High-dose Antithymocyte Globulin Research Articles

High-dose individualized antithymocyte globulin with therapeutic drug monitoring in high-risk cord blood transplant

BackgroundGraft-versus-host disease (GvHD) and rejection are main limitations of cord blood transplantation (CBT), more so in patients with severe inflammation or previous rejections. While rigorous T-cell depletion with antithymocyte globulin (ATG) is needed to prevent GvHD and rejection, overexposure to ATG leads to slow T-cell recovery after transplantation, especially in CBT. ObjectiveTo evaluate high-dose, upfront ATG with individualized dosing and therapeutic drug monitoring (TDM) in pediatric CBT for patients at high risk for GvHD and rejection. Study designHeavily inflamed patients and patients with a recent history of rejection were eligible for individualized high-dose ATG with real-time TDM. The ATG dosing scheme was adjusted to target a post-CBT exposure of <10 AU*day/mL, while achieving a pre-CBT exposure of 60–120 AU*day/mL; exposure levels previously defined for optimal efficacy and safety in terms of reduced GvHD and rejection, respectively. Main outcomes of interest included efficacy (target exposure attainment) and safety (incidence of GvHD and rejection). Other outcomes of interest included T-cell recovery and survival. ResultsTwenty-one patients were included ranging from 2 months to 18 years old, receiving an actual median cumulative dose of ATG of 13.3 mg/kg (range 6–30 mg/kg) starting at a median 15 days (range 12–17) prior to CBT. Dosing was adjusted in 14 patients (increased in 3 and decreased in 11 patients). Eighteen (86%) and 19 (91%) patients reached the target pre-CBT and post-CBT exposure, respectively. Cumulative incidence for acute GvHD was 34% (95% CI 23–45) and 5% (95% CI 0–10%) for grade 2–4 and grade 3–4, respectively; cumulative incidence of rejection was 9% (95% CI 2–16%). Overall survival was 75% (95% CI 65–85%). ConclusionIndividualized high-dose ATG with TDM is feasible and safe for patients with hyperinflammation in a CBT setting. We observe high target ATG exposure attainment, good immune reconstitution (despite very high doses of ATG) and acceptable rates of GvHD and rejection.

Evaluating anti-thymocyte globulin induction doses for better allograft and patient survival in Asian kidney transplant recipients

Anti-thymocyte globulin (ATG) is currently the most widely prescribed induction regimen for preventing acute rejection after solid organ transplantation. However, the optimal dose of ATG induction regimen in Asian kidney recipients is unclear. Using the Korean Organ Transplantation Registry, we performed a retrospective cohort study of 4579 adult patients who received renal transplantation in South Korea and divided them into three groups according to the induction regimen: basiliximab group (n = 3655), low-dose ATG group (≤ 4.5 mg/kg; n = 467), and high-dose ATG group (> 4.5 mg/kg; n = 457). We applied the Toolkit for Weighting and Analysis of Nonequivalent Groups (TWANG) package to generate high-quality propensity score weights for intergroup comparisons. During four-year follow-ups, the high-dose ATG group had the highest biopsy-proven acute rejection rate (basiliximab 20.8% vs. low-dose ATG 22.4% vs. high-dose ATG 25.6%; P < 0.001). However, the rates of overall graft failure (4.0% vs. 5.0% vs. 2.6%; P < 0.001) and mortality (1.7% vs. 2.8% vs. 1.0%; P < 0.001) were the lowest in the high-dose ATG group. Our results show that high-dose ATG induction (> 4.5 mg/kg) was superior to basiliximab and low-dose ATG induction in terms of graft and patient survival in Asian patients undergoing kidney transplant.

Cost-Effectiveness of Low-Dose Antithymocyte Globulin Versus Other Immunotherapies for Treatment of New-Onset Type 1 Diabetes.

Objective: Several immunotherapies have shown efficacy in slowing C-peptide decline in new-onset type 1 diabetes. Although most of these biologic drugs are expensive, they offer the opportunity to reduce downstream disease management costs and risk of complications. The objective of this study is to examine the cost-effectiveness of immunotherapies versus no treatment for patients with new-onset type 1 diabetes. Methods: Using Markov microsimulation modeling and efficacy data from immunotherapy trials, we examined the cost-effectiveness of six immunotherapies for new-onset type 1 diabetes, namely, low-dose (2.5 mg/kg) antithymocyte globulin (ATG), high-dose (6.5 mg/kg) ATG, abatacept, alefacept, rituximab, and teplizumab, versus no treatment. Effectiveness was measured by quality-adjusted life-years (QALYs). Costs were estimated from a health system perspective. Results: Low-dose ATG treatment saves US$10,270, on average, over a patient's lifetime and generates 0.09 additional QALYs compared with no treatment. These cost savings arise as low-dose ATG generates downstream savings in disease management costs that more than offset its cost. In contrast, treatment with other immunotherapies yields smaller QALY gains (0.02-0.05 additional QALYs) and increases lifetime costs by US$9500-US$168,380 relative to no treatment, with incremental cost-effectiveness ratios that exceed the willingness-to-pay threshold of US$100,000 per QALY. Conclusions: Low-dose ATG treatment is both less costly and more effective relative to other immunotherapies and no treatment for new-onset type 1 diabetes.

A clinical study of lyophilized intravenous human immunoglobulin containing high-titer cytomegalovirus-neutralizing antibody for the treatment of cytomegalovirus viremia after allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection increases the risk of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, some patients do not respond to ganciclovir (GCV) or foscarnet sodium. Lyophilized intravenous human immunoglobulin containing high-titer CMV-neutralizing antibody (CMV-IVIG) is another option to further improve treatment safety and efficacy. This study was designed to evaluate the efficacy of the combination of CMV-IVIG with traditional antiviral drugs (GCV or foscarnet sodium) at different time points for the treatment of post-allo-HSCT CMV viremia and to determine the clinical value of CMV-IVIG as a preemptive treatment strategy. The clinical data of 73 patients who received allo-HSCT and concurrent CMV-IVIG to treat post-allo-HSCT CMV viremia at our hospital between January and September 2020 were retrospectively analyzed. The patients were divided into two groups based on the total dose of antithymocyte globulin (ATG) used in the pretransplant preconditioning regimen, i.e., the low-dose ATG group (rabbit ATG ≤6 mg/kg, porcine ATG ≤40 mg/kg) (n=19) and the high-dose ATG group (rabbit ATG >6 mg/kg, porcine ATG >40 mg/kg) (n=54). Real-time quantitative polymerase chain reaction (RQ-PCR) was performed to measure the peripheral CMV-DNA level. Patients with CMV viremia (CMV-DNA >1,000 copies/mL) received concurrent CMV-IVIG therapy [early (within 3 days after the diagnosis of CMV viremia) or late (after 3 days)]. The CMV-DNA conversion rate, the median time of CMV-DNA conversion, and the two-week response rate and reactivation rate were analyzed for different ATG doses and different time points of CMV-IVIG use. The overall response rate to CMV-IVIG combined with traditional anti-CMV drugs was 100%. Early use of CMV-IVIG significantly reduced the median time of CMV-DNA conversion (14 vs. 21 days, P=0.000), especially in the high-dose ATG group (14 vs. 23 days, P=0.000). For patients with post-allo-HSCT CMV viremia, early use of CMV-IVIG effectively accelerates CMV-DNA conversion and peripheral CMV clearance, significantly improves the two-week response rate, and reduces the two-week reactivation rate. These effects are more pronounced in the high-dose ATG group.

Comparing Beta Cell Preservation Across Clinical Trials in Recent-Onset Type 1 Diabetes.

Several immunotherapies have demonstrated endogenous insulin preservation in recent-onset type 1 diabetes (T1D). We considered the primary results of rituximab, abatacept, teplizumab, alefacept, high-dose antithymocyte globulin (ATG), low-dose ATG, and low-dose ATG ± granulocyte-colony–stimulating factor trials in an attempt to rank the effectiveness of the agents studied. C-peptide 2-h area under the curve means were modeled using analysis of covariance. The experimental treatment group effect for each study, compared with its internal control, was estimated after adjusting for baseline C-peptide and age. Percentage increase in C-peptide over placebo and the absolute difference within study were calculated to compare and contrast effect size among interventions. Low-dose ATG (55% and 103%) and teplizumab (48% and 63%) ranked highest in C-peptide preservation at 1 and 2 years, respectively. Low-dose ATG and teplizumab show the greatest impact on C-peptide preservation among recent new-onset T1D studies; these should be further explored as core immunotherapies in the T1D prevention setting.

Clinical features and treatment outcomes in patients with T-cell large granular lymphocytic leukemia: A single-institution experience

AimLarge granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder associated with failure of hematopoiesis and autoimmune diseases. This study describes the clinical features and treatment responses of 108 patients with T-cell large granular lymphocyte leukemia (T-LGLL). MethodsClinical data were collected from T-LGLL patients treated at an anemia treatment center within the hematology and blood diseases unit of a single hospital from January 2009 to April 2019. ResultsThe majority of patients (78 %) were symptomatic at the time of presentation. Splenomegaly was observed in 41 % of cases, while hepatomegaly and lymphadenopathy were rare (6 % and 7 %, respectively). Cyclosporine (CsA) monotherapy was used as first-line therapy for 16 patients, with an overall response rate (ORR) of 56 %. CsA in combination with steroids was administered in 83 patients, with an ORR of 48 %. Among patients experiencing relapse or resistance to first-line therapy, 10 received antithymocyte globulin (ATG) therapy, with an ORR of 50 %; an additional 9 patients received a modified regimen of high-dose cyclophosphamide (CTX) therapy, yielding an ORR of 78 %. ConclusionsThis study provides new information regarding the clinical features and therapeutic strategies for T-LGLL, which can be used to improve clinical decision making for T-LGLL patients. The data presented here indicate the CsA is an effective option for the treatment of T-LGLL, while modified regimens of high-dose CTX or ATG are safe and effective choices for patients with CsA refractory disease.

Unmanipulated haploidentical hematopoietic stem cell transplantation using very low-dose antithymocyte globulin and methylprednisolone in adults with relapsed/refractory acute leukemia

Allogeneic hematopoietic stem cell transplantation (HSCT) could be the only curative therapy for patients with relapsed/refractory acute leukemia (RRAL). Many reports have described unmanipulated haploidentical HSCT (HID-HSCT) using high-dose antithymocyte globulin (ATG). However, the transplant outcomes of HID-HSCT using very low-dose ATG (thymoglobulin, 2-2.5mg/kg) and methylprednisolone (mPSL, 1mg/kg) for patients with RRAL have not been reported. We compared the outcomes of 46 patients with RRAL who underwent HID-HSCT using very low-dose ATG (thymoglobulin) and mPSL with the outcomes of 72 patients who underwent non-HID-HSCT. Patient characteristics differed regarding conditioning intensity (myeloablative; 19.6% in HID-HSCT vs. 61.1% in non-HID-HSCT, P < 0.001) and having undergone multiple HSCT (26.1% vs. 11.1%, P = 0.045). However, we found no significant differences in the 1-year overall survival (OS, 31.7% vs. 29.1%; P = 0.25), disease-free survival (DFS, 20.5% vs. 23.7%; P = 0.23), cumulative incidence of relapse (CIR, 40.0% vs. 42.8%; P = 0.92), non-relapse mortality (NRM, 39.5% vs. 33.5%; P = 0.22), or 100-day grade II-IV acute graft-versus-host disease (32.6% vs. 34.7%; P = 0.64) following HID-HSCT vs. non-HID-HSCT, respectively. Subgroup analysis stratified by disease and intensity of conditioning regimen demonstrated the same results between HID-HSCT and non-HID-HSCT. Furthermore, multivariate analysis showed that HID-HSCT was not an independent prognostic factor for OS (hazard ratio (HR) = 0.95 [95% confidence interval (CI), 0.58-1.58]), DFS (HR = 1.05 [95%CI, 0.67-1.68]), CIR (HR = 0.84 [95%CI, 0.48-1.47]), or NRM (HR = 1.28 [95%CI, 0.66-2.46]). In summary, transplant outcomes for RRAL were comparable in the HID-HSCT and non-HID-HSCT groups. HID-HSCT using very low-dose ATG and mPSL for RRAL may be a viable alternative to non-HID-HSCT.

164-OR: Efficacy of New-Onset Type 1 Diabetes (T1D) Intervention Trials: Variation in 1-Year Outcomes

Mean 2-hour area under the curve (AUC) C-peptide levels at 1 year remain the primary endpoint for most intervention trials in subjects with new onset T1D. Comparisons of efficacy across T1D intervention trials with positive outcomes remain limited. We performed a cross-trial comparison of the NIH TrialNet sponsored new-onset rituximab, abatacept, low-dose anti-thymocyte globulin (ATG) (2.5mg/kg), and ATG/GCSF trials and the NIH ITN sponsored high-dose ATG (6.5mg/kg), alefacept, and teplizumab trials. One-year study group mean AUC C-peptides were adjusted for baseline C-peptide and age and compared to their internal control to retain individual study effects. To further compare differences in AUC C-peptide across trials, rituximab was used as a reference and percent increase compared to rituximab effect was calculated. Using this methodology, low-dose ATG (164%) and teplizumab (171%) provided for the largest relative improvements in AUC C-peptide. Percent effects over rituximab of the other studies were: high-dose ATG (-43%), alefacept (33%), abatacept (37%) and ATG/GCSF (45%). Despite the limitations of meta-like analyses and the notable differences in control group AUC C-peptides, these data strongly support the notion that low-dose ATG and teplizumab provide for the greatest relative preservation of AUC C-peptide amongst recently completed new onset T1D interventions. Disclosure M.J. Haller: Advisory Panel; Self; Pancreum, SAB Biotherapeutics. M.N. Greco: None. D. Schatz: None. M.A. Atkinson: None. T.M. Brusko: Advisory Panel; Self; Caladrius Biosciences, Inc. Board Member; Self; OneVax, LLC. L.M. Jacobsen: None. K.C. Herold: Consultant; Self; Provention Bio, Roche Pharma. S.E. Gitelman: Advisory Panel; Self; Avortes, Intermountain Therapeutics, Lilly Diabetes, Tolerion, Inc. Research Support; Self; Caladrius Biosciences, Inc., Janssen Research &amp; Development. Other Relationship; Self; Novo Nordisk Inc. J. Krischer: None. B.N. Bundy: None. Funding The Leona M. and Harry B. Helmsley Charitable Trust; National Institutes of Health; JDRF; Sanofi; Amgen Inc.; McJunkin Family Charitable Foundation

Role of thymoglobulin in matched sibling allogeneic hematopoietic stem cell transplantation with busulfan and fludarabine conditioning in myeloid malignanicies.

In vivo T-cell depletion using anti-thymocyte globulin (ATG) is widely used in allogeneic hematopoietic stem cell transplantation (HSCT) for prophylaxis of GvHD. We investigated the influence of thymoglobulin dose (an ATG) on GvHD following matched sibling donor (MSD) HSCT with a busulfan and fludarabine preparative regimen. Medical records of 180 patients who received MSD HSCT with a conditioning regimen of busulfan, fludarabine, and ATG (BuFluATG) were reviewed retrospectively. The median age was 53 years (range 18-68). Initial diagnoses were acute myeloid leukemia (73.3%) and myelodysplastic syndrome (26.7%). Forty-four and 68 patients (24.4 and 37.7%) experienced acute and chronic GvHD of any grade, respectively. High-dose (⩾4.5 mg/kg) ATG was independently associated with decreased risk of acute GvHD (hazard ratio=0.36, 95% confidence interval (CI): 0.15-0.84, P=0.019) compared to low-dose ATG (<4.5 mg/kg). Although ATG dose was associated with the risk of acute GvHD, it was not associated with the risk of chronic GvHD in our study. A higher dose (⩾4.5 mg/kg) of ATG decreases the risk of acute GvHD but had no significant impact on disease-free survival in MSD HSCT patients conditioned with BuFluATG. The optimal dose of ATG should be further investigated in a large prospective study context.

Alemtuzumab Induction and Delayed Acute Rejection in Steroid-Free Simultaneous Pancreas-Kidney Transplant Recipients.

The optimal immunosuppressive regimen in simultaneous pancreas-kidney transplant (SPKT) recipients that prevents acute rejection episodes (AREs) and allows optimal outcome remains elusive. This cohort study assessed incidence and time to AREs in 73 consecutive SPKT recipients receiving alemtuzumab induction and steroid-free maintenance with tacrolimus and mycophenolate mofetil. A cohort with single high-dose antithymocyte globulin (ATG; n = 85) and triple therapy served as controls. In addition, we provided mechanistic insights in AREs after alemtuzumab depletion, including composition and alloreactivity of lymphocytes (flow cytometry and mixed lymphocyte reaction) plasma alemtuzumab levels (enzyme-linked immunosorbent assay), and maintenance drug exposure. Overall number of AREs at 3 years was significantly lower with alemtuzumab versus ATG induction (26.0% vs 43.5%; adjusted hazard ratio, 0.38; P = 0.029). Most AREs (94.6%) with ATG occurred within the first month, whereas 84.2% of AREs with alemtuzumab occurred beyond 3 months. Patients with and without an ARE in the steroid-free alemtuzumab group showed no differences in composition of lymphocytes, or in alemtuzumab levels. Of note, more than two thirds of these AREs were preceded by empiric tacrolimus and/or mycophenolate mofetil dose adjustments due to viral infections, leukopenia, or gastrointestinal symptoms. Alemtuzumab induction resulted in a significant lower incidence of AREs. Empiric dose adjustments beyond 3 months in the absence of steroids carry a significant risk for subsequent rejection in SPKT recipients.

Anti-Thymocyte Globulin for Graft-Versus-Host Disease Prophylaxis in Patients with Acute Myeloid Leukemia Undergoing Reduced Intensity Conditioning Allogeneic Stem Cell Transplantation in First Complete Remission: A Survey on Behalf of the EBMT Acute Leukemia Working Party

Introduction: Anti-thymocyte globulin (ATG) prophylaxis was shown to be effective in reducing both acute and chronic graft-versus-host disease (GVHD) following allogeneic stem cell transplantation (allo-SCT). In previously published studies, the addition of ATG to the pre-allo-SCT conditioning regimen resulted in substantial reduction in GVHD incidence, but theoretically ATG could be associated with increased relapse rate, especially if administered at high doses (> 6 mg/kg of ATG Thymoglobulin or >15mg/kg of ATG Fresenius) to patients with high-risk acute myeloid leukemia (AML). To further address this question, we assessed transplantation outcome in adult AML patients who underwent reduced intensity conditioning (RIC) allo-SCT from a matched sibling donor (MSD) while in complete remission (CR1), with or without ATG. We used the registry data of the Acute Leukemia Working Party of the European Society forBlood and Marrow Transplantation (EBMT).Patients and Methods: Inclusion criteria were adult patients with AML (de novo or secondary) who underwent RIC allo-SCT from an MSD in CR1 between the years 2000 and 2014. RIC conditioning was defined as per the EBMT criteria. Only patients presenting with intermediate- or poor-risk cytogenetics or secondary AML were included in the analysis. Patients who received any kind of T cell depletion including Campath were excluded. Patients were considered to receive high-dose ATG if they received > 6mg/kg of ATG-Thymoglobulin or >15mg/kg of ATG-Fresenius. All other patients were considered as a control.Results: Two hundred and five of the 1750 patients, included in the analysis, received high-dose ATG. The median patient age at diagnosis was 56 years and the median donor age was 55 years for both groups. Fifty four percent of the patients were males. The graft properties were similar in the high-dose ATG and control groups, with female-to-male transplants in 48/205 (23.4%) and 378/1545 (24.6%) patients, respectively. Peripheral blood was the source of grafts in 193/205 (94.1%) and 1413/1545 (91.5%) patients, respectively (p=NS). Poor cytogenetics was reported in 28/205 (13.7%) patients who were given high-dose ATG and in 247/1545 (16%) patients from the control group. Secondary AML was more prevalent among patients from the high-dose ATG group compared to the control group [84/205 (41%) vs 425/1545 (27.5%; p=0.0003]. The median follow-up was 44.6 (0-177) months.The 3-year overall survival (OS) and leukemia-free survival (LFS) were almost identical in both groups, amounting to 54.9 and 46.3 months, respectively for patients who received high-dose ATG and to 54 and 49.5 months, respectively for patients in the control group (p=0.978 and 0.376 ). Differences in relapse incidence (RI) (40.9% vs 34.4%; p=0.1) and non-relapse mortality (NRM) (11.7% vs 16%; p=0.152) were insignificant. The composite endpoint of GVHD-free/relapse-free survival (GRFS) was similar in the groups after one and three years of follow-up (42.9 vs 49.7 and 31.1 vs 36 months, respectively; p=0.152 and p=0.124). In multivariate analysis, the use of ATG prophylaxis was not found to be associated with any of the evaluated parameters. In contrast, OS, LFS, RI, NRM and GRFS were all affected by patient age and presence of secondary AML. Poor cytogenetics correlated with inferior OS, LFS, RI and GRFS. Donor CMV positivity was associated with lower OS, LFS and GRFS and a higher rate of NRM.Conclusions: In AML patients presenting with intermediate- or poor-risk cytogenetics who are referred to RIC allo-SCT from an MSD in CR1, the addition of ATG to conditioning regimens does not influence the outcome. The composite GRFS endpoint is mainly affected by AML risk factors (hazard ratio of 1.43 for secondary AML and 1.37 for poor-risk cytogenetics; p=0.0001). The role of ATG prophylaxis in AML patients undergoing RIC allo-SCT should be prospectively evaluated. DisclosuresMaertens:Gilead: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy; Astellas: Consultancy, Speakers Bureau; Merck Sharp & Dohme: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Anti-Thymocyte Globulin Kills Leukemic Stem Cells in Vitro

Background: Although curative treatment for acute myeloid leukemia, the success of allogeneic hematopoietic cell transplantation (HCT) is limited due to leukemia relapse and graft-versus-host disease (GvHD). Rabbit anti-thymocyte globulin (ATG) used for GvHD prophylaxis does not increase relapse (Walker et al: Lancet Oncol 2016). The non-increase in relapse could be because ATG has a direct anti-leukemic effect (Dabas et al: BBMT 2016). Multiple studies have suggested that a high number of leukemic stem cells (LSCs, also called leukemia initiating cells) remaining after therapy is associated with relapse. Therefore, targeting LSCs may be important for treating or preventing relapse. We demonstrated ATG's cytotoxic effect against acute myeloid leukemia (AML) blasts (Dabas et al: BBMT 2016). However, ATG's effect on LSCs has not been evaluated. In this study, we investigated in vitro ATG-induced complement-independent cytotoxicity (CIC, presumably direct induction of apoptosis) and complement-dependent cytotoxicity (CDC) against LSCs. This was also compared to ATG-induced CIC and CDC against healthy hematopoietic stem cells (HSCs).Methods:Frozen peripheral blood mononuclear cells (PBMNCs) from 15 patients newly diagnosed with AML were used as the source of LSCs. Frozen mononuclear cell apheresis samples from 15 healthy stem cell donors were used as the source of HSCs. We measured by flow cytometry CIC and CDC induced by ATG at 10 mg/L (the median peak concentration achieved with 4.5 mg/kg ATG given over day -2, -1 and 0) and 50 mg/L. CIC was induced by incubating cells with ATG in the presence of heat-inactivated (complement-depleted) fetal bovine serum for 4 hours. As a negative control for the calculation of background, cells were cultured under the same conditions except without ATG. CDC was induced by incubating cells with ATG in the presence of human serum as a source of complement for 15 minutes. As a negative control (for background), cells were incubated with ATG in the presence of heat-inactivated human serum (no complement). The LSCs and HSCs were phenotypically defined as CD45dim/neg, side scatterlow, CD34+, CD38neg and negative for CD14, CD16, CD19, CD56, CD235a and CD41a. For CIC, dying/dead cells were identified as Annexin V positive (Annexin V+). For CDC, dead cells were identified as 7-amino-actinomycin D positive (7AAD+).Results: ATG induced both CIC and CDC of LSCs at the concentration of 50 mg/L but not 10 mg/L. For CIC, the median percent Annexin V+ LSCs after incubation with 50 mg/L ATG was 22.3% vs 18.4% for background (p=0.0043, Wilcoxon matched pairs test). For CDC, the median percent 7AAD+ LSCs after incubation with 50 mg/L ATG was 37.2% vs 2% background (p=0.0004, Wilcoxon matched pairs test). Next, we compared the cytotoxicity of 50 mg/L ATG against LSCs versus healthy HSCs. For CIC, a significantly greater percent of LSCs than HSCs was killed (P=0.0363, Mann-Whitney rank sum test) (Figure 1). Similarly, for CDC, there was a trend toward a greater percent of LSCs than HSCs killed (P=0.0971) (Figure 2).Conclusion: ATG kills LSCs in vitro via both CIC and CDC. However, the degree of the killing is minor and only observed at a higher ATG concentration than typically achieved in patients. Our data also suggest that if high dose ATG was used in patients (resulting in ≥50 mg/L concentration), LSCs could be killed to a greater degree than healthy HSCs. [Display omitted] [Display omitted] DisclosuresDaly:Sanofi-Genzyme: Other: Advisory Board.

Antithymocyte Globulin at Clinically Relevant Concentration Kills Leukemic Blasts

Background/Rationale: Success of allogeneic hematopoietic cell transplantation (HCT) is limited due to graft-versus-host disease (GvHD) and leukemia relapse. Contrary to small molecule immunosuppressive drugs like cyclosporine or methotrexate, rabbit antithymocyte globulin (ATG) used for GvHD prophylaxis with myeloablative HCT conditioning does not increase relapse. The mechanism of the anti-GvHD effect of ATG has been studied extensively, however, the reason for ATG not increasing relapse is not known. We hypothesized that ATG has an anti-leukemic activity. Here we investigated the anti-leukemic activity of ATG in vitro at concentrations achieved in vivo with our standard ATG dose of 4.5 mg/kg (median 10 mg per liter of serum; range, 1-25). We focused on complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC).Methods: Blood or bone marrow from patients newly diagnosed with acute myeloid leukemia (AML, n=25) or acute lymphoid leukemia (ALL, n=4), obtained before induction chemotherapy, was used as the source of leukemic cells. We measured CDC and CIC of ATG, specifically against leukemic blasts at clinically achieved concentrations (1-25 mg/L) and an achievable concentration (50 mg/L). The leukemic blasts were identified as CD45dim/neg and side scatter low (SSclow) cells in case of AML-M0, M1 or M2 (according to British-American-French classification) or ALL. In case of AML-M4 or M5, we gated on CD45dim/neg and SSclow/intermediate cells. These cells could contain both leukemic and normal blasts and some post-blast (maturing/mature) cells. We minimized the fraction of normal blasts and post-blast cells included by introducing a "dump channel". The dump channel consisted of antibodies against antigens expressed by cells of lineages other than the leukemia lineage (except for antigens aberrantly expressed by the leukemic cells) and by post-blast cells. For most AML patients, the dump channel consisted of CD10, CD19, CD41a, CD235a, CD14 and CD16. For most ALL patients, the dump channel consisted of CD117, CD33, CD41a, CD235a, CD14 and CD16. Thus, leukemic blasts were defined as mononuclear cells that were CD45dim/neg and SSclow (or SSclow/intermediate for M4/M5) and did not express dump channel antigens. CDC was induced by incubation of the patient specimen with human serum (source of complement). CIC was induced by incubation of the patient specimen with heat-inactivated serum (no complement). For CDC dead cells were identified as 7-amino-actinomycin D positive (7AAD+). For CIC dead/dying cells were identified as Annexin V positive (Annexin V+).Results: ATG induced death of leukemic blasts both via CDC and CIC. Median 0.3%, 3.0%, 12.6% and 41.7% blasts were killed with 1, 10, 25 and 50 mg/L ATG, respectively, via CDC. Median 2.1%, 8.0%, 11.4% and 12.6% blasts were killed after 4 hour incubation with 1, 10, 25 and 50 mg/L ATG, respectively, via CIC. For both CDC and CIC, the percents were significantly higher than background (p<0.05). There was a high variability in the sensitivity of the leukemic blasts to ATG. The adjusted (background subtracted) percent of blasts killed ranged from 2.6% to 93.8% via CDC (Figure 1) and from 1.5% to 69.9% via CIC (Figure 2). When we arbitrarily defined a patient with resistant leukemic blasts as <10% (adjusted) leukemic blasts dead after exposure to 50 mg/L ATG, there were 3/28 patients with resistant leukemic blasts in case of CDC (Figure 1) and 9/23 patients in case of CIC (Figure 2).Conclusion: ATG at clinically relevant concentrations kills primary leukemic blasts in vitro. Some acute leukemias are highly sensitive and others relatively resistant to ATG. Whether this applies to in vivo needs to be determined. If yes, and given that the killing of leukemic cells is ATG concentration-dependent, conditioning regimens with high dose ATG could be developed for patients with leukemia sensitive to ATG to maximize the concurrent anti-GvHD and anti-relapse effects. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Renal Allograft Outcome and Infectious Complications Following Antithymocyteglobulin and Alemtuzumab Used as Induction in Kidney Transplantation.

Background: Antithymocyte globulin (ATG) and Humanized Monoclonal Anti-CD52 Antibody, Alemtuzumab (Targeted all T-cells depleting antibodies) were used as induction regimens for kidney transplantation. However, both drugs have been questionable in threatened Infectious complications which would be the main barriers to patients and allograft disease-free survival. Methods: Sixty-eight patients (Cadeveric donor 38, Living donor 30) who have been performed transplantation between July 1996 - 2009 were retrospective study analyzed kidney allograft outcome and infectious complications after induction therapy between ATG 200-400 mg, ATG 500-700 mg 1 given intravenously and alemtuzumab 30 mg given subcutaneously. Immunosuppressive agents were Tacrolimus(88.2%), Cyclosporin A(8.8%), Mycophenolate mofetil(52.9%), Azathioprine(35.2%), Everolimus(0.3%) and corticosteroids. Standard perioperative antimicrobial prophylaxis consisted of ceftriaxone in combination with Fluconazole, Trimethoprim-sulfamethoxazole and Acyclovir in high risk patients. Results: Patient and kidney graft survival at thirteen years was 95.58%. The rejection rate in low dose ATG group was 20.58%, high dose ATG was 32.14 (p-value = ns). There was no incidence of rejection in alemltuzumab group. Three grafts from ATG group were lost from rejection. The mild grade infection rate 65 events/302 patient years, moderate grade 51 event/302 patient years, severe infection 8 event/302 pateient years with no difference in terms of prevalence according to the three groups. Conclusion: ATG and alemtuzumb are effective for use as induction regimens. Infectious complications are comparable between low dose ATG, high ATG and alemtuzumab.

High Serum Level of Antithymocyte Globulin Immediately before Graft Infusion Is Associated with a Low Likelihood of Chronic, But Not Acute, Graft-versus-Host Disease

Rabbit antithymocyte globulin (ATG) is administered during transplant conditioning to decrease the risk of both acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD). Here we evaluated the relationship between the serum concentration of ATG (capable of binding to lymphocytes) immediately before graft infusion (day 0) or on day +7 or +28 post-transplantation and the development of aGVHD or cGVHD. We studied 180 patients whose conditioning included 4.5 mg/kg antithymocyte globulin (ATG; Thymoglobulin). For aGVHD, we found no association with ATG levels on day 0. Nevertheless, high day +7 and +28 ATG levels were associated with a low likelihood of aGVHD. For cGVHD, high ATG levels at all 3 time points (days 0, +7, and +28) were associated with a low likelihood of cGVHD. In conclusion, high-dose ATG administration at the time of graft infusion appears to inhibit the development of cGVHD, but not aGVHD; however, higher ATG levels on days +7 and +28 are associated with lower rates of both aGVHD and cGVHD.

Expansion of Memory-Type CD8+ T Cells Correlates With the Failure of Early Immunosuppression Withdrawal After Cadaver Liver Transplantation Using High-Dose ATG Induction and Rapamycin

We report on a pilot study investigating the feasibility of early immunosuppression withdrawal after liver transplantation (LT) using antithymocyte globulin (ATG) induction and rapamycin. LT recipients received 3.75 mg/kg per day ATG from days 0 to 5 followed by rapamycin-based immunosuppression. In the absence of acute rejection (AR), rapamycin was withdrawn after month 4. Immunomonitoring included analysis of peripheral T-cell phenotypes and clonality, cytokine production in mixed lymphocyte reaction, and characterization of intragraft infiltrating cells. Ten patients were enrolled between October 2009 and July 2010. In the first three patients, complete withdrawal of immunosuppression after month 4 led to AR. No further withdrawals of immunosuppressive were attempted. Two AR occurred in the remaining seven patients. ATG induced profound T-cell depletion followed by CD8(+) T-cell reexpansion exhibiting memory/effector-like phenotype associated with progressive oligoclonal T-cell expansion (Vβ/HPRT ratio) and gradually enhanced anti-cytomegalovirus and anti-Epstein-Barr virus T-cell frequencies. Patients developing AR were characterized by decreased TCAIM expression. AR were associated with increased donor-specific production of interferon (IFN)-γ and interleukin (IL)-17, increased intragraft expression of IFN-γ mRNA, and significant CD8(+) T-cell infiltrates colocalizing with IL-17(+) cells. High-dose ATG followed by short-term rapamycin treatment failed to promote early operational tolerance to LT. AR correlates with expansion of memory-type CD8(+) T cells and increased levels of IFN-γ and IL-17 in mixed lymphocyte reaction and in the graft. This suggests that resistance and preferential expansion of effector memory T-cell in lymphopenic environment could represent the major barrier for establishment of tolerance to LT in approaches using T-cell-depleting induction.

A high antithymocyte globulin dose increases the risk of relapse after reduced intensity conditioning HSCT with unrelated donors

The study included 110 consecutive patients with hematological malignancies receiving fludarabine-based reduced intensity conditioning (RIC) and hematopoietic stem cell transplantation (HSCT) from matched unrelated donors. The median age was 55 yr (range 11-68) and all but 15 patients received peripheral blood stem cell grafts. Antithymocyte globulin (ATG) (Thymoglobulin, Genzyme) at a total dose of 6 mg/kg (n = 66) or 8 mg/kg (n = 44) was given to all patients according to protocol. The ATG dose did not affect time-to-neutrophil or platelet engraftment. The incidences of acute GVHD grades II-IV were 34% and 18% (p = 0.11) and of chronic GVHD were 40% and 26% (p = 0.46) in patients receiving 6 and 8 mg/kg of ATG, respectively. The five-yr relapse-free survival (RFS) was 61% and 36% (p = 0.14) in patients, given low and high ATG dose, respectively. In patients given low-dose ATG, the incidence of relapse was lower compared to those given high-dose ATG, 19% vs. 41% (p = 0.04). In multivariate analysis, age >50 yr (p < 0.001), absence of acute (p < 0.001) and chronic GVHD (p = 0.001) were correlated to relapse, and low-dose ATG was associated with improved RFS (p < 0.05). A high dose (8 mg/kg) of ATG in RIC HSCT with unrelated donors increased the risk for relapse and reduced the RFS.

Antithymocyte Globulin Induction Therapy in Heart Transplantation: Prospective Randomized Study of High vs Standard Dosage

Background In cardiac transplantation, high-dose antithymocyte globulin (ATG) induction therapy as short-term rejection prophylaxis has not been used. Objective To evaluate the efficacy and safety of intraoperative use of single high-dose ATG induction therapy after heart transplantation. Patients and Methods Fourteen patients received single high-dose ATG therapy plus shortened standard therapy (group1), and 16 patients received ATG standard therapy (group2). Results No perioperative deaths were reported. During follow-up, 3 deaths were recorded. Five- year patient survival was 92.8% in groupl vs 85.7% in group2 ( P = .34). The mean (SD) number of acute rejection episodes per patient was 2.5 (2.2) in the high-dose ATG group vs 2.7 (2.5) in the standard therapy group ( P = .83), with 5-year freedom from acute rejection of 45.5% in group 1 vs 35.6% in group 2 ( P = .85). Infections were observed in 6 patients in group1 and in 8 patients in group2 ( P = .69). Malignant disease was diagnosed in 1 patient in the high-dose group and 3 patients in the standard therapy group ( P = .35). Chronic allograft vasculopathy was recognized in 4 patients (28%) in group1 and 8 (50%) in group2 ( P = .05). Five-year actuarial freedom from allograft vasculopathy was 69.2% in the high-dose ATG group vs 50.0%% in the standard therapy group ( P = .35). Conclusions High-dose ATG for prevention of rejection episodes is safe and efficacious, with a lower rate of early and late complications, in particular, graft vasculopathy.

Stable Mixed Donor-Donor Chimerism After Double Cord Blood Transplantation

Double cord blood transplantation (DCBT) has been used increasingly and has proven to be both safe and efficacious. In chimerism analysis, previous studies have[ indicated single unit predominance early after DCBT. In the present study, we evaluated the chimeric pattern in T-, B- and myeloid cells using PCR-based chimerism analysis in seven patients after DCBT: five patients had acute leukemia and two had lymphoma. Five patients received myeloablative conditioning and two patients were given reduced intensity conditioning. All patients received antithymocyte globulin (ATG) before DCBT. Three of the six evaluable patients showed donor–donor mixed chimerism in all cell lineages at 90 days after DCBT. Interestingly, two patients in long-term follow-up showed mixed donor chimerism in all cell lineages at 25 and 35 months after DCBT, respectively. Both patients are doing clinically well. Neither of the two developed GVHD after DCBT. In conclusion, in this study donor–donor mixed chimerism was common after high dose ATG and DCBT. Further studies are warranted concerning the immunological consequences of the phenomenon of donor–donor mixed chimerism after DCBT. Double cord blood transplantation (DCBT) has been used increasingly and has proven to be both safe and efficacious. In chimerism analysis, previous studies have[ indicated single unit predominance early after DCBT. In the present study, we evaluated the chimeric pattern in T-, B- and myeloid cells using PCR-based chimerism analysis in seven patients after DCBT: five patients had acute leukemia and two had lymphoma. Five patients received myeloablative conditioning and two patients were given reduced intensity conditioning. All patients received antithymocyte globulin (ATG) before DCBT. Three of the six evaluable patients showed donor–donor mixed chimerism in all cell lineages at 90 days after DCBT. Interestingly, two patients in long-term follow-up showed mixed donor chimerism in all cell lineages at 25 and 35 months after DCBT, respectively. Both patients are doing clinically well. Neither of the two developed GVHD after DCBT. In conclusion, in this study donor–donor mixed chimerism was common after high dose ATG and DCBT. Further studies are warranted concerning the immunological consequences of the phenomenon of donor–donor mixed chimerism after DCBT.

Stable mixed donor–donor chimerism after double cord blood transplantation

Double cord blood transplantation (DCBT) has been used increasingly and has proven to be both safe and efficacious. In chimerism analysis, previous studies have indicated single unit predominance early after DCBT. In the present study, we evaluated the chimeric pattern in T-, B- and myeloid cells using PCR-based chimerism analysis in seven patients after DCBT: five patients had acute leukemia and two had lymphoma. Five patients received myeloablative conditioning and two patients were given reduced intensity conditioning. All patients received anti-thymocyte globulin (ATG) before DCBT. Three of the six evaluable patients showed donor-donor mixed chimerism in all cell lineages at 90 days after DCBT. Interestingly, two patients in long-term follow-up showed mixed donor chimerism in all cell lineages at 25 and 35 months after DCBT, respectively. Both patients are doing clinically well. Neither of the two developed GVHD after DCBT. In conclusion, in this study donor-donor mixed chimerism was common after high dose ATG and DCBT. Further studies are warranted concerning the immunological consequences of the phenomenon of donor-donor mixed chimerism after DCBT.