Protocol or for cause biopsies were performed in six of seven patients in IVIG+placebo and five of six in IVIG+rituximab, with no additional patients showing evidence for ABMR. One patient in the IVIG+placebo and one in the IVIG+rituximab group experienced Banff 1A cellular rejection at 1 year. An analysis of all biopsies performed in both groups is shown in Table 3. Of note, no TG was seen on protocol biopsies for the patients treated with IVIG+rituximab. Assessment of renal function in both groups showed mean serum creatinine levels of 1.7±0.5 mg/dL and 2.0±0.6 mg/dL for IVIG+placebo and 1.1±0.4 mg/dL and 1.1±0.4 mg/dL for IVIG+rituximab at 6 M (P=0.026) and 12 M (P=0.046), respectively. Thus, treatment with IVIG+rituximab was associated with significantly improved renal function posttransplant at 6 M and 12 M, likely a result of the impact of ABMR in the two IVIG+placebo patients with surviving allografts.
Adverse events (AEs) were minimal, including minimal infusion-related reactions to IVIG+rituximab and IVIG alone that were similar to those previously described (12, 15, 16, 19). Six patients (86%) developed SAEs (eight episodes) in the IVIG+placebo group versus three patients (50%) in the IVIG+rituximab group (P=ns). Of note, one patient (IVIG+rituximab) died at 12 months from a Nocardia brain abscess. This was likely a complication of the immunosuppressive protocol. These are summarized in Table 1.
Desensitization therapies have emerged as part of a multipronged effort to increase transplants among HS patients. Unfortunately, neither desensitization or paired exchange paradigms have increased the number of patients undergoing transplantation in the United States (UNOS/OPTN, 2013) (3, 4). Despite these discouraging observations, there is progress in both areas that will likely improve the numbers of transplants performed for sensitized patients. Toward this end, new UNOS allocation policies will give special advantage to HS patients awaiting DD transplantation (25). Thus, continued development of desensitization therapies is critical because HS patients remain on the UNOS wait list for years with increased morbidity and mortality compared to desensitized and transplant patients (14, 19). Our group has extensive experience with both HD IVIG alone (12) and IVIG+rituximab as desensitization agents for DD recipients (15, 16, 19).
This placebo-controlled trial was undertaken to determine the benefit of rituximab as a desensitization agent when used with IVIG. Unfortunately, the number of SAEs experienced prohibited the completion of this trial and limited the assessments that can be made from it. However, there are still important lessons learned. There was a trend toward increasing numbers and severity of early ABMR episodes within the first month posttransplant in patients desensitized in the IVIG+placebo group (3/7 placebo vs. 0/6 rituximab, P=0.06). We also noted a trend toward lower DSA levels posttransplant in the IVIG+rituximab group. This observation suggests that the effect of rituximab in desensitization is likely aimed at depletion of memory B cells that rapidly become activated and increase DSA production when the alloantigen stimulus of the allograft is seen. Data to support this contention have recently been presented by Zachary et al. (26) who detected alloreactive B cells in DSA-negative patients with a sensitization history. A similar group of patients who were treated with rituximab before transplantation showed no alloreactive B cells. Kohei et al. (27) also showed that treatment of ABOi transplant patients with rituximab resulted in long-term prevention of de novo DSA production and low levels of ABMR compared to a cohort of non-ABOi LD transplant recipients not receiving rituximab. They concluded that rituximab administration at the early phase could prevent chronic B-cell activation, de novo DSA production, and chronic ABMR. In addition, Lynch et al. (28) recently showed that non-sensitized living donor transplant recipients rapidly developed detectable alloreactive B cells secreting HLA class I specific DSAs within 1 month posttransplant. The authors conclude that cryptic B-cell responses to allografts are more common than previously thought.
We have reported on the criteria used for acceptance of LD and DD transplants using the IVIG+rituximab protocol (19, 29, 30). Our experience indicates these ranges do not engender a high risk for rebound DSA development, ABMR, or graft loss from ABMR in IVIG+rituximab-treated patients. Since implementation of this protocol in 2005, we have consistently seen posttransplant ABMR rates at 24%. Here, it appears that IVIG+placebo was not sufficient to sustain low levels of DSA, ABMR, and graft loss when compared to IVIG+rituximab despite having a similar “acceptable” DSA and crossmatch starting point. DSA levels rapidly rebounded in two of three patients in the IVIG+placebo group who experienced ABMR, but declined over time in the IVIG+rituximab group (Fig. 2A and B). The risk for ABMR was also likely greater in the IVIG+placebo group because of higher numbers with previous transplants. Kannibhiran et al. (11) recently reported a similar finding in a survey of 487 patients. One hundred forty-three (29%) were DSA positive at transplant. The described features associated with ABMR included previous transplants (P<0.003) and the sum of all DSA scores (P<0.0001). Van Der Hooge et al. (31) reported on a placebo-controlled trial of rituximab as an induction agent for kidney transplant recipients. Two hundred eighty patients were examined (138 randomized to rituximab and 142 placebo). Overall, there was no difference in the graft rejection rates. However, when data for high-risk patients (repeat transplants and HLA sensitized) were analyzed, there was a significant reduction in rejection episodes for those who received rituximab (17.9% vs. 41.1%, P=0.039). The authors conclude that a single dose of rituximab given as an induction agent at transplant significantly reduces rejection rates in sensitized patients.
Alloreactive B cells can rapidly emerge in DSA-negative patients posttransplant (26, 28). Detecting these cells is an especially difficult problem as traditional markers for sensitization are not present (DSA) and assays for alloreactive B cells are just emerging (26, 28). However, rituximab can deplete these memory B cells with consistent prevention of ABMR episodes. Rituximab can also alter T-cell responses by reducing the antigen-presenting cell and cytokine production capabilities of B cells (32, 33).
Data from this abbreviated trial do have significant limitations in terms of making relevant conclusions. These include a possibility of type 1 statistical errors because of the low numbers of entered patients and random distribution of more patients with previous transplants to the IVIG+placebo group. However, clinically important trends were observed, most importantly, the prevention of ABMR episodes, TMA, and transplant glomerulopathy in the IVIG+rituximab group. In addition to assessment of DSA levels and crossmatch results at transplant, one should also assess sensitization history (especially repeat transplants) as risk for clandestinely sensitized B cells potentially mediating ABMR. One patient death from a Nocardia brain abscess is sobering, but unusual as rituximab is not associated with significant increased risk for infection or malignancy in sensitized transplant patients (34). Finally, this work is supportive of a role for rituximab as a desensitization agent because it appears to engender long-term effects on alloimmunity and alloreactive B cells that are likely beneficial to the allograft, and may be independent of effects on PRA levels.
MATERIALS AND METHODS
Patients and Study Design
The study was designed as a multiple-center, phase I and II, exploratory study that used a blinded, placebo-controlled format. The study aimed to examine the safety and efficacy of human polyclonal IVIG 10%, when given at (2.0 g/kg [maximum 140 g/dose]×2) on days 1 and 30, +placebo (0.9% normal saline) (×1) versus IVIG (2.0 g/kg [maximum 140 g/dose]×2) on days 1 and 30, and +rituximab (1 g×1) administered on day 15 to reduce DSA to a level that was permissive for DD transplantation. Patients were randomized 1:1 by computer. The investigators were blinded to randomization and treatments. The study entered subjects actively listed on the UNOS list (18–65 years) who were (HS) defined as panel reactive HLA antibody (PRA) positive at greater than 50% with assessment of DSA and CMX values at time of transplant (19). Rates of transplantation were compared between the two groups as the primary end point. If acceptable crossmatches and DSA levels were seen, the patients proceeded to DD transplantation. An acceptable CMX in the context of desensitization is defined as a negative complement-dependent cytotoxicity, at least at a 1:2 dilution of sera. A negative flow cytometry crossmatch against donor T and B cells (FCMX) OR a positive T- and B-cell FCMX∼250 channel shifts (CS) or less (deemed an “acceptable” crossmatch) for T and B cells (negative: <100 mean channel shifts [MCS] for B cell and <50 MCS for T cell) (15, 16, 19). Transplant patients received an additional dose of IVIG at transplantation (within 10 days) and an additional dose of rituximab (1 g) or placebo at 6 months posttransplant. Patients who were desensitized and did not undergo transplantation within 6 months after desensitization completed the study. Criteria for acceptance of DD offers after desensitization and decision to proceed with transplantation as well as details of immunosuppression and posttransplantation management were previously described (15, 16, 19). Campath-1H was used as the induction agent in all study patients (24). All patients received immunosuppression with tacrolimus, mycophenolic acid, and prednisone, and all patients were monitored and prophylaxed for infectious complications as previously described (19, 23). Patients who experienced ABMR or AR were managed per center protocol as previously described (19). The study design is shown in Figure 1A.
Entered subjects were followed to determine the proportion who received a transplant within 6 months of study completion. This was the primary end point. We hypothesized that addition of rituximab to the IVIG desensitization protocol would increase rates of transplantation by exerting an additive effect on IVIG induced reduction in DSA levels. In addition, we examined multiple parameters including risk for ABMR, graft and patient survival, allograft function, infectious complications, and DSA levels at specific time points posttransplant. Implantation biopsies were performed in all transplant patients. In those who sustained a viable and functioning kidney allograft for 12 months, repeat study biopsies were performed. All subjects were evaluated on an intent-to-treat basis.
Assessments during therapy include routine laboratory tests; measurement of PRA, DSA, and DSA-relative intensity scale (RIS) levels; vital signs assessments with each infusion; electrocardiograms; and collection of data related to AE and SAE. All AEs and SAEs were recorded, graded, and reported to the Cedars-Sinai Medical Center Institutional Review Board (IRB), the study sponsor (Roche-Genentech), and FDA Medwatch. Safety stopping rules included assessment of infusion-related SAEs and occurrence of ABMR posttransplant in three or more transplant subjects.
This study was approved by the IRB at Cedars-Sinai Medical Center. The study was conducted in accordance with the Declaration of Helsinki, with the ethical guideline based on federal regulations and the common rule. Cedars-Sinai Medical Center also has a Federal Wide Assurance. The study was an investigator-initiated trial (NCT01178216, FDA IND#: 109067, S.C.J., MD, Principal Investigator). The study was designed, conducted, and evaluated solely by the investigators after approval and funding by Genentech-Roche Inc. The study was a phase I and II randomized double-blinded placebo controlled format with all investigators blinded to study drug administration. Data was maintained in a confidential manner until the study was terminated. In accordance with the study protocol, a data safety monitoring board was established to review safety concerns and report on any AEs or SAEs quarterly. In addition, safety-stopping points were included in the protocol. The study protocol and supporting documentation is available in the supplemental digital content submitted with the article. The data analysis and manuscript preparation was completed entirely by the investigators.
Statistical analysis was performed using the paired Student t test. Different parameters, including rates of transplantation by treatment group, patient and graft survival, mean serum creatinines (SCrs), primary or multiple transplants, acute rejection and ABMR episodes, and transplant by DSA status, were analyzed. Differences were considered significant at the two-sided 0.05 level. The relationship between DSA levels, DSA-RIS scores, graft loss, and patient and graft survival between the treatment groups were analyzed using chi-square methods.
The authors thank the members of the Kidney Transplant and Transplant Immunotherapy Program, the Transplant Immunology Laboratory, the Renal Pathology Group, and HLA Laboratory at Cedars-Sinai Medical Center for their hard work and dedication to our patients.
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IVIG; Rituximab; Desensitization; Donor-specific HLA antibodies
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