Graft-versus-leukemia Effect Research Articles

Microrna-31 Promotes T-Cell Pathogenicity in Chronic Graft-Versus-Host Disease

MicroRNAs (miRs) are short, non-coding RNAs that repress gene expression at post-transcriptional level. Certain miRs play critical roles on T-cell responses in inflammation and autoimmunity. To understand the biology of miRs in T-cell allo-responses, we measured expression profiles of miRs in donor T cells in allogeneic vs. syngeneic recipients following bone marrow transplantation (BMT). We found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in vivo(Fig. A). MiR-31 was shown to regulate autoimmunity and cancer. However, its roles in regulating T-cell response remain largely undetermined. Recent study shows miR-31 represses peripheral Treg differentiation in EAE development. In contrast, miR-31 inhibits CD8 T-cell function in anti-virus response. We hypothesize that miR-31 is required for CD4 T-cell pathogenicity in chronic graft-versus-host disease (cGVHD), but is dispensable for CD8 T-cell cytolytic function in graft-versus-leukemia (GVL) activity.To study how miR-31 regulates T-cell allo-response, we used mice with miR-31 conditional knock out (KO) in T cells (miR-31fl/fl CD4Cre). No differences were observed in the baseline composition of naïve WT (miR-31fl/fl) and miR-31 KO splenocytes. After being transferred into allogeneic recipients, KO T cells had impaired ability to proliferate, reflected by fewer divided (% CFSElow) cells and reduced donor T-cell expansion in recipient spleens (Fig. B). To further evaluate how miR-31 contributes to T-cell pathogenicity in cGVHD, we used a murine model of BMT (B6 → BALB/c), in which low dose spleonocyes can induce scleroderma manifestation. Significant reduction of clinical scores (Fig. C), including improved skin scores and body weight loss, were found in the recipients of miR-31 KO grafts compared to those with WT grafts. This finding was validated in another haploidentical BMT model of scleroderma (B6 → B6D2F1), indicating miR-31 is required for T-cell to induce cGVHD. Consistently, the pathological damage was dramatically reduced in skin, gut and lung in the recipients of miR-31 KO grafts (Fig. D).Mechanistically, miR-31 deficiency in donor T cells resulted in significantly reduced numbers of CD4 T cells that produce IFNγ, IL-4/5 or IL-17 in recipient spleen and mesenteric lymph nodes. In absence of miR-31, fewer effector and central memory cells were generated from donor CD4 T cells. Further, T cells that are capable of localizing to B-cell follicles (CXCR5+PD-1hi) were significantlyreduced in spleen (Fig. E) and peripheral lymph nodes in recipients of miR-31 KO grafts. Interestingly, among these cells, more foxp3+ follicular T-regulatory (Tfr) but fewer foxp3- follicular T-helper (Tfh) cells were observed in the recipients of KO grafts (Fig. F), indicating miR-31 contributes to the unbalance of Tfr and Tfh cells during cGVHD development. Consistently with the role of miR-31 in regulating Tfr and Tfh cells, we found Blimp1 is the predicted targets of miR-31, which is critically involved in the homeostasis of Tfr and Tfh cells. On donor B-cell compartment, lower levels of CD86 and MHC-II expression were observed in the recipients of miR-31 KO grafts. In addition, germinal center B cells and plasma cells were also reduced in those recipients, indicating miR-31 indirectly regulates B-cell activation and differentiation through controlling T-cell pathogenicity.For translational purpose, we evaluated how miR-31 impacts T cell-mediated GVL effect. Using B6 → BDF1 BMT model, we consistently found attenuated skin manifestation in recipients of KO T cells. Importantly, in the ongoing experiment, miR-31 KO T cells were capable of controlling P815(mastocytoma) growth as efficiently as WT T cells (Fig. G), suggesting miR-31 may be dispensable for T cells to mediating GVL activity after allogeneic BMT.Taken together, the current work reveals that miR-31 plays pleotropic roles in enhancing CD4 T-cell pathogenicity in cGVHD. MiR-31 represents a promising therapeutic target for the control of cGVHD while sparing GVL effects after allogeneic hematopoietic stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Donor Type in Patients with Acute Myeloid Leukemia Given Allogeneic Hematopoietic Cell Transplantation after Low-Dose TBI Based Regimen: A Study from the Acute Leukemia Working Party of the EBMT and Eurocord

Backround. Low-dose total body irradiation (TBI)-based nonmyeloablative conditioning regimens are widely used in acute myeloid leukemia (AML) patients in first or second complete remission (CR) at the time of transplantation. With these regimens the burden for tumor eradication relies mostly on immune-mediated graft-versus-leukemia (GVL) effects that can be impacted by donor type. Here we assessed the impact of donor type in a large cohort of AML patients transplanted in CR after low-dose TBI-based nonmyeloablative conditioning regimens.Methods. Inclusion criteria included >= 18 years of age, de novo or secondary AML in CR1 or CR2, transplantation between 2004 and 2015, conditioning with fludarabine + 2 Gy TBI with or without pre- or post-transplant cyclophosphamide, no in vitro or in vivo T cell depletion of the graft (besides post-transplant cyclophosphamide), and either an HLA-matched sibling donor (MSD), a 10/10 HLA-matched unrelated donor (MUD), an HLA-haplo-identical donor (Haplo) or a single or double umbilical cord blood (CBT).Results. Data from 1357 patients receiving grafts from MSD (n=594), MUD (n=442), Haplo (n=77) or given single or double CBT (n=244) were included. The proportion of patients transplanted in CR1 was 87%, 80%, 66% and 60% in MSD, MUD, Haplo and CBT groups, respectively (P<0.001) while median patient age at transplantation was 58, 63, 57 and 55 years, respectively (P<0.001). The proportion of patients with secondary AML was 16%, 22%, 21% and 20% in MSD, MUD, Haplo and CBT groups (P=0.04). Median follow-up was 49, 25, 17 and 48 months, respectively (P<0.001). Graft rejection occurred in 2% of each MSD and MUD recipients, 7% of haplo patients and 8% of CBT recipients (P<0.001). Grade III-IV acute GVHD was more frequent in CBT (16%) than in MUD (9%), haplo (8%) or MSD (7%) recipients (P<0.001). In contrast, chronic GVHD was less frequent in CBT (27%) and in haplo (30%) patients than in MSD (50%) and MUD (52%) recipients (P<0.001). Two-year OS and GRFS were 60% and 30% in MSD patients, 56% and 41% in CBT recipients, 54% and 24% in MUD recipients, and 42% and 35% in haplo patients, respectively (P=0.02 for OS and P=0.03 for GRFS ; Figure 1). Two-year incidences of relapse and nonrelapse mortality (NRM) were 33% and 12% in MSD patients, 29% and 20% in MUD recipients, 34% and 17% in CBT recipients, and 37% and 21% in haplo patients, respectively (P=0.7 for relapse and P<0.001 for NRM). In multivariate analyses, in comparison to MSD patients, each other donor type was associated with a significantly higher NRM. Interestingly, CBT was associated with a higher incidence of grade III-IV acute GVHD (HR 2.9, P=0.001) but a lower incidence of chronic GVHD (HR=0.4, P<0.001) than MSD resulting in a significantly worse GVHD-free relapse free survival (GRFS) the first 100 days (HR=1.5, P=0.02) but significantly better GRFS beyond day 100 (HR=0.6, P=0.001). Finally, there was a suggestion for lower OS in haplo patients (HR 1.5, P=0.08). Factors associated with worse OS included intermediate and adverse-risk cytogenetics and secondary AML. AML relapse, GVHD and infections were the primary causes of death of 24%, 6% and 4% of MSD recipients, 21%, 7% and 7% of MUD recipients, 22%, 5% and 14% of haplo recipients, and 25%, 8% and 5% of CBT recipients, respectively.Conclusions. In this large cohort of AML patients transplanted in CR following low-dose TBI-based nonmyeloablative conditioning regimen there were no significant differences in OS and LFS between MSD, MUD and UCB while there was a suggestion for lower LFS and OS with haplo due to a higher incidence of death from infection. Since this regimen relies nearly exclusively on GVL effects for tumor eradication, our data suggest that the intensity of GVL effects is comparable with these four donor types. Further studies including more haplo patients are needed to confirm these results. [Display omitted] DisclosuresSavani:Jazz Pharmaceuticals: Speakers Bureau. Mohty:Sanofi: Honoraria, Speakers Bureau.

Early HHV-6 Reactivation in CMV-Seronegative Cord Blood Transplant Recipients Is Associated with Inferior Relapse-Free and Overall Survival

IntroductionHHV-6 reactivation has been associated with transplant complications such as graft failure, acute GVHD, and CMV reactivation. However, the causal relationships and the involved mechanisms remain largely speculative. In addition, as the immunosuppressive settings that promote HHV-6 reactivation may also increase the risk of reactivation of other viruses (e.g. CMV), determining the independent effect of HHV-6 is difficult. In this retrospective study, we focused on early reactivations (until day 30 post-transplant) and their impact on relapse because of the well-established immunosuppressive effect of HHV-6 on the innate and adaptive immune system, which could theoretically weaken the graft-versus-tumor (GvT) effect, resulting in more relapse and worse survival.MethodsTo eliminate potential confounders and address the independent effect of HHV-6 reactivation, we restricted our population to CMV-seronegative recipients of UCB (2011-2017) and excluded those with DNA virus reactivations other than HHV-6 (CMV, adenovirus, EBV, and BK virus). HHV-6 reactivation was defined as ≥1 positive quantitative DNA PCR on whole blood within 30 days post-transplant. The absence of HHV-6 reactivation was defined as ≥1 negative PCR in the first 2 weeks and ≥1 negative PCR in the second 2 weeks post-transplant. A subset of patients had research blood samples collected on day 30, which we used to measure functionally relevant T-cell subsets by flow cytometry.Results54 patients were included (41 with and 13 without HHV-6 reactivation; Table 1). The groups were similar except more malignant disorders in those with HHV-6 reactivation, P= 0.02). The HHV-6 reactivation group had a lower RFS (38 [22-54]% vs. 88 [39-98]%, P= 0.03; Figure 1A), lower OS (46 [29-61]% vs. 92 [54-99]%, P= 0.01; Figure 1B), and more relapse (31 [15-47]% vs. 0%, P= 0.07; Figure 1C) at 2 years plus a trend for higher 2-year NRM (28 [14-43]% vs. 8 [0-23]%, P= 0.10; Figure 1D). For the latter two results, only patients with malignant hematological disorders were included. Neutrophil recovery, graft failure, acute GVHD grade II-IV, and chronic GVHD were similar in both groups.Next, we evaluated day 30 blood T-cell immunophenotype focusing on 3 markers with potential relevance to T-cell function: (i) CD57 (associated with T-cell replicative senescence), (ii) NKG2a (associated with resistance to immunotherapy in solid tumors), and (iii) KIR2DL2/3 (known to prematurely end the immune synapse and inhibit sustained TCR-induced gene transcription resulting in decreased IFN-γ production). The relative abundances of T cells expressing CD57, NKG2a,or KIR2KL2/3 were all significantly higher in the group with HHV-6 reactivation (P < 0.05). Interestingly, the abundance of NKG2c+CD57+ cells among NKG2a+ NK cells (so called “adaptive” NK cells) was marginally significantly higher in the group with HHV-6 reactivation (P= 0.07).ConclusionsPrevious studies have reported higher NRM in patients who reactivate more DNA viruses post-transplant (Hill J, et al. Blood 2017). We demonstrated an independent association between early HHV-6 reactivation and worse RFS/OS, attributed in part to relapse. This was consistent with the observed skewed T-cell repertoire in the group with HHV-6 reactivation, which showed markers of decreased immune effector function, possibly reflecting an attenuated GvT effect. Although the relative abundance of adaptive NK cells was somewhat higher in the group with HHV-6 reactivation (reminiscent of CMV reactivation), this did not seem to rescue the GvT effect which was apparently impaired due to T-cell defects. If confirmed in larger cohorts, rigorous prophylaxis and/or HHV-6 vaccination may be explored to decrease the risk of relapse and improve survival. [Display omitted] DisclosuresMiller:Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

Interim Results of a Phase 2 Clinical Trial Using Mb-IL21 Ex Vivo Expanded NK Cells to Enhance Graft Versus Leukemia Effect for Patients with Myeloid Malignancies after Haploidentical Transplantation

▪Background: Disease relapse has emerged as the most common cause of treatment failure after transplantation. Natural killer (NK) cells have potent antitumor and antiviral effects. We hypothesized that multiple infusions of ex vivo expanded NK cells administered in early post-transplant will enhance graft-versus-leukemia (GVL) effect and began a phase 1/2 clinical trial in haploidentical transplant patients (clinicaltrials.gov NCT01904136).Methods: Results from the phase 1 clinical trial were recently reported. The aim was to determine the maximum tolerated dose of ex vivo expanded NK cells with a K562 expressing mb-IL21 feeder cell system in patients with myeloid malignancies (AML/MDs/CML). Patients received conditioning with melphalan 140mg/m2, fludarabine 160mg/m2 and 2GyTBI, and GVHD prophylaxis with post-transplant cyclophosphamide, tacrolimus and mycophenolate. All patients had bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor and infused fresh on Day -2, and cryopreserved on Day +7 and +28 (up to 90). Having an NK-cell alloreactive donor or certain KIR genotype was not a requirement to participate in this study, although these characteristics were evaluated in all patients. In the phase 1 study dose escalation patients received NK cells starting at 1x105 to 1x108/Kg/dose for 3 doses. No dose limiting toxicities were observed in the phase 1 study. 1x108/kg/dose was chosen for the phase 2 trial.Results: Twenty patients were treated to date, 13 on the phase 1 study and 7 on the phase 2 part. The median age was 45 years (range 18-59), the median follow-up survivors was 16.5 months. Twelve patients (60%) were females. The median HCT-CI was 2 (range 0-8). Ten patients (50%) had AML (9 in CR1 and 1 beyond CR2), 3 (15%) had MDS (2 with induction failure, 1 untreated), and 7 (35%) had advanced CML (4 in CP2 after progression to blast-phase of which 2 in CNS, 1 in CP1, 1 clonal evolution). Of the patients with AML 4 patients with AML had poor-risk cytogenetics and 6 had intermediate with high-risk mutations except one (2 had FLT3, 2 had NRAS, 1 had ASXL1) and 3 patients had primary induction failure. Two MDS patients had complex cytogenetics and one diploid with high percent of bone marrow blasts. DRI was very high/high in 10 patients and intermediate/low in 10 patients. All patients received all 3 NK cell infusions as planned except one patient. Eight patients received the maximum dose of 1x108/kg/dose.One patient had early death due to hemorrhage. Nineteen patients were evaluable for engraftment and all achieve primary engraftment after a median time of 19 days (range 16-42). Nine of 12 female patients (75%) had donor-specific anti-HLA antibodies. Chimerism was 100% donor in all patients except one who had mixed chimerism. The cumulative incidence (CI) of all aGVHD at Day100 and 1 year post-transplant was 26.1% and 48% (3 patients had late acute GVHD), exclusively grade 2 and exclusively GI tract. No grade 3 or grade 4 aGVHD, and no chronic GVHD was observed. Also, none of the patients developed liver GVHD. The CI of TRM at 1 and 2 years post-transplant was 5.6% and 16.7%, respectively. Only one patient relapsed to date for a CI of relapse at 1 and 2 years on 5.6%. The only patient who relapsed was an advanced FLT3+AML patient (beyond CR2) who received the lowest NK cells dose. None of the patients who received ≥1x105/kg dose relapsed. The median overall survival and progression-free survival (PFS) were not reached. The 1-year and 2-years PFS was 84.4% and 74%, respectively (Figure 1). Eight of 19 patients (42%) reactivated CMV and 4 patients (21%) had grade 1 transient BKV cystitis.Conclusions: Multiple infusions of high doses of donor-derived ex vivo expanded NK cells with a K562 feeder cell system and mbIL-21 administered early post-transplant are safe and are associated with low relapse rate and low incidence of viral reactivation. A randomized trial is needed to confirm these results. [Display omitted] DisclosuresOran:Celgene: Research Funding; AROG: Research Funding; Astex: Research Funding. Lee:Courier Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy; Miltenyi: Speakers Bureau.

The Management of Relapsed Hodgkin´s Lymphoma after Haploidentical Stem Cell Transplantation: Donnor Lymphocyte Infusion and Brentuximab Vedotin

IntroductionDespite the high cure rate of Hodgkin lymphoma (HL) with conventional radiochemotherapy, HL is refractory in 15% to 30% of patients. In those patient who relapse or refractory to chemotherapy Allogeneic transplantation (alloSCT) will be an option, despite the promising results the rate of relapse is higher. In this situation there is not standardized treatment, we hypothesized that treatment with Brentuximab Vedotin (BV) in combination with donor lymphocyte infusion (DLI) would be a promising approach for relapsed HL after alloSCT that selectively targets lymphoma cells and enhances the graft versus leukemia (GvL) response by the induction of immunogenic cell death.Material and methodBetween 2014-2016 we studied 6 patients who were relapses of HL after haploSCT, we designed a treatment algorithm combining BV infusions during 6 cycles (1.8 mg/kg per day every 21 days) with donor DLI)administration in escalate dosis 1x10e4/kg CD3, increasing 1 log for dosis in an alternating regimen after the third cycles of BV. We stop the DLI when GVHD appear or toxicity, we perform a PET-CT after the first 3 cycles BV, after ILD + 6 cycles BV and 3 month after finish the treatment designed. We analisis safety, complication an side effect of the combination. The aim of this study was to retrospectively evaluate the efficacy and safety of BV + DLI in HL relapsing or progressing after allo-SCT.ResultsA total of 4 patients (66%) were nodular sclerosis histological subtype and 34% (2 patients) was linfocitic predominance. The median age was 32 years (22-48). Median follow-up was 837 days (405-975). The median of treatment lines received before transplantation was 6 (4-7). Only 2 patients (34%) had reached haploTPH in complete remission.Peripheral blood was used as progenitors source in 66% (6). The median time to relapse was 286 days (range 180-463). 5 patients (83%) received BV before alloSCT, only 2 (34%) achieved CR.All the patients relapse after the alloSCT, then we decide began the designed treatment, after the first 3 cycles of BV, 17% (1 patients) was in CR. After that we associated DLI in all the cases, the median was 8 (3-13) plus another 3 cycles of BV, after this in the reevaluation CR was observed in 50% (3 patients). When combined radiotherapy (RT) in selected cases, CR rate was 100% in the PET-CT 3 month after finish the treatment designed. The combination DLI + BV was without important toxicity. Acute GVHD was observed in 83% (5 patients) the 100% was grade I-II. 66% (4 patients) developed chronic GVHD, 100% was limited extension. Treatment with BV + DLI resulted in an PFS of 100%.ConclusionsThe combination treatment was well tolerated, confirming a manageable safety profile for BV plus DLI after alloSCT, is an effective and safe therapy to achieve disease control in HL relapsing after alloSCT. We assumed that the combination of BV with DLI will have the potential to further increase the GvL effect. Despite this findings, we need multicentric studies to perform standarized treatments. DisclosuresNo relevant conflicts of interest to declare.

Graft-Vs-Leukemia Effect in Acute Lymphoblastic Leukemia: Mild Acute Graft-Vs-Host Disease Protects Against Relapse and Improves Survival after Allogeneic Transplantation: A CIBMTR Analysis

Introduction: Graft-versus-host disease (GVHD) associated with allogeneic donor hematopoietic cell transplantation (alloHCT) provides protection against leukemia relapse, but more severe GVHD may be detrimental and increase mortality.Methods: We examined the net effect of GVHD on overall survival (OS) of 5215 patients with acute lymphoblastic leukemia (ALL) treated with alloHCT in 232 centers between the years 2000 and 2014 and reported to the CIBMTR. We examined three cohorts: a) adults in complete remission (CR1 and 2; n=2593) treated with myeloablative (MAC) or reduced intensity conditioning (RIC), b) pediatric ALL aged 1-18 in CR1/CR2 (n=1619) treated by MAC and c) patients of all ages with advanced ALL (active disease or CR≥3; n=1003) who received MAC. Transplant outcomes were compared by GVHD presence and severity: no GVHD (reference group), grade I-II acute GVHD (aGVHD), grade III-IV aGVHD, plus each with or without chronic GVHD (cGVHD).Results: Patients had B-cell (80%) or T-cell ALL (15%); 26% were Ph+. MAC was used in most patients (93%), followed by HLA-identical sibling (28%), adult HLA-matched unrelated donor (31%) or umbilical cord blood (24%) grafts. 28% received in vivo T cell depletion. Cumulative incidence of relapse and 5-year overall survival (OS) in the three cohorts are shown in table. In multivariate analysis adjusted for age, white blood cell count, donor source, KPS, GVHD prophylaxis and conditioning, aGVHD (both grade I-II and III-IV) and cGVHD yield protection from relapse as compared to no GVHD (HR 0.49-0.69) in both adult and pediatric CR cohorts (Table). Patients with advanced ALL with cGVHD or with grade III-IV aGVHD also had a lower, but still excessive risk of relapse. However, in all 3 cohorts, increased transplant-related-mortality (TRM) accompanied grade III-IV aGVHD (HR 3.91, 3.69, 2.69) and abrogated the protection from relapse resulting in inferior OS (Table). Importantly, adults in CR1/2 with grade I-II aGVHD without cGVHD had a significantly better OS compared to no GVHD (HR 0.83). A similar net survival benefit of grade I-II aGVHD occurred in children in CR1/CR2 (HR 0.76). CGVHD alone or in combination with grade I-II aGVHD augmented protection from relapse (HR varied 0.51-0.67), but had a neutral effect on OS in the two CR cohorts. Patients with advanced ALL had improved OS if they developed cGVHD with or without grade I-II aGVHD.Conclusions: Our data confirm the potent GVL effect of alloHCT in ALL in CR and demonstrate that the net beneficial effect of aGVHD on OS in ALL is confined to patients who experience grade I-II aGVHD and no cGVHD. This favorable net effect extends from pediatric to adult patients who are in remission. Those with advanced disease experience modest survival benefit from cGVHD. Nevertheless, the benefit of relapse protection is abrogated by increased mortality in patients with grade III-IV aGVHD and is weaker in those without any GVHD. Immunity-modulating interventions post-HCT and more effective GVHD therapies may better harness the potential graft-versus leukemia effect in ALL. [Display omitted] DisclosuresFlowers:Pharmacyclics: Consultancy. De Lima:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding. Bachanova:Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

NOTCH Inhibition Separates GVL from GVHD Via Modulating Host Dendritic Cell and Donor T-Cell Activation

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can eradicate chemorefractory leukemia and reduce the relapse rate of high risk cases through the graft versus-leukemia (GVL) activity. However, graft-versus host disease (GVHD) limited the overall clinical benefit of allo-HSCT. Efforts are made to separate GVL from GVHD but few approaches are confirmed effective. Notch is a highly conserved signaling system important for lymphocyte effector and memory differentiation. Previous studies have shown that inhibition of Notch signaling could reduce aGVHD ex vivo and in vivo by downregulating both activated antigen presenting cells (APCs) and CD8+ T cells. It is less established whether Notch inhibition could also maintain GVL activity while reducing GVHD. The aim of the current work was to further study whether GVL activity could be maintained or enhanced by Notch inhibition.Methods: Murine models of GVHD and GVL were established (C57BL/6→BALB/c). γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) was used to block Notch1 signaling. The recipients were monitored daily for survival, clinical signs of GVHD and tumor size. GVHD histopathology were systematically examined and evaluated based on the scoring system. In order to study the mechanism of Notch inhibition on both GVHD and GVL, phenotypes of APCs and T cells in recipients were analyzed by flow cytometry. Expression of FasL by PCR, levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by ELISA and cytotoxicity of T cells were compared in recipients with or without Notch inhibition.Results: Reduced GVHD was observed in Notch inhibited recipients, which is similar to the results of previous study. However, in GVL model, both the median time when tumor volume is over 0.5cm3 and the change of tumor volume had no difference with or without Notch inhibition. Suppressed differentiation of CD8+ cytotoxic T cells (CTL) and decreased levels of TNF-α did not result in attenuated GVL activity. Further mechanism studies showed that Notch inhibition up-regulates CD11c+CD80+ DCs and central memory T cells (CD4+CD62L+ and CD8+CD62L+ T cells) while suppressing CD8+ CTL differentiation. We then compared levels of TNF-α and IFN-γ which are important in mediating aGVHD. Levels of TNF-α were decreased but IFN-γ were increased rather than decreased after Notch inhibition. We hypothesized that the contradictory results may ascribe to the difference between TNF-α and IFN-γ in inducing GVHD and GVL. Reduced TNF-α and increased IFN-γ levels could be the causes for down-regulated donor CTL activity. Moreover, FasR-FasL interaction plays a critical role in GVL instead of aGVHD. FasL expression were similar in in DAPT groups and the controls, suggesting similar GVL activity in recipients with or without Notch inhibition.Conclusions: Notch inhibition reduces GVHD and does not impair GVL effect. Increased number of central memory T cells and higher levels of interferon-γ (IFN-γ) could be the causes for enhanced GVL. DisclosuresLi:Guangzhou Science and Technology Project (No. 2015100010211): Research Funding; National Natural Science Foundation of China (91642111): Research Funding; Guangdong Provincial Basic Research Program (No. 2015B020227003: Research Funding; Guangdong Provincial Applied Science and Technology Research & Development Program (No. 2016B020237006): Research Funding.

Pharmacological Control of Recipient NK Cell Function By Venetoclax Promotes Engraftment and Limits GVHD Following Reduced Intensity Conditioning without Impacting the Graft Versus AML Effect

BackgroundThe outcome of allogeneic haematopoietic stem cell transplantation (alloHSCT) is a complex interaction between conditioning regimen intensity (efficacy vs toxicity), recipient immunology (rejection vs engraftment) and donor immunology (rejection vs engraftment, and GVHD vs GVL). We must find means to lower conditioning toxicity, promote donor engraftment and limit GVHD in order to improve alloHSCT outcomes.Aims: Optimize the use of BCL2 inhibitors to modify recipient NK cell function in mouse models of alloHSCT in order to minimize GVHD severity and onset, and retain GVL responses.Hypothesis: Therapeutic targeting of recipient NK cell frequency or function pre-transplant will allow reduced intensity conditioning (RIC) and promote both donor T cell engraftment and GVL whilst reducing the risks of GVHD.MethodsMHC-mismatched mouse model of alloHSCT: Donor BM and T cells from BALB/c (H2Kd) mice were injected into irradiated C57BL/6 (H2Kb) recipients. On day 0, C57BL/6 WT, or Bcl2fl/fl (Bcl2 deleted in NKp46+NK1.1+ cells) recipients were irradiated with a RIC dose of 2 x 400 rad, before i.v. injection of 7.5e6 BALB/c BM + 1e6 T cells. C57BL/6 WT mice were also treated on day -2 and -1 by oral gavage with 100 mg/kg Venetoclax (ABT-199) or vehicle, before receiving alloHSCT. Mice were monitored for onset of GVHD and tested for early engraftment (day 7-14 post-transplant), and late engraftment (up to day 50).Graft-versus leukaemia (GVL) AML model:8e5mixed lineage leukaemia cells (MLL-AF9) were injected i.v. on day 0 into C57BL/6 WT recipients. On days 8 and 9 mice were treated by oral gavage with 100 mg/kg Venetoclax or vehicle, before receiving alloHSCT on day 10. Mouse survival and AML burden in the BM was measured on day 30.ResultsWe found that at both a myeloablative radiation dose (1200 rad) and a RIC non-myeloablative dose (800 rad), recipient NK cells were significantly more radio-resistant than either CD8+ T or myeloid cells. However, when a RIC dose of 800 rad was used during alloHSCT, the rejection threshold was not overcome and engraftment failed. We investigated both genetic and pharmacological models of recipient NK cell suppression during alloHSCT with RIC, to promote donor cell engraftment, reduce GVHD, and retain GVL.Conditional deletion of Bcl2 in NK cells results in a 90% loss of NK cells in vivo . Bcl2fl/fl alloHSCT RIC recipients showed robust donor engraftment, but absence of the pro-inflammatory cytokine storm and substantially less GVHD as determined by clinical scores and gut histology, compared to WT recipients. Pharmacological inhibition of BCL2 in WT recipients recapitulated the transplant findings in Bcl2fl/fl recipients. We found that BCL2 inhibition by Venetoclax, a BCL2 antagonist approved in the treatment of AML, resulted in NK cell apoptosis in human cells . We extended our observations in Bcl2fl/fl recipients to show that pharmacological inhibition of BCL2 in WT mice with just two doses of Venetoclax resulted in rapid depletion of NK cells. Over 35% of alloHSCT WT recipient mice pre-treated with Venetoclax developed long-term full donor engraftment even in the setting of RIC, with minimal GVHD, and retained potent GVL effects against pre-established AML.ConclusionsVenetoclax depletion of NK cells in WT alloHSCT recipients in combination with RIC was: 1) well-tolerated, 2) associated with low rates of GVHD, 3) resulted in long-term donor haematopoietic cell engraftment, and 4) maintained a GVL response. Recipient NK cell inhibition may therefore represent a means by which to deliver alloHSCT more safely. DisclosuresRitchie:Amgen Inc.: Honoraria.

Downregulation of MHC Class II in Relapsed AML Cells after Allogeneic Transplantation

The evolutionary model of Acute Myeloid Leukemia (AML) progression suggests that malignant clones survive initial therapy and acquire new genetic and epigenetic changes, ultimately resulting in therapy-resistant disease. Allogeneic hematopoietic stem cell transplant (HCT) is a potent therapy that provides benefit in part through an immune-mediated Graft-vs-Leukemia (GVL) effect, although relapse after HCT remains a major clinical problem. Recent studies have demonstrated the gain and loss of specific AML-associated mutations at the time of relapse, consistent with a model of clonal evolution; however, a comprehensive screen of genetic changes in AML relapsed after HCT has not yet been reported. We hypothesized that the distinctive, immune-mediated GVL effect imposed by HCT may result in distinct patterns of tumor evolution in relapse after this therapy.To test this, we performed enhanced exome sequencing of matched diagnosis and relapse tumors to identify somatic mutations in 15 patients who relapsed after matched related or unrelated donor HSCT. For comparison, we analyzed 20 cases of AML relapsed after chemotherapy alone. In the majority of samples, new somatic variants were identified, and copy number analysis demonstrated gain and loss of genomic regions, as has been previously reported. However, compared to cases relapsed after chemotherapy alone, there were no gene mutations or copy number changes that were specific to relapse after HCT. Specifically, we found no changes in genes involved in antigen presentation, cytokine production, or immune checkpoints, and an unbiased pathway analysis revealed no enrichment for mutations in immune response pathways. Overall, the spectrum of mutations found at relapse after HCT did not differ from that observed in relapse after chemotherapy only.Since no canonical genetic changes were observed in relapsing AML after HCT, we speculated that clonal evolution of AML after HCT may instead occur through selection of randomly occurring epigenetic changes in AML cells that allow them to escape immune surveillance. To test this hypothesis, we performed RNA sequencing on purified AML blasts (CD45 positive, side scatter low) from presentation and relapsed AML sample pairs from both post-HSCT and post-chemo relapses. Interestingly, in the post chemotherapy relapse cases, only 8 genes were identified with expression changes that met pre-specified cutoffs for significance (FDR<0.5). In contrast, in the post relapse cohort, 34 genes were found to be significantly upregulated, and 187 genes were significantly downregulated, compared to the same sample at presentation. Gene pathway analysis revealed significant dysregulation of immune response genes (the most dysregulated pathway, p<10-35) as well as pathways encompassing cell adhesion/motility and the innate immune response.Four classical MHC Class II genes were among the 187 genes that were downregulated after post-HCT relapse: HLA-DPA1 (mean fold change -11.9 in 6/7 cases), HLA-DPB1 (mean fold change -6.5 in 6/7 cases), HLA-DQB1 (mean fold change -9.7 in 6/7 cases), and HLA-DRB1 (mean fold change -11.9 in 6/7 cases) (Figure 1). In these same 6 cases, there was a strong (but not significant) trend towards downregulation in the remaining MHC Class II genes: HLA-DQA1, HLA-DRB3, and HLA-DRA, as well as CIITA, a transcriptional activator believed to act as the “master regulator” of MHC Class II genes (Figure 1). Several other genes involved in MHC Class II antigen processing and presentation were significantly downregulated post-HCT (FDR<.05), including HLA-DMA, HLA-DMB, and CD74 . In contrast, there was no significant downregulation of MHC Class I genes observed after allo-relapse, raising the possibility that loss of MHC Class II presentation may play a particularly important role in immune evasion after HCT. To validate these findings, we performed flow cytometry for Class II and Class I genes on banked post-HCT relapse samples, and this confirmed significant downregulation of MHC Class II (but not Class I) expression on post-HCT relapse samples (Figure 2). Taken together, these results suggest that loss of MHC Class II-mediated antigen presentation may be an important mechanism of immune escape after HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Lysosomal Acid Lipase Is a Potential Therapeutic Target for Controlling Gvhd after Allogeneic Hematopoietic Cell Transplantation in Mice

Graft-versus-host disease (GVHD) is a main barrier for the success of allogenic hematopoietic cell transplantation (allo-HCT). Lysosomal acid lipase (LAL) mediated cell-intrinsic lipolysis to generate free fatty acids and cholesterol in the cells. It plays an essential role in the development, proliferation and function of T cells. LAL deficiency decreases Th1 and Th2 differentiation while inducing the generation of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs). Treg and MDSC are suppressive and Th1/Th2/memory CD8 cells are pathogenic in GVHD. LAL is also required for CD8 memory T cells development. Due to its importance in alloreactive T cell metabolism, we hypothesize that LAL is a valid potential target for the control of GVHD.Using murine models of allo-HCT, we show here that transfer of reconstituted donor LAL-/- T-cells is associated with significantly less GVHD, compared with LAL+/+ donors in MHC-mismatched (B6->BALB/c or FVB->B6) (Figure 1A) and haploidentical (B6->BDF1) model (Figure 1B). To investigate the effect of pharmacologic LAL inhibition, Orlistat, a specific LAL inhibitor was chosen. Oral treatment of transplanted recipients with Orlistat significantly reduced GVHD severity and improves mortality of recipient mice in B6-BDF1 model. Mechanistically, LAL-/- donor T cells significantly reduced proliferation in allogeneic recipients compared to WT counterpart. Furthermore, LAL deficiency in donor T cells also reduced T cell activation and Th1 differentiation after transplantation. Strikingly, we observed that LAL-/- T-cells or Orlistat treatment largely preserved graft-versus-leukemia (GVL) activity against P815 mastocytoma in B6->BDF1 model (Figure 1, C&D).Taken all together, our current study provides evidences that LAL can be a valid target for the treatment of GVHD while maintaining GVL effect. Lipid lipase inhibitors could be a potential pharmacological agents for treating GVHD. Our findings thus explore a novel therapeutic strategy for the control of GVHD by altering lipid metabolism in donor T cells.*Hung Nguyen and Sandeepkumar Kuril contributed equally to this work [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide Contributes to Impaired Graft-Versus-Leukemia Effects and Regulates Graft-Versus-Host Disease in the Murine Stem Cell Transplant Model

IntroductionPost-transplant cyclophosphamide (PTCy) for controlling alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT) is an effective prophylaxis for acute and chronic graft-versus-host disease (GVHD). HLA-haploidentical HSCT using PTCy is a promising option for patients with hematological malignancy who require allo-HSCT and lack an immediately available HLA-identical related or unrelated donor. In contrast, a relatively high relapse rate was reported in HLA-haploidentical HSCT using PTCy with non-myeloablative conditioning compared to HLA-matched related or unrelated HSCT using conventional GVHD prophylaxis. The effects of GVHD have been shown to correlate partially with those of graft-versus-leukemia (GVL), and therefore, PTCy treatment might contribute to the increase in relapse. However, the rationale for the high relapse rate in HSCT using PTCy compared to conventional HSCT remains poorly understood. To address this issue, we explored the effect of PTCy on survival, leukemia progression and immune reconstruction in a murine transplant model.Material & MethodsTwelve-week old C57BL/6 (B6) mice were irradiated with 5 Gy in one fraction on day 0 and transplanted with 1.0 × 107 spleen cells and 1.0 × 107 bone marrow cells from Balb/c mice. All mice were purchased from Japan SLC Inc. and maintained in a pathogen-free environment. Mice consumed normal chow and autoclaved hyperchlorinated water (pH 4) after the transplantation. Mice were not treated (control) or received cyclophosphamide 100 mg/kg at days 3 and 4 intraperitoneally. A leukemia cell line that was derived from the B6 bone marrow and transformed with the BCR/ABL fusion gene (BA-1) was used for leukemogenic experiments. One million BA-1 cells were intravenously infused into the recipients 25 (±4) days after transplantation with no additive treatment. We continuously examined weight, GVHD score, peripheral blood cell counts, and peripheral blood cell prolife of the recipient mice, and finally investigated spleen and bone marrow cells. The chimeric balances among host-residual H-2Db+ cells and donor graft-derived H-2Dd+ cells in CD3+ T cells were monitored. To evaluate the function of activated T cells in the spleen, CD62L+, CD44+, and PD-1+ marker levels in CD8+ and CD4+ cells were quantitatively monitored at various time points. GVHD scoring was based on weight loss, posture, activity, fur texture, and skin integrity (severity score 0 to 2 for each variable, maximum index 10) as previously reported. Leukemia progression was defined by the presence of 0.05% BA-1 cells in peripheral blood mononuclear cells as detected by GFP expression. All experimental procedures were approved by an institutional review committee of the Osaka City University.ResultsControl B6 recipients developed severe weight loss and GVHD after transplantation and died of GVHD at around day 60, whereas the recipients with PTCy had significantly improved weight, GVHD score, and survival compared to control mice (figure1). These results were comparable to those of previous reports. In the leukemogenic experiment, the control recipients showed significantly longer survival than the ones with PTCy (figure2). Recipients with PTCy had a higher rate of leukemia progression compared to the control at day 30 after BA-1 injection, although there were no significant differences in the chimeric balances among donor graft-derived H-2Dd+ T cells both before and at day 7, 14, 21, 28, and 35 after BA-1 injection. Prior to BA-1 injection, CD62L+ CD44- naïve CD8+/CD4+ T cell number was low, and CD62L- CD44+ effector memory CD8+/CD4+ T cell number was high in the spleen of the control recipients compared to that in PTCy recipients. Moreover, the numbers of CD44+ PD-1+ memory CD8+/CD4+ T cells were also overwhelmingly high in the spleen of control recipients. These results showed that use of PTCy after allo-HSCT promoted T cell tolerance to alloantigens by eliminating antigen-exposed T cells and reduced the probability of rejection in leukemia as a consequence.ConclusionOur data indicated that PTCy treatment after allo-HSCT contributes to impaired GVL effects by reducing the number of antigen-exposed T cells and improves GVHD regulation in a murine stem cell transplant model. [Display omitted] DisclosuresHino:Novartis: Research Funding.

The Anti-Viral Compound Cosalane Attenuates Acute Graft-Versus-Host Disease in Murine Hematopoietic Stem Cell Transplant Models

Introduction : Acute graft-versus-host disease (aGVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). Mechanistically, aGVHD depends on the trafficking of donor conventional T cells (Tcons) into host secondary lymphoid tissues (SLT) where they are activated by host antigens. Previously our group demonstrated that donor Tcons deficient in the lymphoid trafficking receptor CC-Chemokine Receptor 7 (CCR7) generated attenuated aGVHD responses but retained their graft-versus-leukemia (GVL) potential (Coghill JM et al. Blood. 2010; 115(23): 4914-22). This suggested that the receptor might be targeted therapeutically. Since there were no drugs affecting the CCR7 axis, we undertook a high throughput screen (HTS) of over 200,000 compounds to identify small molecule antagonists of the receptor. This effort demonstrated that cosalane, a compound originally developed as a novel HIV therapeutic but with additional activity against numerous other enveloped viruses including cytomegalovirus and herpes simplex virus, also possessed an intrinsic ability to block human and murine CCR7 signaling in response to both of its ligands. As a result, we examined the ability of cosalane to modulate aGVHD in vivo in mouse HSCT models.Results : Cosalane did not substantially alter aGVHD outcomes when dosed intravenously post-transplant to murine recipients in a standard C57BL/6 “B6” (H-2b) into C57BL/6-DBA2 F1 “B6D2” (H-2bxd) haplotype matched HSCT model. However, when donor Tcons were incubated with cosalane in vitro for one hour prior to transplant, aGVHD was significantly attenuated and survival was prolonged compared to cells incubated with DMSO vehicle (Figure 1A, *P=0.004 for curve comparison between BM with Tcons and BM with Tcons/cosalane groups by log rank test). Cosalane exposure did not appear to result in any cytotoxicity, and did not affect Tcon activation and expansion in vitro in response to CD3 stimulation in the presence of supplemental IL-2. Furthermore, when the entire stem cell product (bone marrow cells plus Tcons) was incubated with cosalane prior to transplant, myeloid engraftment was unaffected and survival times were once again improved with no evidence of bone marrow aplasia (Figure 1B, **P=0.001 for curve comparison by log rank test).While cosalane was originally identified as an inhibitor of CCR7, its effects on donor Tcon trafficking into host lymphoid sites post-transplant were surprisingly modest. No major differences in donor Tcon accumulation within the host inguinal lymph nodes, mesenteric lymph nodes, or spleen were noted on either transplant day +7 or day +14. A strong trend for reduced Tcon numbers was noted, however, within the host colon and liver by the second week post-transplant, implying an additional mechanism of action beyond its activity against CCR7.Cosalane's effects did not appear to be strain dependent, as survival times were also improved in a completely MHC mismatched B6 into BALB/c (H-2d) model system, although to a lesser extent. In addition, cosalane pretreatment of human peripheral blood mononuclear cells resulted in prolonged survival times in a mouse xenogeneic aGVHD transplant model. We went on to examine cosalane's effects on beneficial GVL effects using a well described luciferase transduced p815 (H-2d) murine mastocytoma model. Here survival times were prolonged in B6D2 mice receiving cosalane treated B6 Tcons/tumor versus those receiving DMSO treated B6 Tcons/tumor (who all succumbed to aGVHD) and those receiving bone marrow alone/tumor (who all died from malignancy). We have found cosalane's effects on aGVHD to be dose dependent in a B6 into B6D2 system (treatment range 13uM-39uM), and additional work is ongoing to optimize its administration so as to maximize anti-inflammatory effects while sparing anti-tumor immunity.Conclusions : The anti-viral compound cosalane is able to attenuate aGVHD in multiple allogeneic and xenogeneic model systems after a brief incubation with the donor Tcon and/or bone marrow product in vitro prior to transplant. Although identified in our HTS as a CCR7 antagonist, its effects on lymphoid trafficking after HSCT have thus far been limited, and additional mechanistic studies are currently ongoing. Given its known anti-viral effects against numerous pathogens common to the HSCT setting, we are also exploring its anti-infective properties in murine model systems. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced dose of PTCy followed by adjuvant \u03b1-galactosylceramide enhances GVL effect without sacrificing GVHD suppression

Posttransplantation cyclophosphamide (PTCy) has become a popular option for haploidentical hematopoietic stem cell transplantation (HSCT). However, personalized methods to adjust immune intensity after PTCy for each patient’s condition have not been well studied. Here, we investigated the effects of reducing the dose of PTCy followed by α-galactosylceramide (α-GC), a ligand of iNKT cells, on the reciprocal balance between graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect. In a murine haploidentical HSCT model, insufficient GVHD prevention after reduced-dose PTCy was efficiently compensated for by multiple administrations of α-GC. The ligand treatment maintained the enhanced GVL effect after reduced-dose PTCy. Phenotypic analyses revealed that donor-derived B cells presented the ligand and induced preferential skewing to the NKT2 phenotype rather than the NKT1 phenotype, which was followed by the early recovery of all T cell subsets, especially CD4+Foxp3+ regulatory T cells. These studies indicate that α-GC administration soon after reduced-dose PTCy restores GVHD-preventing activity and maintains the GVL effect, which is enhanced by reducing the dose of PTCy. Our results provide important information for the development of a novel strategy to optimize PTCy-based transplantation, particularly in patients with a potential relapse risk.

Immunomodulatory Therapies for the Treatment of Graft-versus-host Disease.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a potentially curative therapy for patients suffering from hematological malignancies, and its therapeutic success is based on the graft-versus-leukemia (GvL) effect. Severe acute and chronic graft-versus-host disease (GvHD) are life-threatening complications after allo-HCT. To date, most of the approved treatment strategies for GvHD rely on broadly immunosuppressive regimens, which limit the beneficial GvL effect by reducing the cytotoxicity of anti-leukemia donor T-cells. Therefore, novel therapeutic strategies that rely on immunomodulatory rather than only immunosuppressive effects could help to improve patient outcomes. Treatments should suppress severe GvHD while preserving anti-leukemia immunity. New treatment strategies include the blockade of T-cell activation via inhibition of dipeptidyl peptidase 4 and cluster of differentiation 28-mediated co-stimulation, reduction of proinflammatory interleukin (IL)-2, IL-6 and tumor necrosis factor-α signaling, as well as kinase inhibition. Janus kinase (JAK)1/2 inhibition acts directly on T-cells, but also renders antigen presenting cells more tolerogenic and blocks dendritic cell-mediated T-cell activation and proliferation. Extracorporeal photopheresis, hypomethylating agent application, and low-dose IL-2 are powerful approaches to render the immune response more tolerogenic by regulatory T-cell induction. The transfer of immunomodulatory and immunosuppressive cell populations, including mesenchymal stromal cells and regulatory T-cells, showed promising results in GvHD treatment. Novel experimental procedures are based on metabolic reprogramming of donor T-cells by reducing glycolysis, which is crucial for cytotoxic T-cell proliferation and activity.

A novel clinically relevant graft-versus-leukemia model in humanized mice.

The prognosis for acute myeloid leukemia (AML) relapse post allogeneic hematopoietic stem cell transplantation (alloSCT) is dismal. Novel effective treatment is urgently needed. Clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. The mechanisms that mediate immune escape of leukemia (thus causing GVL failure) remain poorly understood. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. Here, using our large, longitudinal clinical tissue bank that include AML cells and G-CSF mobilized donor hematopoietic stem cells (HSCs), we successfully established a novel GVL model in humanized mice. Donor HSCs were injected into immune-deficient NOD-Cg-Prkdcscid IL2rgtm1Wjl /SzJ (NSG) mice to build humanized mice. Immune reconstitution in these mice recapitulated some clinical scenario in the patient who received the corresponding HSCs. Allogeneic but HLA partially matched patient-derived AML cells were successfully engrafted in these humanized mice. Importantly, we observed a significantly reduced (yet incomplete elimination of) leukemia growth in humanized mice compared with that in control NSG mice, demonstrating a functional (but defective) GVL effect. Thus, for the first time, we established a novel humanized mouse model that can be used for studying human GVL responses against human AML cells in vivo. This novel clinically relevant model provides a valuable platform for investigating the mechanisms of human GVL and development of effective leukemia treatments.

Oral Proteasome Inhibitor Ixazomib for Switch-Maintenance Prophylaxis of Recurrent or Late Acute and Chronic Graft-versus-Host Disease after Day 100 in Allogeneic Stem Cell Transplantation

Graft-versus-host disease (GVHD) is a frequent complication in the first year after allogeneic stem cell transplantation (allo-HCT). Recipients of reduced-intensity (RI) or nonmyeloablative (NMA) conditioning combined with calcineurin inhibitor (CNI)-based GVHD prophylaxis frequently develop GVHD in the context of immunosuppression taper. Ixazomib is an oral proteasome inhibitor with a wide safety profile that has demonstrated immunomodulatory properties, inhibition of pro-inflammatory cytokines, and anti-tumor activity. We hypothesized that switch-maintenance GVHD prophylaxis using ixazomib would facilitate CNI taper without increased GVHD frequency and severity while maintaining graft-versus-tumor (GVT) effect and an acceptable safety profile. We conducted an open-label, prospective, single-center pilot study in patients with hematologic malignancies who received an RI or NMA conditioning and CNI-based GVHD prophylaxis that were within day 100 to 150 after HCT (n=18). Patients were treated with ixazomib once weekly on a 28-day cycle (3 weeks on, 1 week off). Treatment was safe; most adverse events were grade 1 or 2, with cytopenia and elevation in transaminases the most common. Five patients were removed from the study because of toxicity or side effects. Only 5 of 18 patients developed GVHD during the study, and its severity was driven by acute manifestations while chronic involvement was mild. The cumulative incidence of grade II-IV acute and chronic GVHD at 1-year after HCT was 33% (95% confidence interval [CI], 13-55). No patients died during the study, and only 1 had malignant relapse. An additional patient relapsed after completion of the study but within 1 year after HCT. The probability of progression-free survival and GVHD-free/relapse-free survival (composite endpoint) at 1 year were 89% (95% CI, 75-100) and 78% (95% CI, 61-100), respectively. Immune reconstitution analysis showed a rapid and sustained recovery in T-cell subpopulations and B cell reconstitution, and vaccine response in a subset of patients demonstrated continuing or de novo positive protective antibody titers. This study demonstrated low incidence of recurrent and late acute and chronic GVHD within 1 year after HCT possible associated with switch-maintenance GVHD prophylaxis using ixazomib. This approach allowed for CNI taper while preserving GVT effect, without aggravating GVHD. Our findings support further development of this approach and provide a proof-of-concept for switch-maintenance GVHD prophylaxis.

Preliminary analysis of a phase 1/2 study of NEXI-001 donor-derived multi-antigen-specific CD8+ T-cells for the treatment of relapsed acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT).

2538 Background: Allogeneic HCT is a potentially curative therapy for many patients with AML that relies on a graft-versus-leukemia (GvL) effect. Patients who relapse after allogeneic HCT have a poor prognosis and few treatment options. Donor lymphocyte infusion (DLI) can achieve a GvL effect in some patients, however, efficacy is frequently associated with the development graft-versus-host disease (GVHD). There is a substantial need for treatment approaches that enhance the benefit of GVL while decoupling toxicities associated with GVHD. Methods: We report ongoing results from a first-in-human study (NCT04284228) of a non-genetically engineered, donor-derived adoptive cellular therapy product, NEXI-001, which contains multiple populations of CD8+ T cells that recognize different HLA 02.01-restricted peptides from the WT1, PRAME, and Cyclin A1 antigens. NEXI-001 contains T cell memory subtypes that combine anti-tumor potency with long-term persistence. Results: At the time of this analysis, 7 patients with relapsed AML after allogeneic HCT were enrolled. Five Patients were treated with single infusions of NEXI-001 at three different dose levels: 50, 100 and 200 million. Currently, the median follow-up is 5 months. Significantly, GVHD, cytokine release syndrome, neurotoxicity, or NEXI-001-related adverse events were not observed. NEXI-001 treatment resulted in reductions in red blood cell and platelet transfusions and increased donor chimerism. Decreases in myeloblasts in bone marrow and peripheral blood and reduction in the size of an extramedullary myeloid sarcoma were suggestive of an anti-leukemia effect (Table). Correlative studies indicate that NEXI-001 CD8+ cells undergo a rapid proliferation after infusion and are also associated with a robust hostlymphocyte recovery that occurs as quickly Day 3 after infusion. NEXI-001 infused CD8+T cells are detectable by multimer staining in peripheral blood of patients and proliferate over time. TCR sequencing analyses determined that infused NEXI-001 cells contain T cell clones that were undetectable in the peripheral blood of patients at baseline but were detected in blood and bone marrow and persist over time. Conclusions: NEXI-001 has the potential to enhance GvL effect without the associated toxicities of GVHD, cytokine release syndrome, and neurotoxicity. Due to these encouraging results, the trial will proceed with an evaluation of repeated NEXI-001 dosing Clinical trial information: NCT04284228. [Table: see text]

Clinical effects of administering leukemia-specific donor T cells to patients with AML/MDS after allogeneic transplant

AbstractRelapse after allogeneic hematopoietic stem cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusion of unselected donor lymphocytes (DLIs) enhances the graft-versus-leukemia (GVL) effect. However, because the infused lymphocytes are not selected for leukemia specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease (GVHD), related to the concurrent transfer of alloreactive lymphocytes. Thus, to minimize GVHD and maximize GVL, we selectively activated and expanded stem cell donor–derived T cells reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, and NY-ESO-1). Products that demonstrated leukemia antigen specificity were generated from 29 HCT donors. In contrast to DLIs, leukemia-specific T cells (mLSTs) selectively recognized and killed leukemia antigen–pulsed cells, with no activity against recipient's normal cells in vitro. We administered escalating doses of mLSTs (0.5 to 10 × 107 cells per square meter) to 25 trial enrollees, 17 with high risk of relapse and 8 with relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD seen. We observed antileukemia effects in vivo that translated into not-yet-reached median leukemia-free and overall survival at 1.9 years of follow-up and objective responses in the active disease cohort (1 complete response and 1 partial response). In summary, mLSTs are safe and promising for the prevention and treatment of AML/MDS after HCT. This trial is registered at www.clinicaltrials.com as #NCT02494167.

The Role of Myeloid-Derived Suppressor Cells (MDSCs) in Graft-versus-Host Disease (GVHD)

Background: Myeloid-derived suppressor cells (MDSCs) are implicated in the complex interplay involving graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) in hematologic malignancies. Methods: A review of literature through PubMed was undertaken to summarize the published evidence on the pathophysiology and clinical implications of MDSCs in allo-HCT. Literature sources published in English since 1978 were searched, using the terms Natural Suppressor (NS) cells, MDSCs, GVHD, and allo-HCT. Results: In vivo studies demonstrated that MDSCs derived from mobilization protocols could strongly suppress allo-responses mediated by T cells and enhance T-Reg activity, thus inhibiting GVHD toxicity. However, the influence of MDSCs on the GVL effect is not fully defined. Conclusions: The induction or maintenance of MDSC suppressive function would be advantageous in suppressing inflammation associated with GVHD. Pathways involved in MDSC metabolism and the inflammasome signaling are a promising field of study to elucidate the function of MDSCs in the pathogenesis of GVHD and translate these findings to a clinical setting.

Graft-versus-Host Disease Prophylaxis with Post-Transplantation Bendamustine in Patients with Refractory Acute Leukemia: A Dose-Ranging Study

The prognosis of acute leukemia refractory to induction chemotherapy or immunotherapy is dismal. Salvage allogeneic hematopoietic stem cell transplantation (HSCT) is widely used option for these patients, but only 10% to 15% of patients are cured by the procedure. Preclinical studies indicate that substitution of post-transplantation cyclophosphamide with bendamustine (PTB) in a prophylaxis regimen may be associated with an augmented graft-versus-leukemia (GVL) reaction. The aim of this study was to establish the optimal dose of PTB and evaluate the antileukemic effect of HSCT with this type of graft-versus-host disease (GVHD) prophylaxis. In the prospective trial (NCT02799147), PTB was administered in doses of 140, 100, and 70 mg/m2 on days +3 and +4. Myeloablative conditioning with fludarabine and oral busulfan was provided to all patients. The first 12 patients received single-agent PTB, and subsequent patients received combination therapy with tacrolimus and mycophenolate mofetil (MMF). Inclusion criteria were acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL) refractory to at least one induction course of chemotherapy or target therapy and ≥5% clonal blasts in the bone marrow. The study cohort comprised 22 patients with AML and 5 with ALL. Seven patients were enrolled in the 140 mg/m2 group (due to a stopping rule), and 10 each were enrolled in the 100 mg/m2 and 70 mg/m2 groups. Primary refractory disease was documented in 41% of the patients, and secondary refractory was documented in 59%. The median blast count in the bone marrow at the start of the conditioning was 18% (range, 6% to 97%). Transplantation was performed with a matched sibling donor in 5 patients, a matched or mismatched unrelated donor in 15, and a haploidentical donor in 7. Engraftment was documented in 93% of the patients, including 89% with complete remission and 63% without measurable residual disease. After PTB prophylaxis, we observed an unusual complication, a cytokine release syndrome (CRS), in 70% of the patients, including grade 3 to 5 CRS in 44%. The most frequent clinical symptoms included high fever in 67% of patients, abnormal liver function tests in 67%, pancreatitis in 63%, skin vasculitis in 56%, enterocolitis in 48%, inflammation of oral mucosa in 37%, disseminated intravascular coagulation in 37%, and central nervous system toxicity in 26%. The development of CRS was associated with use of an HLA-mismatched donor (75% versus 20%; P=.0043). Classic acute GVHD was documented in 44% of the patients. Grade II-IV acute GVHD was associated with grade 3 to 5 CRS (67% versus 25%; P=.031). Moderate and severe chronic GVHD in the 100-day survivors were more often observed after single-agent PTB than after the combination immunosuppression (100% versus 18%; P=.002). A relatively low relapse rate was observed for this patient population. Three-year overall survival was 28% (95% confidence interval [CI], 13% to 46%), and event-free survival was 29% (95% CI, 13% to 46%). Nonrelapse mortality was 46% (95% CI, 25% to 64%), and the cumulative incidence of relapse was 26% (95% CI, 11% to 44%). No relapses were documented after day +100. There were no statistically significant differences among the dose groups (P=.3481); however, survival was higher in the 100 mg/kg group. Survival was higher in patients with AML compared with those with ALL (35% versus 0%; P=.0157). PTB represents a promising option to augment the GVL effect in refractory AML; however, the high CRS-associated mortality necessitates additional studies to reduce the risk of this complication. Thus, routine clinical application of PTB cannot be currently recommended. Combination immunosuppression with tacrolimus and MMF partially ameliorates these complications, at least in the setting of HLA-matched allografts. Biological mechanisms of CRS and GVL after PTB require further elucidation.