A Head-to-head Comparison of De Novo Sirolimus or Everolimus Plus Reduced-dose Tacrolimus in Kidney Transplant Recipients: A Prospective and Randomized Trial.

BACKGROUND: Mammalian target of rapamycin inhibitors (mTORi), sirolimus (SRL) and everolimus (EVR), have distinct pharmacokinetic/pharmacodynamics properties. There are no studies comparing the efficacy and safety of de novo use of SRL versus EVR in combination with reduced-dose calcineurin inhibitor. METHODS: This single-center prospective, randomized study included first kidney transplant recipients receiving a single 3 mg/kg antithymocyte globulin dose, tacrolimus, and prednisone, without cytomegalovirus (CMV) pharmacological prophylaxis. Patients were randomized into 3 groups: SRL, EVR, or mycophenolate sodium (MPS). Doses of SRL and EVR were adjusted to maintain whole blood concentrations between 4 and 8 ng/mL. The primary endpoint was the 12-mo incidence of the first CMV infection/disease. RESULTS: There were 266 patients (SRL, n = 86; EVR, n = 90; MPS, n = 90). The incidence of the first CMV event was lower in the mTORi versus MPS groups (10.5% versus 7.8% versus 43.3%, P < 0.0001). There were no differences in the incidence of BK polyomavirus viremia (8.2% versus 10.1% versus 15.1%, P = 0.360). There were no differences in survival-free from treatment failure (87.8% versus 88.8% versus 93.3%, P = 0.421) and incidence of donor-specific antibodies. At 12 mo, there were no differences in kidney function (75 ± 23 versus 78 ± 24 versus 77 ± 24 mL/min/1.73 m 2 , P = 0.736), proteinuria, and histology in protocol biopsies. Treatment discontinuation was higher among patients receiving SRL or EVR (18.6% versus 15.6% versus 6.7%, P = 0.054). CONCLUSIONS: De novo use of SRL or EVR, targeting similar therapeutic blood concentrations, shows comparable efficacy and safety. The reduced incidence of CMV infection/disease and distinct safety profile of mTORi versus mycophenolate were confirmed in this study.

Donor-Specific HLA Antibodies Are Associated with Graft Failure and Delayed Hematologic Recovery after Unrelated Donor Hematopoietic Cell Transplantation.

Graft failure (GF) is one of the major concerns after allogeneic hematopoietic cell transplantation (allo-HCT) and remains a significant cause of morbidity and mortality. Although previous reports have associated the presence of donor-specific HLA antibodies (DSAs) with an increased risk of GF after unrelated donor allo-HCT, recent studies have failed to confirm this association. We sought to validate the presence of DSAs as a risk factor for GF and hematologic recovery in the unrelated donor allo-HCT setting. We retrospectively evaluated 303 consecutive patients who underwent their first unrelated donor allo-HCT at our institution between January 2008 and December 2017. DSA evaluation was performed using 2 single antigen bead (SAB) assays; DSA titration with 1:2, 1:8, and 1:32 dilutions; C1q-binding assay; and absorption/elution protocol to assess possible false-positive DSA reactivity. The primary endpoints were neutrophil and platelet recovery and GF, and the secondary endpoint was overall survival. Multivariable analyses were performed using Fine-Gray competing risks regression and Cox proportional hazards regression models. The median patient age was 14 years (range, 0 to 61 years), 56.1% were male, and 52.5% underwent allo-HCT for nonmalignant disease, Eleven patients (3.63%) were DSA-positive, including 10 with preexisting DSAs and 1 with post-transplantation de novo DSAs. Nine patients had 1 DSA, 1 patient had 2 DSAs, and 1 patient had 3 DSAs, with a median mean fluorescent intensity (MFI) of 4334 (range, 588 to 20,456) and 3581 (range, 227 to 12,266) in LABScreen and LIFECODES SAB assays, respectively. Overall, 21 patients experienced GF, including 12 with primary graft rejection, 8 with secondary graft rejection, and 1 with primary poor graft function. The cumulative incidence of GF was 4.0% (95% confidence interval [CI], 2.2% to 6.6%) at 28 days, 6.6% (95% CI, 4.2% to 9.8%) at 100 days, and 6.9% (95% CI, 4.4% to 10.2%) at 365 days. In the multivariable analyses, DSA-positive patients had significantly delayed neutrophil recovery (subdistribution hazard ratio [SHR], .48; 95% CI, .29 to .81; P = .006) and platelet recovery (SHR, .51; 95% CI, .35 to .74; P = .0003) compared to patients without DSAs. In addition, only DSAs were significant predictors of primary GF at 28 days (SHR, 2.78; 95% CI, 1.65 to 4.68; P = .0001). The Fine-Gray regression also demonstrated that the presence of DSAs was strongly associated with a higher incidence of overall GF (SHR, 7.60; 95% CI, 2.61 to 22.14; P = .0002). DSA-positive patients with GF had significantly higher median MFI values than DSA-positive patients who achieved engraftment in the LIFECODES SAB assay using neat serum (10,334 versus 1250; P = .006) and in the LABScreen SAB at 1:32 dilution (1627 versus 61; P = .006). All 3 patients with C1q-positive DSAs failed to engraft. DSAs were not predictive of inferior survival (HR, .50; 95% CI, .20 to 1.26; P = .14). Our results validate the presence of DSAs as a significant risk factor for GF and delayed hematologic recovery after unrelated donor allo-HCT. Careful pretransplantation DSA evaluation may optimize unrelated donor selection and improve allo-HCT outcomes.

Untreated Donor-Specific HLA Antibodies Are Associated With Graft Failure and Poor Survival After Haploidentical Transplantation With Post-Transplantation Cyclophosphamide in Pediatric Patients With Nonmalignant Disorders.

Donor-specific HLA antibodies (DSAs) have been recognized as a major risk factor for graft failure (GF) in adult patients with malignancies undergoing haploidentical transplantation with post-transplantation cyclophosphamide (haplo-PTCy). However, the impact of DSAs after pediatric haplo-PTCy for nonmalignant disorders (NMDs) has been poorly reported. We sought to investigate whether preexisting DSAs adversely affect pediatric haplo-PTCy outcomes. We retrospectively analyzed 59 pediatric patients (≤21 years) who received their first haplo-PTCy for NMDs from January 2008 to December 2017. DSA testing was performed using single antigen beads, and mean fluorescence intensity (MFI) >1000 was considered positive, and MFI <1000 and >500 was considered potentially positive, based on HLA epitope reactivity patterns. Primary endpoints were neutrophil and platelet recovery and GF, whereas secondary endpoints included event-free and overall survival. Multivariable analyses were performed using Fine-Gray competing risk regression or Cox proportional hazards regression models. The median age was 10 years, and 66.1% were male. Main indications for haplo-PTCy were Fanconi anemia (n = 33) and severe aplastic anemia (n = 11). All patients received bone marrow as the graft source, and most patients (91.5%) received fludarabine-based conditioning. Overall, 15 patients (25.4%) had DSAs >500 MFI. Four patients had false-positive DSAs with median MFI of 1762. Of the 11 patients with true-positive DSA reactivity, 5 had 1 DSA, 5 had 2 DSAs, and 1 had 3 DSAs, with median MFI of 2372 (range 527-24,200). Four patients received desensitization therapy with rituximab and plasmapheresis, whereas 7 patients were untreated. All patients with treated DSAs achieved donor engraftment. In the multivariable analyses, untreated DSAs were associated with lower neutrophil recovery (subdistribution hazard ratio [SHR] = 0.15; 95% confidence interval [CI], 0.03-0.63; P = .001), increased GF (SHR = 20.57; 95% CI, 6.57-64.43; P < .001), inferior event-free survival (hazard ratio [HR] = 10.09; 95% CI, 3.37-30.22; P < .001), and poor overall survival (HR 5.56; 95% CI, 1.92-16.12; P = .002). Both treated DSAs (SHR = 0.26; 95% CI, 0.10-0.68; P = .006) and untreated DSAs (SHR = 0.13; 95% CI, 0.04-0.37; P < .001) adversely affected platelet recovery. Our results indicate that the presence of DSAs is an independent predictor of poor outcomes after pediatric haplo-PTCy for NMDs. Therefore DSA-positive donors should be avoided whenever possible, and when a DSA-negative donor is unavailable, desensitization therapy must be performed to enhance the likelihood of donor engraftment and improve transplantation outcomes.

High soluble HLA-DQB2 levels in posttransplant serum are associated with kidney graft dysfunction.

HLA-DQB2 is a gene of limited polymorphism, with unknown function that presents at least two transcript variants: v1, which encodes the full-length beta-chain, and v2, which lacks exon 4 and could give rise to a soluble protein. We previously showed a strong correlation between high v2 expression in preimplantation biopsies (PIB) of kidneys from young (18- to 49-year olds) but not from old, deceased donors and 1-year posttransplant low (estimated glomerular filtration rate < 45 ml/min/1.73 m2 ) graft function (GF). In this study, we aimed to investigate the impact of posttransplant soluble HLA-DQB2 (sDQB2) serum levels, v1 expression in PIB, and recipient HLA-DQB2 rs7453920 A/G polymorphism on GF. sDQB2 was evaluated by enzyme-linked immunosorbent assay in sera from 114 recipients, collected at least 1 year (median 2.1 years) after transplantation. Higher sDQB2 levels were observed in recipients of kidneys from young, but not from old, donors that had a ≥30% decline in GF within 1 year after blood collection for sDQB2 determination. Among the 15 recipients of kidneys from young donors with sDQB2 ≥ 1.52 ng/ml, 40% presented a ≥30% decline in GF, whereas this occurred in none of the 43 recipients with lower sDQB2 levels (p = 0.007; OR: 36.5). Expression of HLA-DQB2 variant 1, measured by reverse transcription-polymerase chain reaction (RT-PCR) in 92 PIB from young or old donors, did not significantly differ between transplants with high or low 4-year GF. HLA-DQB2 rs7453920 single nucleotide polymorphism (SNP) frequencies did not significantly differ between recipients with low or high 4-year GF. We conclude that HLA-DQB2 variant 1 expression in PIB and recipient rs7453920 SNP polymorphism are not associated with graft outcome. On the other hand, the association, in transplants of kidneys from young donors, between high posttransplant serum sDQB2 levels and decline in GF is a very interesting finding that deserves a validation study in a larger cohort.

The influence of the antithymocyte globulin dose on clinical outcomes of patients undergoing kidney retransplantation.

Optimizing antithymocyte globulin (rATG) dosage is critical for high immunological risk patients undergoing a repeat kidney transplant. This natural retrospective cohort study compared clinical outcomes of two successive cohorts of consecutive recipients of retransplants receiving 5 x 1 mg/kg (rATG-5, n = 100) or a single 3 mg/kg (rATG-3, n = 110) dose of rATG induction therapy. All patients had negative complement-dependent cytotoxicity crossmatch and no anti-HLA A, B, DR donor-specific antibodies (DSA). The primary endpoint was efficacy failure (first biopsy-proven acute rejection, graft loss, or death) at 12 months. There was no difference in the cumulative incidence of efficacy failure (18.0% vs. 21.8%, HR = 1.22, 95% CI 0.66-2.25), respectively. There were no differences in 3-years freedom from biopsy proven acute rejection, and patient, graft, and death-censored graft survivals. There were no differences in the incidence of surgical complications (25.0% vs. 18.2%; p 0.151), early hospital readmission (27.8% vs. 29.5%; p = 0.877) and CMV infections (49% vs. 40%; p = 0.190). There were also no differences in the incidence (59.6% vs. 58.7%, p = 0.897) and duration of delayed graft function but a stable difference in estimate glomerular filtration rate was observed from month 1 (54.7±28.8 vs. 44.1±25.3 ml/min/1.73 m2, p = 0.005) to month 36 (51.1±27.7 vs. 42.5±24.5, p = 0.019). Mean urinary protein concentration (month 36: 0.38±0.81 vs. 0.70±2.40 g/ml, p = 0.008) and mean chronic glomerular Banff score in for cause biopsies (months 4-36: 0.0±0.0 vs. 0.04±0.26, p = 0.044) were higher in the rATG-3 group. This cohort analysis did not detect differences in the incidence of efficacy failure and in safety outcomes at 12 months among recipients of kidney retransplants without A, B, and DR DSA, receiving induction therapy with a single 3 mg/kg rATG dose or the traditional 5 mg/kg rATG.

Prospective randomized study comparing everolimus and mycophenolate sodium in de novo kidney transplant recipients from expanded criteria deceased donor.

The optimal immunosuppressive regimen for recipients of expanded criteria donor (ECD) kidneys has not been identified. In this single-center study, 171 recipients of ECD kidney transplants were randomized to receive antithymocyte globulin induction, and delayed introduction of reduced dose tacrolimus, prednisone and everolimus (r-ATG/EVR, n = 88), or mycophenolate (r-ATG/MPS, n = 83). No cytomegalovirus (CMV) pharmacological prophylaxis was used. The primary endpoint was the incidence of CMV infection/disease at 12 months. Secondary endpoints included treatment failure [first biopsy-proven acute rejection (BPAR), graft loss, or death] and safety. Patients treated with EVR showed a 89% risk reduction (13.6 vs. 71.6%; HR 0.11, 95% CI 0.06-0.220, P < 0.001) in the incidence of first CMV infection/disease. Incidences of BPAR (16% vs. 5%, P = 0.021), graft loss (11% vs. 1%, P = 0.008), death (10% vs. 1%, P = 0.013), and treatment discontinuation (40% vs. 28%, P = 0.12) were higher in the r-ATG/EVR, leading to premature study termination. Mean glomerular filtration rate was lower in r-ATG/EVR (31.8 ± 18.8 vs. 42.6 ± 14.9, P < 0.001). In recipients of ECD kidney transplants receiving no CMV pharmacological prophylaxis, the use of everolimus was associated with higher treatment failure compared with mycophenolate despite the significant reduction in the incidence of CMV infection/disease (ClinicalTrials.gov.NCT01895049).

Validation of phage display method for protease inhibitor selection using synthetic hybrid peptides.

A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa)) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1'-P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH2; Ki = 14 µM) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases ≈20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH2; Ki = 259 µM). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.