Depleted Stem Cell Transplantation Research Articles

Outcomes of Unrelated Partial T Cell Depleted Stem Cell Transplantation for Patients with Hemoglobinopathies

Outcomes of Unrelated Partial T Cell Depleted Stem Cell Transplantation for Patients with Hemoglobinopathies

Predictors of Covid-19 Vaccination Response After In-Vivo T-Cell–Depleted Stem Cell Transplantation

Covid-19 vaccination is recommended in allogeneic transplant recipients, but many questions remain regarding its efficacy. Here we studied serologic responses in 145 patients who had undergone allogeneic transplantation using in vivo T-cell depletion. Median age was 57 (range 21-79) at transplantation and 61 (range 24-80) at vaccination. Sixty-nine percent were Caucasian. One third each received transplants from HLA-identical related (MRD), adult unrelated (MUD), or haploidentical-cord blood donors. Graft-versus-host disease (GVHD) prophylaxis involved in-vivo T-cell depletion using alemtuzumab for MRD or MUD transplants and anti-thymocyte globulin for haplo-cord transplants. Patients were vaccinated between January 2021 and January 2022, an average of 31 months (range 3-111 months) after transplantation. Sixty-one percent received the BNT162b2 (bioNtech/Pfizer) vaccine, 34% received mRNA-1273 (Moderna), and 5% received JNJ-78436735 (Johnson & Johnson). After the initial vaccinations (2 doses for BNT162b2 and mRNA-1273, 1 dose for JNJ-7843673), 124 of the 145 (85%) patients had a detectable SARS-CoV-2 spike protein (S) antibody, and 21 (15%) did not respond. Ninety-nine (68%) had high-level responses (≥100 binding antibody units [BAU]/mL)m and 25 (17%) had a low-level response (<100 BAU/mL). In multivariable analysis, lymphocyte count less than 1 × 109/ mL, having chronic GVHD, and being vaccinated in the first year after transplantation emerged as independent predictors for poor response. Neither donor source nor prior exposure to rituximab was predictive of antibody response. SARS-CoV-2 vaccination induced generally high response rates in recipients of allogeneic transplants including recipients of umbilical cord blood transplants and after in-vivo T cell depletion. Responses are less robust in those vaccinated in the first year after transplantation, those with low lymphocyte counts, and those with chronic GVHD.

Effects of Various ATG Doses on Rejection Rate and Immune Recovery in Haploidentical T and B Cell Depleted Stem Cell Transplantation

T and B cell depletion of haploidentical stem cells with CD3/CD19 coated magnetic microbeads prevents from GvHD and allows to coinfuse donor NK cells and other accessory cells. Additional in vivo depletion of the graft by serotherapy is not necessary. Thus, a dosing scheme of ATG is needed, which provides a rejection prophylaxis by depleting lymphocyte subsets of the patients but which does not harm the graft, in particular cotransfused NK cells. We retrospectively analyzed the impact of various Grafalon (anti-human T-lymphocyte immunoglobulin = ATLG; formerly ATG-Fresenius) doses given at day -12 on engraftment rate and on posttransplant immune recovery.A total of 50 pediatric patients received either 3x5 (n=7), 3x10 (n=34) or 3x20mg/kg (n=9) ATLG starting at day -12 to day -9, followed by fludarabine (160 mg/m2, d -8 to -5), thiotepa (10 mg/m2 d -4) and melphalan (140 mg/m2 d -3 to -2). Diagnoses were: ALL/AML n=10, relapsed solid tumors n=33, nonmalignant diseases n=7. Most patients received MMF until day 30-60. ATLG serum levels were measured in 24 patients by flow cytometry (T cell specific rabbit IgG). Apoptosis/necrosis of donor NK cells was assessed with Annexin V/PI staining.A median number of 16x106 CD34/kg and 65x106 NK cells/kg with 29x103 T cells/kg were infused. Median time to ANC>500/µl was 9.5 days. Graft rejection occurred in 3/7 patients with 15mg (42%), in 7/34 patients with 30mg (20%) and in 1/9 patients with 60mg ATLG (11%). All rejectors could be rescued by reconditioning and 2nd stem cell donation or by infusion of autologous back ups. Acute GvHD grade 0-I occurred in 47 patients (94%), GvHD grade II and III-IV was observed in 1 and 2 patients (2%, 4%), respectively. Chronic GvHD occurred in 9 patients (18%). TRM at 1 year was 10% for the entire group. Immune recovery of CD3+ T cells was influenced by the various amounts of ATLG given in the 3 subgroups (15 vs 30 vs 60mg/kg) with a median number of 98 vs 10 vs 2 cells/µl at day +30, 95 vs 100 vs 2 cells/µl at day +90 and 75 vs 218 vs 239 cells/µl at day +180 (figure 1a). Recovery of CD56+ NK cells was fast and reliable and reached peaks of 459 vs 342 vs 155 cells/µl at day +30. Later on NK cells dropped to normal levels of 152 vs 167 vs 104 cells/µl at day +90 and 118 vs 172 vs 99 cells /µl at day +180. ATLG serum levels reached mean peak values of 9.6 µg/ml ±4.7 vs 22.1±13.8 µg/ml vs 53.7 ±13.9 µg/ml at day -8 and dropped to 1.4 ±1µg/ml vs and 3.0 ±1.98 µg/ml and 11.5 ±5.8 µg/ml at day 0 (15 vs 30 vs 60mg/kg, figure 1b). In vitro incubation of isolated NK cells from healthy donors with comparable ATLG doses (1 or 10 µg/ml) resulted in 26 (41)% apoptosis and 0.2 (0.2)% necrosis after 24 hours. Thus, vitality of 73 (59)% of NK cells were preserved.Conclusions: Our aim was to reduce the rejection rate and to improve immune recovery in patients receiving CD3/19 depleted haploidentical peripheral stem cells, in particular without hampering donor NK cells in the grafts. Administration of 15, 30 or 60 mg/kg ATLG was started at an early time point (day -12) of the regimen. Higher ATLG doses correlated with higher serum levels. Peak levels at day -8 provided a rejection prophylaxis. At day 0, various lower levels were achieved and minimized further in vivo depletion. Increased ATLG doses were associated with a lower rejection rate. On the other hand, recovery of T cells was slower after 60mg/kg ATLG in the early posttransplant phase with lower CD3 counts at day 30 and 90. Since all rejectors were successfully regrafted, it may be reasonable to balance between rejection risk and impaired immune recovery and to use an average dose (30mg). Further studies should investigate if serotherapy might be given even earlier than in this analysis. [Display omitted] DisclosuresLang:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Martinius:Neovii Biotech: Employment. Handgretinger:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding.

HLA-haploidentical TCRαβ+/CD19+-depleted stem cell transplantation in children and young adults with Fanconi anemia

AbstractWe report on the outcome of 24 patients with Fanconi anemia (FA) lacking an HLA matched related or unrelated donor, given an HLA-haploidentical T-cell receptor αβ (TCRαβ+) and CD19+ cell-depleted hematopoietic stem cell transplantation (HSCT) in the context of a prospective, single-center phase 2 trial. Sustained primary engraftment was achieved in 22 (91.6%) of 24 patients, with median time to neutrophil recovery of 12 days (range, 9-15 days) and platelet recovery of 10 days (range, 7-14 days). Cumulative incidences of grade 1 to 2 acute graft-versus-host disease (GVHD) and chronic GVHD were 17.4% (95% confidence interval [CI], 5.5%-35.5%) and 5.5% (95% CI, 0.8%-33.4%), respectively. The conditioning regimen, which included fludarabine, low-dose cyclophosphamide and, in most patients, single-dose irradiation was well tolerated; no fatal transplant-related toxicity was observed. With a median follow-up of 5.2 years (range, 0.3-8.7 years), the overall and event-free survival probabilities were 100% and 86.3% (95% CI, 62.8%-95.4%), respectively (2 graft failures and 1 case of poor graft function were considered as events). The 2 patients who experienced primary graft failure underwent a subsequent successful HSCT from the other parent. This is the first report of FA patients given TCRαβ+/CD19+-depleted haplo-HSCT in the context of a prospective trial, and the largest series of T-cell–depleted haplo-HSCT in FA reported to date. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Haploidentical α/β T-cell and B-cell depleted stem cell transplantation in severe mevalonate kinase deficiency.

Mevalonic aciduria represents the most severe form of mevalonate kinase deficiency (MKD). Patients with mevalonic aciduria have an incomplete response even to high doses of anti-cytokine drugs such as anakinra or canakinumab and stem cell transplantation (SCT) represents a possible therapy for this severe disease. We report the first two children affected by severe MKD who received haploidentical α/β T-cell and B-cell depleted SCT. Both patients received a treosulfan-based conditioning regimen and one received a second haploidentical-SCT for secondary rejection of the first. Both patients obtained a stable full donor engraftment with a complete regression of clinical and biochemical inflammatory signs, without acute organ toxicity or acute and chronic GvHD. In both, the urinary excretion of mevalonic acid remained high post-transplant in the absence of any inflammatory signs. Haploidentical α/β T-cell and B-cell depleted SCT represents a potential curative strategy in patients affected by MKD. The persistence of urinary excretion of mevalonic acid after SCT, probably related to the ubiquitous expression of MVK enzyme, suggests that these patients should be carefully monitored after SCT to exclude MKD clinical recurrence. Prophylaxis with anakinra in the acute phase after transplant could represent a safe and effective approach. Further biological studies are required to clarify the pathophysiology of inflammatory attacks in MKD in order to better define the therapeutic role of SCT.

Feasibility and Outcomes of T-Cell Depleted Hematopoietic Stem Cell Transplantation in Patients with Relapsed or Refractory AML and High Risk MDS

Feasibility and Outcomes of T-Cell Depleted Hematopoietic Stem Cell Transplantation in Patients with Relapsed or Refractory AML and High Risk MDS

Successful Salvage Haploidentical Alpha-Beta T Cell–Depleted Stem Cell Transplantation After Busulfan-Based Myeloablation in a Patient With IPEX Syndrome: A Case Report

BackgroundX-linked immunodysregulation syndrome with polyendocrinopathy and enteropathy (IPEX) is caused by FOXP3 gene mutations that block the generation of regulatory T lymphocytes. We report an 18-month-old boy with classic IPEX who underwent 2 hematopoietic stem cell transplantations (HSCTs). MethodsThe first HSCT from an unrelated 8/10 HLA-matched umbilical cord blood donor (UCB) was performed after a conditioning regimen consisting of treosulfan, fludarabine, thiotepa, and thymoglobulin. Due to complete rejection of the UCB transplant, a second transplantation from a 6/10 HLA-matched mother was performed after alpha-beta T-cell depletion. The second conditioning regimen consisted of busulfan, fludarabine, a single dose of cyclophosphamide 1 g/m2, and Grafalon (Neovii Pharmaceuticals, Rapperswil, Switzerland). The T-cell depletion product contained 15.06 x 106 CD34+ cells per kilogram body weight (BW) and 4.19 x 105 alpha-beta T lymphocytes per kilogram BW. Due to acute graft rejection, the boy was treated with thymoglobulin, and full donor chimerism in both T lymphocytes and mononuclear cells was achieved. The immunosuppressive therapy was stopped 1 year after transplantation. To date, the patient remains free from graft-vs-host disease (GVHD) and immunosuppression. ConclusionsHSCT after busulfan-based reduced-toxicity conditioning in patients with IPEX syndrome is feasible and well tolerated and can result in full donor engraftment. Monitoring of chimerism and aggressive therapy in cases of graft rejection are warranted due to the high reactivity of residual autologous T lymphocytes. T-cell depletion reduces the risk of GVHD and the need for steroid therapy, which is especially challenging in patients with diabetes.

First Results of a Prospective I/II Clinical Trial in Adult Patients Using TCR Alpha/Beta Depleted Stem Cell Transplantation from Matched Related and Unrelated Donors

Introduction: We report the first analysis of a multicenter prospective single-arm phase I/II study that assesses the safety and feasibility of transplantation of TCRalpha/beta depleted stem cells from matched related or unrelated donors using the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a non-myeloablative conditioning in adult patients (trial nr NL48606.000.14). The conditioning regimen consisted of ATG (Thymoglobulin®) 1.5mg/kg i.v. days -12 to -9; fludarabine i.v. 40 mg/m2 days -5 to -2 and busulfan i.v. (Busivex®) days -5 to -2 (cumulative AUC of 80-90mg*h/L), followed by 28 days of mycophenolic acid. All 35 subjects were eligible to address the primary endpoint which is the incidence of acute GVHD at day 100.Results: 2 centers enrolled 10 female and 25 male patients (median age 59 years, range 19 - 69 years), including 9 AML, 4 ALL, 2 CML, 5 MM, 1 NHL, 10 MDS, 4 MPN. Diseases were in CR (n=17), VGPR (n=1), PR (n=5) or non-remission (n=12). 4 subjects had one or more previous allogeneic SCTs. Donors included 4 MRD, 24 10/10 matched MUD and 7 9/10 matched MUD. 8 male patients received stem cells of female donors. The median number of CD34+ cells and αβ TCR cells/kg was 6.1x 10^6 (range, 1.9-10) and 14.5x10^3 (range, 0-136), respectively. One primary graft failure was observed. Primary engraftment of ANCs > 500 cells/μL was reached at a median of 14 days (range 9 - 48 days) and of platelets > 20 at a median of 17 days (range 10 - 99) days.The median time of follow-up of this first analysis was 190 days (range 110-400 days). 3/35 patients developed acute GVHD grade III-IV during the first 100 days, 9/35 patients developed acute GVHD grade II-IV and 16/35 patients grade I-IV at day 100. At day 100 the cumulative incidence (CI) of CMV was 31%, of EBV 36% and of BK cystitis 20%. The combined CI of CMV/EBV/BK infections with a CTC-AE grade III-V was 53% at day 100.Immune reconstitution was rapid with a median of 227 (range 34 - 1626) CD3+ cells/µl on day 100 but varied substantially between patients. The median numbers of CD3/CD4+ and CD3/CD8+ at day 100 post transplant were 60 (range 30 - 120) and 124 (range 5 - 1450) cells/µl. The median numbers of NK were 289 (range 32-1140) at day 30 and 128 (range 24 - 1228) cells/µl at day 100 post transplant. The median numbers of γδ T cells were 38 (range 4-143) at day 30 and 50 (range 8-556) cells/µl at day 100 post transplant.26 out of 35 patients were alive, 2 patients died of a relapse, 2 of GVHD and 5 of infectious complications. Kaplan Meier estimates of the OS are 88% (+/- 6%) at day 100 and 68% (+/- 9%) at 1Y. Kaplan Meier estimates of the EFS are 77% (+/- 7%) at day 100 and 52% (+/- 10%) at 1Y. 2 patients developed a relapse before day 100 and 5 patients before 1Y. It is noted that the outcome of 5 patients with MM was very poor in this cohort (3 patients developed a relapse and 2 patients died of complications).Conclusion: Allo-SCT of αβ T cell depleted PBMCs form matched related or unrelated donors, in combination with early ATG and a non-myeloablative conditioning regimen, resulted in favorable rates of primary engraftment (34/35), a CI of aGVHD II-IV of 26% with only minimal immuno-suppression and an encouraging early disease control (Fig 1). OS survival was mainly impacted by NRM (Fig 1), by which the majority of complications were of infectious nature. While all patients with MM suffered from NRM or relapse, all other disease categories had a rather favorable outcome. Reducing inter-individual variations in immune reconstitution early after transplantation will be key to further improve outcomes. [Display omitted] DisclosuresMinnema:Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy, Research Funding; Janssen: Consultancy; Servier: Consultancy. Kuball:Novartis: Research Funding; Miltenyi Biotec: Research Funding; Gadeta (www.gadeta.nl): Consultancy, Equity Ownership, Patents & Royalties: on gd T cells and receptors and isolation strategies, Research Funding.

Very Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy for Prevention of Viral Infection after Allogeneic T Cell Depleted Stem Cell Transplantation

▪Background: Reactivation of latent viruses post-allogeneic stem cell transplant (SCT) negatively affects outcomes and increases non-relapse mortality. We have reported over 80% early CMV reactivation rate and significant additional costs in recipients of T cell depleted (TCD) SCT. Ex vivo generated multi-virus specific T cells (MVSTs) are effective as a therapy of active infection, but have not been evaluated as prophylaxis early after transplant. In a Phase I study (NIH 14-H-0182) we transferred MVSTs targeting immunodominant viral proteins of CMV, Epstein-Barr virus (EBV), BK and adenovirus (Ad) immediately post SCT as a novel approach to prevent viral reactivation post-SCT.Methods: All subjects were enrolled in HLA-matched T cell depleted (TCD) transplant protocol (NIH 13-H-0144). MVSTs cells were manufactured from SCT sibling donors by stimulation of elutriated lymphocytes for 14 days with seven overlapping peptide libraries (pepmixes) pulsed onto autologous dendritic cells (DCs) in presence of IL-7, IL-15 and IL-2. MVSTs were infused as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used as follows: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. Three additional subjects received MVST cells manufactured using pepmix-pulsed mononuclear cells as stimulators (Cohort 3A; 1x10e6TNC/kg) under an amended protocol. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT) defined as Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least “probably” or “definitely” related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes of efficacy, immune reconstitution and GVHD biomarkers (ST2, REG3). CDR3 sequencing (ImmunoSEQ) was performed on selected MVST products and peripheral blood samples post MVST and compared to control SCT recipients. GVHD biomarkers were analyzed pre- and post-treatment.Results: Twelve subjects were treated: nine received MVSTs generated using DCs and three subjects using mononuclear cells (cohort 3A). Median time from SCT to MVST administration was 13 days (range D +2 to +52 post-SCT). Median time to MVST for subjects in Cohort 3A was 3 days (range 2-7). There were no immediate infusion-related adverse events or DLT at the highest dose level. De novo grade II-III aGVHD post-MVST infusion was seen in three subjects (one in Cohort 1 and two in Cohort 3A), but GVHD biomarker elevation predated MVST infusion. CMV reactivation post-MVST occurred in 6 out of 12 subjects (50%) vs. 45 out of 52 patients (50% vs 87%) in a historical group of recipients of T cell depleted SCT. In all cases CMV reactivation occurred in the context of high dose steroids and in two subjects MVSTs were derived from CMV seronegative donors with minimal anti-CMV activity. One subject experienced rapidly rising AdV viremia (asymptomatic) and received an additional infusion of MVSTs. We saw self-limiting low level EBV replication in 8 cases and one BK viremia, but no disease. CDR3 sequencing of MVST products and serial peripheral blood from subjects revealed a robust contribution of ex vivo expanded cells to the overall repertoire, in contrast to untreated controls where the repertoire of (sham) MVST cell products generated from the transplant donors did not significantly overlap with the immune repertoire in peripheral blood of TCD-SCT recipients in the early post SCT period. Only at day +180 some convergence of repertoires became visible indicating spontaneous immune reconstitution (Figure). Detailed CDR3 analysis of cytokine-captured CMV pp65 and IE-1 specific CD4+ and CD8+ T cells was performed in a representative subject clearly demonstrating the detrimental effect of high-dose steroids on frequency of anti-viral T cells, precipitating CMV reactivation.Conclusions: This is the first report demonstrating safety and feasibility of using MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen and a minimal risk of aGVHD was observed, as there was no correlation with GVHD biomarkers. As revealed by serial CDR3 sequencing, MVSTs robustly contributed to the T cell repertoire. Our results suggest efficacy of this strategy in reducing viral reactivation. A Phase II study is warranted. DisclosuresNo relevant conflicts of interest to declare.

207 - Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells is a Safe Strategy to Prevent Viral Reactivation in Recipients of Allogeneic T Cell Depleted Stem Cell Transplant

207 - Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells is a Safe Strategy to Prevent Viral Reactivation in Recipients of Allogeneic T Cell Depleted Stem Cell Transplant

Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy to Prevent Viral Reactivation in Recipients of Allogeneic T Cell Depleted Stem Cell Transplant

Background: Allogeneic stem cell transplant (SCT) recipients suffer from a defective T cell mediated immunity causing potentially fatal reactivation of latent viruses. After T cell depleted SCT we have observed over 80% CMV reactivation and significant additional costs ($58-74k/patient). Despite aggressive monitoring and pre-emptive therapy, reactivation and/or positive CMV serology brings a significantly higher risk for non-relapse mortality (NRM). Adoptive transfer of ex vivo generated virus specific donor T cells is effective as a treatment of infection post-SCT but it has not been tested as a prophylaxis of early reactivation. Here in a Phase I study we transferred multi-virus specific T cells (MVSTs) immediately post SCT, targeting CMV, Ebstein-Barr virus (EBV), BK and adenovirus (Ad) as a novel strategy to prevent viral reactivation in the recipients of T cell depleted sibling HLA-matched SCT.Methods: Subjects were eligible if enrolled in HLA-matched T cell depleted transplant protocol (13-H-0144) and deemed at risk for CMV reactivation. MVST cells were manufactured from SCT sibling donors. Elutriated lymphocytes were stimulated with autologous dendritic cells (DCs) pulsed with seven overlapping peptide libraries (pepmixes) spanning the length of immunodominant proteins from CMV (pp65 and IE1), EBV (BZLF1 and EBNA1), BK (LT and VP1) and Ad5. Cultures were maintained in G-Rex flasks for 14 days in presence of IL-7, IL-15 and IL-2 (after 72hrs), tested for sterility, phenotype, potency and cryopreserved. MVST cells were thawed and administered intravenously as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used at the following dose levels: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT), (Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least "probably" or "definitely" related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes, including efficacy (Figure) and immune reactivity (for donor/recipient pairs).Results: MVST cells recognized the majority of pepmixes, were polyfunctional and robustly proliferated in response the cognate antigens, but minimally against allogeneic targets- suggesting a limited ability to induce GVHD. CDR3 sequencing of T cell repertoire showed a significant reduction in diversity and a striking dominance of a limited number of clonotypes in the final MVSTs. Nine subjects were enrolled and treated with MVST cells. MVSTs were successfully generated for all subjects, meeting the release criteria. Median time from SCT to MVST administration was 16 days (range D +6 to +52 post-SCT). Two subjects received MVST after day +30 due to cardiac instability and scheduling. There were no immediate infusion-related adverse events or DLT by day 42. One subject in cohort II developed a self-limiting grade I cytokine release syndrome in the setting of low-level EBV reactivation. One patient (cohort 1) developed de novo grade III aGVHD post-MVST infusion. CMV reactivation post-MVST occurred in 4 out of 8 evaluable subjects (50%) who completed D+100 post-SCT vs. 45 out of 52 patients (50% vs 87%; p value=0.031) in a historical cohort of recipients of T cell depleted SCT. In all cases CMV reactivation occurred during treatment with high dose steroids. In two cases MVST were generated from CMV seronegative donors and showed minimal activity against pp65 and IE1. In eight evaluable subjects who reached D+100 post-SCT there was no EBV-related disease, but we saw self-limiting low level EBV replication in 6 out of 8 cases. There were no cases of BK or Ad-related disease or viremia. ELISPOT analysis at D+100 revealed robust reconstitution of anti-viral immunity in analyzed recipients (vs. donors, Figure, B)Conclusions: This is the first report demonstrating that it is safe and feasible to use adoptively transferred allo-MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen. MVSTs had a markedly reduced allo-reactivity and carried a minimal risk of GVHD. Our results also suggest efficacy of this strategy in reducing viral reactivation. A Phase II portion of this study is currently enrolling patients. [Display omitted] DisclosuresSabatino:Kite: Employment, Equity Ownership.

BPX-501 Cells (Donor T Cells Transduced with iC9 Suicide Gene) Treatment Following TCR Alpha Beta Depleted Stem Cell Transplantation in Adults and Children with Hematological Disorders

BPX-501 Cells (Donor T Cells Transduced with iC9 Suicide Gene) Treatment Following TCR Alpha Beta Depleted Stem Cell Transplantation in Adults and Children with Hematological Disorders

Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell–depleted stem cell transplantation

AbstractNatural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL–activated NK cells (aNK-DLI) following HLA-matched, T-cell–depleted (1-2 × 104 T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3+-depleted, CD56+-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL+IL-15Rα+ artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.

Unrelated Cord Blood Versus Non-T Cell Depleted Haploidentical Stem Cell Transplantation: Comparable Results in Adults with Acute Leukemia, a Eurocord and ALWP-EBMT Study

Unrelated cord blood transplantation (UCBT) and related Haploidentical non T-cell depleted stem cell transplantation (Haplo) are alternative options to treat patients with high risk acute leukemia. Both techniques have shown encouraging results.The current retrospective analysis aimed to compare the outcomes of both approaches in adults with ALL and AML receiving either UCBT (n=928) or Haplo (n=518) reported to EBMT centres between 2007 and 2011.Diagnosis was ALL for 716 patients (Haplo=158 and UCBT=558) and AML for 730 (Haplo=360, UCBT=370). Haplo grafts were not T-cell depleted, with 236 patients receiving unmanipulated bone marrow (BM), 253 receiving peripheral blood stem cells (PBSC) and 29 receiving BM+PBSC. For UCBT, 431 patients were treated with a single unit, while 497 received double unit UCBT.Compared with recipients of UCBT, Haplo recipients were more likely to have AML (69% versus 40%, p=<0.001), and were transplanted more recently (2011 versus 2009, p=0.001), had a longer median interval from CR1 to transplant (139 versus 126 days, p=0.07), and had more frequently advanced phase of disease at time of transplant (35% versus 17%, p=0.001).Median follow-up was 19 months and 25 months, (p=0.007) for Haplo and UCBT, respectively. The type of conditioning regimen (MAC or RIC) was similar between both groups, with MAC representing 60% and 56% for Haplo and UCBT, respectively.For Haplo, the most frequently used regimens were Thiotepa-Busulfan-Fludarabine (TBF) for MAC and Treosulfan-based regimens for RIC. Graft versus host disease (GVHD) prophylaxis included CsA+MMF (41%), Sirolimus (15%), while 163 patients received post-transplant Cyclophosphamide (Cy). For UCBT cases, the most common MAC regimens were TBF for 33% of cases, CyFluTBI-12Gy for 27% and CyTBI12Gy for 17%. The most frequently used RIC was CyFluTBI-2Gy in 72% of the cases. GVHD prophylaxis was CsA+MMF in 70% of cases.In univariate analysis, cumulative incidence (CI) of neutrophil engraftment was 92% vs 84% (p=<0.001) for Haplo and UCBT; CI of acute GVHD was 27% vs 32% (p=0.11) and for chronic GVHD it was 30% vs 25% (p=0.06) for Haplo and UCBT. For non-relapse mortality (NRM), relapse incidence (RI) and leukemia-free survival (LFS) outcomes were analyzed separately according to disease status, and no statistically significant differences were observed between Haplo and UCBT.According to disease status, for patients in CR1 (n=610), 2 years RI was 27% versus 28%, p=0.67; NRM 26% versus 29%, p=0.30; and 2 years LFS 48% versus 43%, p=0.16; for Haplo and UCBT, respectively.For patients in CR2 (n=502), 2 years RI was 33% versus 28%, p=0.51; NRM 33% versus 33%, p=0.93; and 2 years LFS 34% versus 39%, p=0.61, for Haplo and UCBT, respectively.For patients in advanced disease status (n=334), 2 years LFS was 11% versus 14%, p=0.92 for Haplo and UCBT, respectively.Infections and GVHD were the most common causes of transplant-related deaths in both groups, (infection 30% in both groups, GVHD 15% and 9%, after Haplo and UCBT, respectively), and 40% of deaths were due to disease recurrence in both Haplo and UCB recipients.In multivariate analysis, UCBT was associated with lower incidence of chronic GVHD (HR= 0.62; p<0.001) and higher NRM (HR= 1.28; p=0.04) when compared to Haplo. No statistically significant differences were observed between Haplo and UCBT for RI (HR= 0.87; p=0.24) and LFS (HR= 1.06; p=0.47). Factors independently associated with lower LFS were disease status at transplant (HR 2.69, p=<0.001), and use of ATG (HR 1.28, p=0.001).In conclusion, in this retrospective analysis, non TCD Haplo and UCBT showed similar RI, NRM, and LFS, highlighting that both approaches are valid for this population of patients. Longer follow-up, more homogeneous transplant strategies (stem cell source, type of conditioning regimen and GVHD prophylaxis) are needed for further evaluation. While waiting for the results of the BMT CTN trial on non TCD Haplo using post-transplant Cy and double UCBT both in the RIC setting, the choice of the specific approach for Haplo or UCBT should be likely based on transplant centre expertise and policy. DisclosuresNo relevant conflicts of interest to declare.

Haploidentical T-cell alpha beta receptor and CD19–depleted stem cell transplant for Wiskott-Aldrich syndrome

Haploidentical T-cell alpha beta receptor and CD19–depleted stem cell transplant for Wiskott-Aldrich syndrome

Prophylactic Donor Lymphocyte Infusion to Prevent Relapse after Allogeneic Stem Cell Transplantation

Donor lymphocyte infusion (DLI) has been used to treat disease relapse after stem cell transplantation, but high incidence of graftversus-host disease (GVHD) after traditional DLI, and low response rates with robust relapse in acute leukemia have limited its wide application. Nonetheless, the promising graft versus leukemia effect elicited by DLI makes it a viable option, especially in the state of minimal residual disease after stem cell transplantation. Prophylactic donor lymphocyte infusion has been explored as a more tolerable, less toxic and efficacious option to prevent relapse. This review will summarize the results of the latest prophylactic DLI studies, and discuss our ongoing prophylactic DLI clinical trial after T cell depleted stem cell transplantation in patients with high risk hematologic malignancies.

Clofarabine, Melphalan, and Thiotepa, Followed by Allogeneic Unmodified Bone Marrow or Peripheral Blood Stem Cell Transplant, by Unmodified Double Cord Blood Transplant, or by CD34+ T-Cell Depleted Stem Cell Transplant for the Treatment of Hematologic Malignancies.

Abstract 3144Based on encouraging results with the use of clofarabine (CLO) for reinduction treatment of acute leukemias, we have developed two allograft protocols for patients with hematologic malignancies with a cytoreductive regimen, using CLO in combination with melphalan (Mel) and thiotepa (Thio). Patients on protocol #1 received unmodified bone marrow (BMT), peripheral blood stem cells (PBSCT), or unmodified double unit cord blood (dCBT). Patients on protocol #2 received CD34+ T-cell depleted stem cells (TCD-SCT). Cytoreduction consisted of CLO 20 mg/m2/day × 5, Thio 10 mg/Kg/day × 1 and Mel 70 mg/m2/day × 2. Graft-versus-host disease (GvHD) prophylaxis consisted of tacrolimus (Tacro) and methotrexate (MTX) with unmodified BMT or PBSCT, tacro and mycophenolate mofetil (MMF) with unmodified dCBT, and none with TCD-SCT. Rabbit ATG at 2.5 mg/Kg × 2 or 3 doses was used for the prevention of rejection with the TCD-SCT.To date, 64 pts were treated with this regimen including: unmodified BMT/PBSCT 27 patients, dCBT 15 patients, and TCD-SCT 22 patients. The median age for patients was 10.2 years (range 0.9–58.7) for unmodified SCT and 41.5 (range 0.6–67.2) for TCD-SCT. This was the second SCT for 13 of 27 pts in the BMT-PBSCT group, 2 of 15 pts in the CBT group, and 4 of 22 pts in the TCD group. Patient diagnoses included acute lymphoblastic leukemia (ALL) (N=36), acute myelogenous leukemia (AML) (N=23), and myelodysplastic syndrome (MDS) (N=5). Patients with ALL or AML in first remission (CR1) or CR2 and MDS in CR1 or refractory anemia (RA) were categorized as having good risk disease (GRD), while all other pts were considered to have poor risk disease (PRD), irrespective of all other factors. There were 15 of 27 pts with PRD in the BMT/PBSCT group, 10 of 15 pts in the CBT group, and 9 of 22 pts in the TCD-SCT group.For the unmodified BMT/PBSCT group, donors were HLA-matched related (N=11), mismatched related (N=1), matched unrelated (N=12), or mismatched unrelated (N=3). All CBT recipients received double-unit grafts from 2 mismatched unrelated donors. For the TCD-SCT group, donors were HLA-matched related (N=8), mismatched related (N=1), matched unrelated (N=4), or mismatched unrelated (N=9).Engraftment occurred in 59 of 61 evaluable pts; three pts died before engraftment. One pt recipient of unmodified BMT/PBSCT suffered a late graft failure, and one pt recipient of CBT suffered an early graft failure in the context of sepsis. Grade 2–4 acute GvHD occurred in 8/26 (31%) evaluable pts in the BMT/PBSCT group, 5/13 (38%) evaluable pts in the CBT group, and 4/20 (20%) evaluable pts in the TCD-SCT group.With a median follow-up of 20.5 months for the unmodified SCT groups and 15.4 months for the TCD group, the overall survival (OS) and disease-free survival (DFS) rates were: 53.7% and 41.0% for the BMT/PBSCT group, 51.3% and 41.5% for the CBT group, and 64.1% and 60.7% for the TCD-SCT group.This cytoreductive regimen represents a promising approach for the transplantation of patients with acute leukemias without the use of total body irradiation. This regimen is also sufficiently immunosuppressive to insure consistent engraftment of T-cell depleted transplants. Lastly, it appears to be relatively well tolerated for younger pts requiring a second SCT. Disclosures:Off Label Use: Clofarabine.

A New Dosing Scheme of ATG-F Prevents Rejection and Maintains Immune Recovery in Haploidentical T and B Cell Depleted Stem Cell Transplantation

AbstractAbstract 4154T and B cell depletion of haploidentical peripheral stem cells with CD3/CD19 coated magnetic microbeads prevents GvHD and allows to coinfuse large numbers of donor NK cells and other accessory cells. The anti CD3 specific OKT3 antibody was routinely used as rejection prophylaxis without affecting CD3 negative cells. However, due to its restricted availability, the substance had to be substituted by polyclonal ATG preparations with a longer half life period and comprising a broader variety of antigen, with the CD56 antigen in particular. We present data with reduced ATG doses given at the beginning of the conditioning regimen in order not to impair cotransfused NK cells and immune recovery. A total of 27 pediatric patients (ALL/AML n=9, relapsed solid tumors n=13, nonmalignant diseases n=5) received either 3×5 mg/kg (n=7) or 3×10 mg/kg (n=20) ATG-F (Fresenius) starting at day -12, followed by fludarabine (160 mg/m2, d -8 to -5) thiotepa (10 mg/m2 d -4) and melphalan (140 mg/m2 d −3 to −2). A median number of 14.4×106 CD34/kg and 62×106 NK cells/kg with 37×103 T cells/kg were infused. Median time to ANC>500 was 9 days in both groups. Graft rejection occurred in 3/7 patients with 15 mg ATG (42%) and in 2/20 patients with 30 mg ATG (10%). After reconditioning with a TLI based regimen, final engraftment was achieved in all patients. Acute GvHD grade II-IV was observed in 1/4 (15 mg) and 1/18 (6%, 30 mg) patients without rejection. Extensive chronic GvHD occurred in 1/4 (15 mg group) and 1/18 (30 mg group) patients. Immune recovery was monitored in the 30 mg group and compared with a historical group receiving OKT3 and the same chemotherapy (n=34). Recovery of CD56+ NK cells was fast with a mean number of 473 vs. 230 cells/μl at day +14, 299 vs. 281 cells/μl at day +30 and 245 vs. 236 cells/μl at day +90. CD3+ T cells reached 12 vs. 16/μl at day +30 and 138 vs. 217/μl at day +90 (30 mg group vs. OKT3 group; no significant differences for all data pairs). ATG serum levels were measured in 9 patients by flow cytometry (amount of ATG binding to the Jurkat cell line, defined as T cell specific rabbit IgG). Median peak levels of 10.5 μg/ml (15 mg group) and 15.0 μg/ml (30 mg group) specific rabbit IgG were reached between day -8 and -6 and dropped to 1.2 and 2.6 μg/ml at day 0. In vitro incubation of NK cells from healthy donors with a comparable dosage of ATG-F (1 or 10 μg/ml) resulted in 26 (41)% apoptosis and 0.2 (0.2)% necrosis (70 (52)% vital cells) after 24 hours. Conclusions:Our aim was to substitute OKT3 by ATG in patients who receive CD3/19 depleted haploidentical peripheral stem cells without hampering donor NK cells infused on day 0 and subsequent immune recovery. Administration of 15 or 30 mg/kg ATG-F was started at an early time point (day -12) of the regimen. Both doses resulted in low serum levels of specific ATG at day 0 and in a fast NK cell recovery. In vitro results suggested, that the majority of NK cells will not be damaged herewith. However, 15 mg/kg seemed to be not effective in preventing graft rejection and use of 30 mg/kg ATG-F has to be recommended. Immune recovery of T and NK cells was comparable to that of a historical control group who received OKT3. This approach will be also of interest for other transplantation strategies in which various components of the grafts and additionally given Tregs or specific T cells have to be preserved. Disclosures:Martinius:Fresenius Biotech: Employment.

Jack of all trades: thymosin α1 and its pleiotropy

Thymosin α1 (Tα1), a thymosin-related 28-mer synthetic amino-terminal acetylated peptide, has gained increasing interest in recent years, due to its pleiotropy. The peptide has been used worldwide as an adjuvant or immunotherapeutic agent to treat disparate human diseases, including viral infections, immunodeficiencies, and malignancies. The peptide can enhance T cell, dendritic cell (DC), and antibody responses, modulate cytokine and chemokine production, and block steroid-induced apoptosis of thymocytes. Its central role in modulating DC function and activating multiple signaling pathways that contribute to different functions may offer a plausible explanation for its pleiotropic action. Additionally, the ability of Tα1 to activate the indoleamine 2,3-dioxygenase enzyme-which confers immune tolerance during transplantation and restrains the vicious circle of chronic inflammation-has been a turning point, suggesting a potential, specific function in immunity. Accordingly, Tα1 has recently been shown to promote immune reconstitution and improve survival of recipients of HLA-matched sibling T cell-depleted stem cell transplants in a phase I/II clinical trial. Thus, Tα1 continues to live up to its promises.

T Cell–Depleted Stem Cell Transplantation for Adults with High-Risk Acute Lymphoblastic Leukemia: Long-Term Survival for Patients in First Complete Remission with a Decreased Risk of Graft-versus-Host Disease

Consolidation with allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides a survival benefit to patients with acute lymphoblastic leukemia (ALL). We have previously reported comparable survival and relapse rates after T cell–depleted (TCD) allo-HSCT compared with unmodified transplantations for acute myelogenous leukemia, myelodysplastic syndrome, and non-Hodgkin lymphoma with significantly decreased graft-versus-host disease (GVHD). We performed a 56-patient retrospective study to evaluate TCD allo-HSCT for the treatment of ALL after myeloablative total body irradiation–based therapy. The 2-year and 5-year overall survival rates for patients with ALL after TCD allo-HSCT were 0.39 (95% confidence interval [CI], 0.26-0.52) and 0.32 (95% CI, 0.19-0.44), respectively, and the 2-year and 5-year disease-free survival rates were 0.38 (95% CI, 0.25-0.50) and 0.32 (95% CI, 0.20-0.44). There was a trend toward improved survival of patients who underwent TCD allo-HSCT in first complete remission compared with those who did so in other remission states. The cumulative incidence of grade II-IV acute GVHD at 1 year was 0.20 (95% CI, 0.10-0.31), and no patients developed grade IV acute GVHD. The cumulative incidence of chronic GVHD in 41 evaluable patients at 2 and 5 years was 0.15 (95% CI, 0.04-0.26), and that of extensive chronic GVHD at 2 and 5 years was 0.05 (95% CI, 0-11.6). We demonstrate OS and DFS rates that compare favorably to unmodified allo-HSCT with lower rates of GVHD.