{'fraction_non_alphanumeric': 0.034162452265455674, 'fraction_numerical': 0.010217772731964083, 'mean_word_length': 5.468624833110814, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Posttransplant reduction in preexisting donor‐specific antibody levels after belatacept‐ versus cyclosporine‐based immunosuppression: Post hoc analyses of BENEFIT and BENEFIT‐EXT

BENEFIT and BENEFIT‐EXT were phase III studies of cytotoxic T‐cell crossmatch–negative kidney transplant recipients randomized to belatacept more intense (MI)‐based, belatacept less intense (LI)‐based, or cyclosporine‐based immunosuppression. Following study completion, presence/absence of HLA‐specific antibodies was determined centrally via solid‐phase flow cytometry screening. Stored sera from anti‐HLA–positive patients were further tested with a single‐antigen bead assay to determine antibody specificities, presence/absence of donor‐specific antibodies (DSAs), and mean fluorescent intensity (MFI) of any DSAs present. The effect of belatacept‐based and cyclosporine‐based immunosuppression on MFI was explored post hoc in patients with preexisting DSAs enrolled to BENEFIT and BENEFIT‐EXT. In BENEFIT, preexisting DSAs were detected in 4.6%, 4.9%, and 6.3% of belatacept MI‐treated, belatacept LI‐treated, and cyclosporine‐treated patients, respectively. The corresponding values in BENEFIT‐EXT were 6.0%, 5.7%, and 9.2%. In both studies, most preexisting DSAs were of class I specificity. Over the first 24 months posttransplant, a greater proportion of preexisting DSAs in belatacept‐treated versus cyclosporine‐treated patients exhibited decreases or no change in MFI. MFI decline was more apparent with belatacept MI‐based versus belatacept LI‐based immunosuppression in both studies and more pronounced in BENEFIT‐EXT versus BENEFIT. Although derived post hoc, these data suggest that belatacept‐based immunosuppression decreases preexisting DSAs more effectively than cyclosporine‐based immunosuppression.

complement-dependent lymphocytotoxic crossmatching became a contraindication to renal transplantation. However, cell-based assays are unable to detect low DSA levels or DSAs that do not activate complement. Thus, patients who are complement-dependent crossmatch-negative may still harbor DSAs. 3 Compared with complement-dependent T-cell crossmatching, solid-phase immunoassays are more sensitive, 4,5 detecting not only low class I antibody levels, but also antibodies against class II antigens and class I and class II allele-specific antibodies. 5,6 Moreover, solid-phase immunoassays are semi-quantitative, permitting the categorization of DSA levels. 5 The phase III Belatacept Evaluation of Nephroprotection and Efficacy as First-Line Immunosuppression Trial (BENEFIT) and BENEFIT-Extended Criteria Donors (BENEFIT-EXT) studies evaluated belatacept, a selective T-cell costimulation blocker approved worldwide to prevent kidney transplant rejection in adults. 7 Kidney transplant recipients enrolled to these similarly designed studies were T-cell lymphocytotoxic crossmatch-negative (determined locally). However, subsequent testing at a central facility of stored sera via solid-phase flow cytometry and single-antigen bead assay revealed that a subset of patients did possess preexisting DSAs.
Importantly, the results from these prespecified exploratory analyses were not intended for use as a requirement for study entry or for patient management (ie, centers were unaware of assay results prior to transplantation). The present post hoc analyses examined the effect of belatacept-based and cyclosporine-based immunosuppression on MFI in the subsets of BENEFIT and BENEFIT-EXT participants with preexisting DSAs. Efficacy and safety at 7 years (84 months) posttransplant in these patients were also evaluated.

| Study design
BENEFIT (NCT00256750) and BENEFIT-EXT (NCT00114777) were 3-year, international, partially blinded, randomized phase III studies. 8,9 Patients undergoing primary transplant with T-cell panel reactive antibodies (PRA) ≥50% or those undergoing re-transplantation with T-cell PRA ≥30% were excluded, as were patients with a locally determined positive T-cell crossmatch with the intended donor.
Patients in BENEFIT received a living or standard-criteria deceased donor kidney. Patients in BENEFIT-EXT received an extendedcriteria donor kidney.
These studies were conducted in accordance with Good Clinical Practice guidelines and the principles outlined in the Declaration of Helsinki. The institutional review boards/ethics committees at participating centers approved the study protocols. All patients provided written informed consent.

| Assessments
The presence of HLA antibodies was assessed in all randomized, transplanted patients at baseline (day 0, the day of transplant), at months 6, 12, 24, 36, 48, 60, and 84, and at the time of clinically suspected episodes of acute rejection. Antibody screening was performed centrally at Emory University using solid-phase flow cytometry screening (FlowPRA, One Lambda, Inc., Canoga Park, CA). HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA, and -DQB1 antigens were identified by molecular typing methods, typically to the two-digit (low resolution) level. HLA-DP typing was not performed, since no study participant had HLA-DP antibodies. Sera from patients with anti-HLA antibodies were subsequently analyzed using Luminex single-antigen bead assay (One Lambda, Inc.) to determine antibody class specificity, the presence/ absence of DSAs, and the mean fluorescence intensity (MFI) of any DSAs present. Sera were not pretreated or diluted prior to singleantigen bead testing. Since this was a post hoc study and because there are no established clinical thresholds for preexisting DSA MFI levels, we wanted to be conservative, considering any patient with DSA MFI levels ≥1000 as preexisting DSA-positive. To minimize variation due to lot/technician differences, all samples-irrespective of the time at which they were drawn-were tested and analyzed at the same time. Singleantigen testing was performed using a modified technique employing a biotin-conjugated secondary antibody followed by phycoerythrinconjugated streptavidin. An MFI value of 1000 in this assay corresponds to a slightly lower MFI value than in the nonmodified (standard) assay.
Details on our modified technique are described in Sullivan et al. 12 The following were analyzed in each study: death or graft loss, renal function (six-variable Modification of Diet in Renal Disease equation 13 ), and acute rejection (central biopsy-proven rejection that was clinically suspected for protocol-defined reasons or clinically suspected for other reasons and treated).

| Statistics
Patients were analyzed according to the intent-to-treat principle.
Descriptive statistics were used to summarize MFI from baseline (pretransplant) to month 24 in the subsets of patients from each study with preexisting DSAs. Any DSA MFI value ≥1000 at baseline was considered positive. Time to death or graft loss was assessed in evaluable patients (patients known to be alive at month 84 or known to have died or experienced graft loss by month 84) and described using Kaplan-Meier event rates. The method of Kaplan-Meier was used to determine time to death or graft loss from randomization to month 84 in the cohorts of patients in each study who did and did not have preexisting DSAs. These cohorts were compared using hazard ratios (HRs) and 95% confidence intervals (CIs), which were calculated via Cox regression; no adjustment was made for multiplicity. Kaplan-Meier cumulative event rates were used to summarize the incidence of acute rejection. Descriptive statistics were used to summarize renal function (month 1 to month 84).

Average MFI of DSAs
In the subset of patients with preexisting DSAs, mean estimated GFR (as observed) between month 3 and month 84 remained essentially stable for those randomized to belatacept MI, increased for those randomized to belatacept LI, and decreased for those randomized to cyclosporine ( Figure 3A).

| D ISCUSS I ON
When the BENEFIT and BENEFIT-EXT studies were undertaken, it was standard practice to assess donor-recipient histocompatibility using only cell-based cytotoxicity assays. When retrospectively examining stored patient sera using solid-phase assays, which are more sensitive than T-cell lymphocytotoxic crossmatching, we discovered  These analyses are limited by the fact that they were conducted post hoc. As a consequence, the numbers of patients analyzed were small, and MFI was not available at all time points for all individuals.
In addition, patients were not randomized according to preexisting DSA status. Moreover, patient sera were only subjected to singleantigen bead testing if the results of the solid-phase flow cytometry screening assay were positive. Because solid-phase antibody screening may be less sensitive than single-antigen bead testing, it is possible that patients with weak preexisting DSAs may have gone undetected. Although they should be interpreted with caution, these preliminary findings suggest that belatacept-based relative to cyclosporine-based immunosuppression may better control the persistence of preexisting DSAs posttransplant, as determined by MFI.
It is intriguing to speculate that belatacept may have the potential to reduce HLA antibodies in sensitized individuals awaiting a kidney transplant, but additional studies are needed to test this hypothesis.

ACK N OWLED G M ENTS
The BENEFIT and BENEFIT-EXT studies were sponsored by Bristol-Myers Squibb. Support for third-party writing assistance for this manuscript was provided by Tiffany DeSimone, PhD, of CodonMedical, an Ashfield Company, part of UDG Healthcare plc, and was funded by Bristol-Myers Squibb.

D I SCLOS U R E
The authors of this manuscript have conflicts of interest to disclose.