01±0.13, respectively. At last follow-up, the mean serum creatinine was 1.42±0.24, 1.58±0.38, and 1.68±0.69, respectively. The median change in serum creatinine (from treatment to last follow-up) in the remission patients is +6.5% (range −14% to +20%), compared with +41% (−29% to +108%) in the relapsed patients (Fig. 3, Mann-Whitney P=0.023).

FIGURE 3

FIGURE 3

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Prognostic Factors

A complete response to bortezomib therapy was not influenced by age at transplant, degree of HLA mismatch, pretreatment immunosuppression, concomitant therapy, posttreatment immunosuppression, time from transplant to antibody appearance, time from antibody appearance to treatment, serum creatinine at time of treatment, initial DSAmax mean fluorescence intensity (MFI), peak DSAmax MFI, and DSA class. A rapid partial response (achieving 50% reduction within 1 month of treatment) was the one variable that was loosely associated with a complete response (odds ratio 8.75, 95% confidence interval 0.88–86.6). Conversely, a high starting DSAmax MFI (>5000 MFI) was found to loosely associate with a lack of complete response (odds ratio 0.3, 95% confidence interval 0.05–1.70).

As with the complete response analysis, age at transplant, degree of HLA mismatch, pretreatment immunosuppression, concomitant therapy, posttreatment immunosuppression, time from transplant to antibody appearance, time from antibody appearance to treatment, serum creatinine at time of treatment, initial DSAmax MFI, peak DSAmax MFI, DSA class, and partial response at 1 month were not found to predict remission. The only factor found to predict continued remission on univariate analysis was the addition of a calcineurin inhibitor (CNI) or mycophenolate mofetil (MMF) at the time of bortezomib therapy. The seven patients treated with a MMF or CNI addition benefited increasing their days of DSA-free survival by 91% (relapse hazard ratio 0.09, 95% confidence interval 0.01–0.76).

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DISCUSSION

In this study, we evaluated the efficacy of bortezomib-based therapy in patients with DSA before evidence of allograft dysfunction. The overall response rate (partial plus complete response) was high (96%). Sixty-nine percent of patients achieved a complete response and this response lasted for an average of 9 months. At last follow-up, the ultimate goal of remission following complete response was achieved in only eight patients. Five have maintained a DSA-free period for over 2 years following complete response. Remission was most likely in patients who had addition of a CNI or MMF at the time of bortezomib therapy. Additionally, in those patients who achieved remission, the change in creatinine from treatment start to last follow-up was significantly better.

The data in this article demonstrate that bortezomib therapy does have a high rate of response. Achievement of a partial or complete response with therapies such as IVIg, rituximab, and plasmapheresis has been shown to occur at a rate of 33% to 60% in acute rejection (1719) and complete response has been shown to be as high as 64% with IVIg+rituximab in preemptive treatment of DSA in lung transplant patients (10). In this study, bortezomib therapy resulted in partial or complete response in 96% of patients and complete response in 69% of patients. This shows that improved or at least equivalent efficacy can be achieved with bortezomib, although comparative studies are warranted to confirm this finding. This is an important preliminary finding given that bortezomib is lower in cost per treatment than IVIg therapy.

However, despite completely removing antibodies in approximately two thirds of patients, not all patients achieved complete response. This indicates that further studies are needed to find the optimal strategy for using bortezomib. One cycle as used in these patients may not be enough. Based on the result from a desensitization trial from the Mayo Clinic, as many as four cycles may be needed to decrease multiple antibodies (14). Also, high DSAmax MFI or class II antibodies may be more resistant to therapy (20).

Our finding that patients with relapse have a greater increase in serum creatinine than those in remission indicates that in addition to removal, sustaining remission may also be important to long-term posttransplant success. From this study, one factor that may improve remission was the use of increased immunosuppression (MMF or CNI) after bortezomib. Bortezomib leads to response in nearly all patients and a complete removal in many, yet its durability varies. In patients with no immunosuppression change after treatment, the period of remission was short. Only those who had a concomitant increase in baseline immunosuppression (MMF or CNI) had persistent remission beyond 1 year. The one exception was a patient with low MFI at baseline who never rebounded despite the lack of immunosuppression change. This durable removal and remission resulted in stability of allograft function. Conversely, in those patients who relapsed, allograft function worsened. This is the same result Hachem et al. found (10), which reinforces the fact that removal, not reduction, should be the goal in treating antibodies.

Several limitations of this trial should be noted. First, comparisons between bortezomib-alone versus bortezomib-combination therapy patients are not statistically powered to show a difference. A larger trial is warranted to prove or disprove these preliminary findings. Second, these patients are preemptively treated with bortezomib and therefore the results of this report may differ from that seen in the acute rejection setting.

In conclusion, the proteasome inhibitor bortezomib induces clinically significant responses, with minimal side effects (11, 12, 2123). Several randomized trials to confirm these findings are ongoing. The results shown in this report along with the future clinical studies should provide guidance on how to manage patients with de novo DSA following transplant.

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MATERIALS AND METHODS

Patients

Study patients were at least 18 years of age and were preemptively treated with bortezomib to treat DSA. Measurable (positive) DSA was defined as at least one DSA specificity above 1000 MFI. In all cases, allograft dysfunction (i.e., serum creatinine increase) was not present at time of HLA antibody detection and therefore biopsies were not conducted to detect subclinical rejection.

All patients were recipients of a living-donor kidney transplant between January 2008 and June 2009 at the Institute of Kidney Diseases and Research Centre-Institute of Transplantation Sciences (IKDRC-ITS), Ahmedabad, India. All patients underwent transplantation under clonal stimulation-deletion protocol described in a previous publication (22). Within 1 week of transplantation, patients were on prednisone alone, or off all immunosuppression. Prednisone dosing ranged from 0 to 20 mg per day. At the time of transplant, all patients were compliment-dependent cytotoxicity, crossmatch, and flow cytometry T- and B-cell crossmatch negative.

All patients were consented to the use of bortezomib. Institutional review board approval was obtained for use of their data.

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Study Design and Treatment

All patients received bortezomib (1.3 mg/m2 of body surface area) as an intravenous bolus (taking 3–5 sec to administer) twice a week for 2 weeks, on days 1, 4, 8, and 11 of a 21-day cycle. All bortezomib doses were accompanied by a single dose of intravenous methylprednisone, 125 mg. In those patients who concomitantly received plasmapheresis (n=14), two to four sessions were conducted during the bortezomib cycle.

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Anti-HLA-Specific IgG Antibody Testing

Anti-HLA antibodies were monitored weekly using single-antigen bead panels by Luminex assay (LABScreen, One Lambda, Canoga Park, CA). LABScreen assay was performed according to the manufacturer's protocol. Briefly, 20 μL of test serum was incubated with the beads for 30 min at room temperature in the dark. All samples were diluted 1:3 in 1× phosphate-buffered saline. Next, samples were washed and 100 μL of 1:100 anti-human-IgG-PE was added. After a second incubation step, samples were washed twice and the samples were read on the LABScan100 flow analyzer (One Lambda). Trimmed mean fluorescence values were obtained from the output file generated by the flow analyzer and normalized using the formula described in the LABScreen single antigen product insert: ([sample-specific florescent value for bead #N−sample-specific fluorescent value for negative control bead]/[background negative control serum fluorescent value for bead #N−background negative control serum fluorescent value for the negative control bead]).

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Definitions

DSAmax is defined as the antibody specificity that arises first posttransplantation. If multiple antibody specificities arise at the same sample point, the antibody with the highest MFI is considered the DSAmax. All other antibodies that arise later or at lower MFI values are considered secondary. DSA removal is defined as decreasing the DSAmax MFI to less than 1000. Complete response is defined as obtaining DSA removal. Partial response is defined as a 50% reduction in DSAmax but not DSA removal. DSA remission (DSA freedom) is defined as the time when all DSA (DSAmax and secondary DSA) are below 1000 MFI. The DSA remission time begins when a complete response is achieved and ends when any DSA (DSAmax, secondary) reappears or a new DSA specificity appears. This DSAmax/secondary DSA reappearance or new DSA appearance is termed DSA relapse. Refractory DSA is defined as a DSAmax that has no change or increase in MFI.

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Assessment of Efficacy

The endpoints evaluated in this study were the DSAmax progression following the use of bortezomib-based therapy. Specifically, the study assessed the overall rate of partial and complete response following bortezomib, the duration of response (i.e., remission), and DSA relapse rate. Evaluation of the endpoints occurred at the end of the cycle and at 1, 3, 6, 9, 12, 18, and 24 months after treatment.

Time-to-event analysis was performed according to the Kaplan-Meier method. The time to any response (partial or complete) was defined as the time from the initial administration of bortezomib to the first time point that the DSAmax MFI dropped by 50% in intensity from its day 0 value. Duration of remission was defined as the time from the achievement of complete response to the time of DSA relapse.

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Statistical Methods

All statistical analyses were performed using Stata/MP version 10.1 (College Station, TX). The P values presented are two-sided and a P value of less than 0.05 was considered statistically significant. Wilcoxon signed-rank test was used to compare median MFI values from baseline to year 1. The comparative analyses between bortezomib-alone versus bortezomib-based combination therapy were summarized descriptively. A univariate analysis to assess the factors associated with remission was performed using logistic regression. The small sample size prohibited use of multivariate logistic regression analysis. Cox proportional hazards modeling was used to evaluate variables as they related to time to relapse (from time of complete response).

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Keywords:

Proteasome; Alloantibodies; Transplantation; Bortezomib

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