Graft-versus-leukemia Research Articles

Siremadlin Monotherapy and in Combination with Donor Lymphocyte Infusion for Patients with Acute Myeloid Leukemia Post Allogeneic Stem Cell Transplantation Who Are in Complete Remission but at High Risk for Relapse

Background: Relapse post allogeneic stem cell transplantation (SCT) remains the main cause of treatment failure (~40%) for patients (pts) with acute myeloid leukemia (AML). Many pts who ultimately relapse post SCT do so within the first year post transplant, and earlier relapse is associated with poorer survival. Therapeutics that prevent early relapse for these pts are urgently needed. Early cellular or pharmacologic intervention or maintenance strategies that enhance the graft-versus-leukemia (GvL) effect after SCT are of renewed interest as they have the potential to reduce relapse risk post SCT. Siremadlin (HDM201) is a novel investigational MDM2 inhibitor with single-agent, anti-AML activity. MDM2 inhibitors, including siremadlin, possess potent immunomodulatory effects in murine solid tumor and AML models. MDM2 inhibition has been shown to synergize with the allogeneic immune response, increase the vulnerability of AML cells to allogeneic donor T cells, and promote the cytotoxicity and longevity of allogeneic donor T cells in murine models. As such, the post-SCT setting is ideal for investigating the immunomodulatory effects of MDM2 inhibition on enhancing the GvL effect (Ho et al. Blood. 2022). Here we describe the design of a Phase Ib/II, open-label study of siremadlin as monotherapy and in combination with donor lymphocyte infusion (DLI) in pts with AML post SCT who are in complete remission (CR) but at high risk for relapse based on the presence of pre-SCT risk factors (NCT05447663). Study Design: The trial is not yet recruiting but aims to enroll ~38 adult pts with AML who underwent first SCT and are currently between Day 60-120 post SCT. Prior to a patient's SCT, they must have a risk factor that puts them at high risk for relapse, including AML in first CR with adverse risk genetics per 2017 European LeukemiaNet risk stratification, therapy-related AML, or secondary AML; or AML in second or greater CR. The SCT must be from an 8/8 human leukocyte antigen (HLA) matched related or unrelated donor at HLA-A, -B, -C, -DRB1 loci using unmanipulated/T-cell replete bone marrow or peripheral blood stem cells as a stem cell source. Post SCT, pts must have achieved CR or CR with incomplete hematologic recovery (CRi) with no evidence of hematologic relapse. Exclusion criteria include prior exposure to an MDM inhibitor, active graft-vs-host disease (GvHD), past history of grade ≥III acute GvHD (aGvHD) or moderate/severe chronic GvHD (cGVHD), recipient of SCT from a haploidentical family donor or cord blood transplant, and prior systemic AML-directed treatments given at any time after SCT (including DLI). This study contains 2 parts, Part 1 is a dose confirmation of siremadlin monotherapy and will enroll ~12 pts. Siremadlin will be tested at a maximum of 3 dose levels with a starting dose of 30 mg/d on Days 1-5 of a 28-day cycle, dose level +1 at 40 mg/d and dose level -1 at 20 mg/d. The safety and tolerability of these dose levels will be assessed to determine the siremadlin recommended dose (RD) for Part 2. Pts in Part 1 of the study will receive siremadlin monotherapy for the entire duration of the treatment and will not receive DLI. Subsequently, ~26 pts will be enrolled in Part 2 and proceed through a priming phase with siremadlin monotherapy at the RD, followed by a combination phase of siremadlin + DLI, and subsequently a maintenance phase with siremadlin monotherapy (Figure). Primary safety endpoints of the study are incidence of dose-limiting toxicities (DLTs) in Part 1 and time to DLT in Part 2. The primary efficacy endpoint is the proportion of pts in Part 2 who are alive and maintained CR/CRi without hematologic relapse ≥6 months after the start of study treatment. Secondary endpoints include number of pts in Part 1 who are alive and maintained CR/CRi without hematologic relapse ≥6 months after the start of study treatment at the RD for Part 2, time to first documented hematologic relapse or death, incidence of relapse at 1 year and 2 years post study treatment, safety, GvHD-free/relapse-free survival, incidence of and time to treatment-emergent grade ≥III aGvHD or moderate/severe cGvHD, and pharmacokinetics. Conclusions: This Phase Ib/II study investigates siremadlin monotherapy and siremadlin in combination with DLI in pts with AML in remission but at high risk of relapse post SCT. The study will evaluate the unique immunomodulatory potential of siremadlin to reduce risk of relapse in this population with high unmet need. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase II Study of PD-1 Blockade Following Fludarabine/Melphalan-Based Autologous Transplantation for Acute Myeloid Leukemia Patients Ineligible for Allogeneic Transplant

Allogeneic transplant (allo) remains the best post-remission therapy for non-favorable risk acute myeloid leukemia (AML) patients, secondary to the potent graft-versus-leukemia (GVL) effect of donor immune cells. Unfortunately, some patients may be ineligible for allogeneic transplant due to psychosocial barriers (e.g. lack of appropriate caregiver support) or unavailability of a suitable donor. Inhibition of the programmed cell death protein-1 (PD-1) pathway is an effective immunotherapeutic anti-cancer strategy for a number of malignancies. We hypothesized that PD-1 blockade following autologous transplant might simulate an allogeneic GVL effect and represent an alternative post-remission strategy in patients with non-favorable risk AML. To this end, we initiated a phase II study whereby AML patients deemed ineligible for allogeneic transplant received a high dose preparative regimen consisting of fludarabine on day -4, -3, -2 (30mg/m2/d) and melphalan on day -1 (180mg/m2 for age ≤60, 140mg/m2 for age >60), followed by autologous stem cell transplantation and post-transplant pembrolizumab starting day +1 (200mg/dose q3weeks x 8 doses) (auto-pembro). Eligible patients were AML patients beyond CR1 or non-favorable CR1 patients, including c-kit-mutated core-binding factor and FLT3-ITD+/NPM1+ AML. Of 32 patients consented/screened for the study, 20 received protocol-directed therapy from January 2017 to July 2021. Reasons for not receiving transplant were mobilization failure [6], failure to achieve CR [2], patient refusal [2], compliance issues [1] and insurance issues [1]. Baseline patient characteristics included a median (range) age of 64 (26-75) years, KPS <80 in 30%, HCT-CI ≥3 in 85% and non-Hispanic white in 45%. Disease characteristics included ELN 2017 favorable, intermediate and adverse risk in 20%, 40% and 40%, disease-risk index low, intermediate and high in 10%, 75% and 15%, secondary/treatment-related in 20%, CR1 status in 80%, and pre-BMT measurable residual disease (MRD) in 30%. Treatment was well tolerated in only 1 non-relapse death. Infections included 5 patients with bacterial infections, 2 with CMV viremia, 1 with localized VZV and 1 patient with HHV6 reactivation. Grade 3 non-hematologic toxicities included febrile neutropenia (30%), nausea/vomiting (25%), diarrhea (25%), hypertension (20%) and stomatitis/esophagitis (15%). There were no study-related grade 4 or 5 toxicities. Possible immune-related adverse events included 4 cases of hypothyroidism, 2 cases of colitis and 1 case each of pneumonitis, erythema multiforme and seronegative arthritis. After a median follow-up of 80 months (range, 15-134), 14 patients remain alive with 10 in continuous remission. Estimated 3-yr OS, DFS, NRM and CIR was 66%, 48%, 5% and 47% respectively. Transplant outcomes in auto-pembro recipients were compared to a contemporaneous cohort of consecutive AML patients receiving allo transplant in first or later CR (n=112) by Cox proportional hazards model, adjusting for significant demographic and risk variables. Compared with allo recipients, auto-pembro recipients were less likely to be non-Hispanic white (45% vs. 69%) and male (35% vs. 59%) but were otherwise similar in regard to baseline patient and disease characteristics. In multivariable analysis, OS and DFS were significantly impacted by age, ELN risk and year of transplant. In contrast, transplant type (auto-pembro vs. allo) had no effect on OS (HR 0.58, p=0.24) or DFS (HR 1.19, p=0.65). Adjusted 3-yr OS and DFS was 76% vs. 66% and 54% vs. 62% respectively (see figure). Although relapse was significant higher following auto-pembro (HR 2.38, p=0.031), it was offset by a corresponding decrease in NRM risk. In conclusion, auto-pembro is a safe and effective consolidation therapy for non-favorable AML patients who are ineligible for allo transplant, with comparable adjusted survival outcomes. Correlative studies are planned to analyze anti-leukemic effect of post-transplant PD-1 blockade following autologous transplant in AML patients. Given these promising results in non-favorable-risk AML patients lacking an allogeneic transplant consolidation option, confirmation in a larger multicenter prospective study is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

T-Cell Replete Unrelated Cord Blood Transplant Give Superior Outcomes to Other Donor Sources in High Risk and Relapsed/Refractory Paediatric Myeloid Malignancy

Introduction: Outcomes of alternate donor haematopoietic stem cell transplant (SCT) in high-risk (HR) and relapsed or refractory (R/R) paediatric acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) have been poor historically. Cord blood (CB) allows T-cell replete transplant, potentially enabling enhanced graft-versus-leukaemia (GVL) in patients with myeloid malignancies. We report the utility of T-replete CB (TRCB) transplant in high-risk paediatric myeloid malignancies in a large multi-centre national analysis, compared with patients transplanted with similar disease and at the same time using other donor sources. Methods: Data was collected from consecutive patients undergoing TRCB transplant for paediatric AML or MDS in 10 UK and Republic of Ireland paediatric bone marrow transplant centres between January 2014 and December 2021. The comparator group consisted of consecutive paediatric patients undergoing either a T-cell depleted (i.e. plus serotherapy) CB SCT or a transplant using any other cell source (matched sibling, matched/mismatched unrelated donor, or haplo-identical donor) at the same centres over the same period for the same indication. Information was gathered directly from the centre using an agreed data proforma and cross-checked for accuracy and completeness against the BSBMT/EBMT data submissions of each centre. Results: Data for 367 patients was analysed in this retrospective analysis which included 112 TRCB recipients and 255 patients in the comparator group. Median follow up was 2 years in the TRCB cohort and 4.5 years in the comparator group. All rates are quoted at 2 years. The median ages were 6.5 years in the TRCB group and 8.9 in the comparator, 91% of each group were AML. The TRCB cohort appeared to be a worse prognosis group with 51.4% of patients flow MRD positive at transplant compared with 26.1% in the comparator group (p <0.001), 46.4% had physician-defined refractory disease at transplant and 21.4% had relapsed following a previous SCT, compared with 8.6% refractory and 5.1% previous SCT in the comparator group (p<0.001 and p<0.001). Within the TRCB cohort Overall survival (OS) and Event Free Survival (EFS) were 64.7% and 64.1%, with EFS of 50% in those that were flow MRD positive at SCT and 79% for flow MRD negative patients (p= 0.009). EFS stratified by clinical disease status was 60.9% for those with primary refractory disease (n=29), 46.5% in those with relapsed refractory disease (n=23), 67% for those in high risk CR1 (n=38) and 79.6% for those in CR2 (n=22). For the 24 patients who had received a previous BMT, EFS was 69%. 67% TRCB recipients developed acute GVHD, of which 30% was grade 3-4, but the rates of chronic GVHD were very low at 5%. To allow for the imbalance in baseline characteristics, a multivariate analysis (MVA) was performed: the TRCB cohort had significantly improved EFS (0.57(0.35-0.91), p=0.019) and time to relapse (0.46(0.26-0.81), p=0.008) with some evidence of improved OS (0.65(0.39-1.07), p = 0.088). The effect appeared similar for MRD positives and negatives (interaction p=0.29 EFS and 0.67 relapse). Figures 1 and 2 highlight the strikingly superior EFS and relapse risk in the TRCB cohort for MRD positive patients in univariate analysis. Transplant-related mortality (TRM) was higher in the TRCB setting, 12% vs 7% (2.05 (1.05, 4.01), p=0.036) but rates of chronic GVHD were significantly lower with TRCB compared to other SCT types, 4.9% vs 19.4% (0.25(0.10, 0.62), p=0.003). Conclusion: This is the largest reported analysis of TRCB outcomes in HR paediatric myeloid malignancy, and we demonstrate excellent results, even in refractory disease. Our data show significantly superior outcomes for patients receiving TRCB compared with other transplant cell sources, owing to a lower relapse risk in the CB setting. This is likely to reflect enhanced GVL effects with TRCB compared with alternate donor sources requiring T-cell depletion. Despite higher TRM in the TRCB setting, when adjusted for the confounding variable of more MRD positive patients in this group, EFS and relapse are highly significant with a strong trend for improved OS. Our data suggests that CB transplant without serotherapy may be the optimal transplant option for children with HR and R/R AML/MDS, particularly with strategies to mitigate the higher TRM. This potential practice change warrants exploration in a large prospective randomised controlled trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Maintenance with Hypomethylating Agents Post-Transplant in Acute Myeloid Leukemia Patients: Safe and Affordable Way to Induce Graft Versus Leukemia Effect

INTRODUCTION Allogenic stem cell transplantation (ASCT) is the only optimistic therapy for poor risk, relapse and/or refractory acute myeloid leukemia (AML) patients for better survival outcome. However, 40% of the patients relapse after ASCT translating into 2-year survival rates below 20% and majority of relapses occurs within 12-18 months of transplant (Devillier, Leukemia & Lymphoma 2013). Hence, it's reasonable to start maintenance therapy as early as possible for such high risk AML. Azacytidine, an hypomethylating agent (HMA) has been shown previously as a potential maintenance drug to be used in post transplant setting (Oran, Blood advances 2020). Recently Keruakous (Hematology/Oncology and Stem Cell Therapy, 2021) had showed that the relapse rate was significantly higher in the control cohort (66.67%) versus in the azacytidine maintenance cohort (25.81%). On the similar concept, we analyzed our AML patients who received maintenance with HMA post transplant. OBJECTIVE To analyze the Relapse free survival (RFS) and overall survival( OS) in AML patients post-transplant with the use of HMA maintenance therapy. METHODS It's a retrospective analysis of AML patients who have undergone ASCT at our centre between May 2020 and April 2022. Maintenance was given to all patients post ASCT irrespective of MRD status post transplant and risk classification pre-transplant. Patients who received maintenance with HMA (either azacytidine@ at 32 mg/m2 daily for 5 days 4 weekly or decitabine @10mg/m2/day for 5 days 6 weekly) and had completed at-least one cycle were included (Oran, Blood advances 2020 and Pusic, Biol of Blood Marrow Transplant 2015). Maintenance was planned for minimum of one year. OS and RFS was calculated by the Kaplan-Meier curves. Events included relapses and deaths. RESULTS Twenty-six patients (AML=24, MDS=1, CMML=1) received maintenance with HMA during the study period. Mean age was 31.5 (14-51) years and 15 (57.69%) patients were male. As per European Leukemia Net (ELN) 2017 classification 5 (19.2%), 12 (46.1%) and 7 (26.9%) patients were categorized into favorable risk, intermediate risk and adverse risk respectively. There were 14 (53.8%) patients in Complete remission (CR1) group, 10 (38.4%) in CR2 group and two(7.6%) were refractory disease. Source of graft was peripheral blood stem cell for all patients. Conditioning regimen was myelo-ablative in 22 (84.6%) patients, whereas 4 (15.3%) patients received reduced intensity. Donors were matched sibling (n=15, 57.69%), haplo-identical (n=6, 23.07%), matched unrelated (n=4, 15.38%) and mismatched unrelated(n=1,3.8%). Azacytidine was started within 100 days of transplant in 22(84.61%) patients. The median of number of cycles of HMA received were 6. The median duration of follow up was 13 months. Seven (26.9%) patients completed 12 cycles. Acute Graft versus host disease (GVHD) was seen in 7 patients (26.9%) involving skin and liver whereas 3 (11.5%) patients had chronic GVHD involving skin and eye. Three (30%) patients developed GVHD prior and 7(70%) developed GVHD post initiation of maintenance. Six (85.7%) patients who relapsed on HMA maintenance had no prior acute or chronic GVHD. Out of all the 7(26.9%) patients who relapsed, 6 patients relapsed within a year and one patient relapsed after a year. One year RFS was 71% (Figure1), Event Free Survival was 67.9% and OS was 81.4% (Figure 2) Only 1 (3.8%) patient had Transplant Related Mortality (TRM), who died of COVID-19 infection. OS at 1 year for those in CR1 and CR2 was 84.6% and 88.9% respectively and it was not statistically significant (P = 0.95). There was no substantial hematological toxicity and resulting drug interruptions during the maintenance. CONCLUSION Early HMA maintenance seems to be an effective strategy post ASCT in AML patients to prevent early relapses and improving overall survival. Development of GVHD for patients on HMA had probably synergistic effect to prevent relapses by mediating graft versus leukemia effect without additional toxicity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel RIPK1 inhibitor reduces GVHD in mice via a nonimmunosuppressive mechanism that restores intestinal homeostasis

AbstractIntestinal epithelial cells (IECs) are implicated in the propagation of T-cell–mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell–recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell–produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

Ruxolitinib on acute graft-versus-host disease prophylaxis after modified donor lymphocyte infusion

ObjectiveTo evaluate the effectiveness of ruxolitinib on acute graft-versus-host disease (aGVHD) prophylaxis and its impact on graft-versus-leukemia (GVL) effect in patients after modified donor lymphocyte infusion (mDLI). MethodsWe retrospectively included patients with relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) who received ruxolitinib prophylaxis between October 2018 and April 2020. The incidence of aGVHD, disease-free survival (DFS), overall survival (OS), and treatment safety were evaluated. ResultsSeventeen patients were followed up for a median time of 8 months (range: 1–26 months). The incidence of aGVHD on Day 30 after mDLI was 41.2% and ranged from Grade 1 to 4; ten of 17 patients (58.8%) achieved a complete response (CR), and two (11.8%) had a partial response (PR). Cytomegalovirus (CMV) reactivation rate was 23.5%, and the median time from mDLI to CMV reactivation was 48.5 days. The mean DFS and OS after mDLI were 1.0 (95% CI 0.0–3.5) and 9.0 (95% CI 1.2–16.8) months, respectively. The causes of death for 10 patients were leukemia relapse (n = 5), aGVHD and septic shock (n = 3), intracranial lesion (n = 1), and COVID-19 (n = 1). ConclusionsWe reported encouraging results of ruxolitinib monotherapy in the prevention of aGVHD and maintenance of GVL for post-transplantation relapsed patients, even though being at high risk with poor initial prognosis.

Mocravimod, a Selective Sphingosine-1-Phosphate Receptor Modulator, in Allogeneic Hematopoietic Stem Cell Transplantation for Malignancy

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the sole curative option for patients with acute myelogenous leukemia. Outcomes are limited by leukemia relapse, graft-versus-host disease (GVHD), and abnormal immune reconstitution. Mocravimod (KRP203) is an oral sphingosine-1-phosphate receptor (S1PR) modulator that blocks the signal required by T cells to egress from lymph nodes and other lymphoid organs. Mocravimod retains T cell effector function, a main differentiator to immunosuppressants. In preclinical models, mocravimod improves survival by maintaining graft-versus-leukemia (GVL) activity while reducing GVHD. In patients undergoing allo-HSCT for hematological malignancies, mocravimod is postulated to prevent GVHD by redistributing allogeneic donor T cells to lymphoid tissues while allowing a sufficient GVL effect in the lymphoid, where malignant cells usually reside. The primary objective of this study was to assess the safety and tolerability of mocravimod in patients undergoing allo-HSCT for hematologic malignancies. Secondary objectives were to determine the pharmacokinetic profiles of mocravimod and its active metabolite mocravimod-phosphate in this patient group, as well as to assess GVHD-free, relapse free survival at 6 months after the last treatment. In this 2-part, single- and 2-arm randomized, open-label trial, we evaluated the safety, tolerability, and pharmacokinetics of mocravimod in allo-HSCT recipients (ClinicalTrials.gov identifier NCT01830010). Patients received either 1 mg or 3 mg mocravimod per day on top of standard of care GVHD prophylaxis with either cyclosporine A/methotrexate or tacrolimus/methotrexate. We found that mocravimod can be safely added to standard treatment regimens in patients with hematologic malignancies requiring allo-HSCT. Mocravimod resulted in a significant reduction of circulating lymphocyte numbers and had no negative impact on engraftment and transplantation outcomes. Our results indicate that mocravimod is safe and support a larger study to investigate its efficacy in a homogeneous acute myelogenous leukemia patient population undergoing allo-HSCT.

The druggable transcription factor Fli-1 regulates T cell immunity and tolerance in graft-versus-host disease.

Graft-versus-host disease (GVHD), manifesting as either acute (aGVHD) or chronic (cGVHD), presents significant life-threatening complications following allogeneic hematopoietic cell transplantation. Here, we investigated Friend virus leukemia integration 1 (Fli-1) in GVHD pathogenesis and validated Fli-1 as a therapeutic target. Using genetic approaches, we found that Fli-1 dynamically regulated different T cell subsets in allogeneic responses and pathogenicity in the development of aGVHD and cGVHD. Compared with homozygous Fli1-deficient or WT T cells, heterozygous Fli1-deficient T cells induced the mildest GVHD, as evidenced by the lowest Th1 and Th17 cell differentiation. Single-cell RNA-Seq analysis revealed that Fli-1 differentially regulated CD4+ and CD8+ T cell responses. Fli-1 promoted the transcription of Th1/Th17 pathways and T cell receptor-inducible (TCR-inducible) transcription factors in CD4+ T cells, while suppressing activation- and function-related gene pathways in CD8+ T cells. Importantly, a low dose of camptothecin, topotecan, or etoposide acted as a potent Fli-1 inhibitor and significantly attenuated GVHD severity, while preserving the graft-versus-leukemia (GVL) effect. This observation was extended to a xenograft model, in which GVHD was induced by human T cells. In conclusion, we provide evidence that Fli-1 plays a crucial role in alloreactive CD4+ T cell activation and differentiation and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising the GVL effect.

Counteracting Immunosuppression in the Tumor Microenvironment by Oncolytic Newcastle Disease Virus and Cellular Immunotherapy.

An apparent paradox exists between the evidence for spontaneous systemic T cell- mediated anti-tumor immune responses in cancer patients, observed particularly in their bone marrow, and local tumor growth in the periphery. This phenomenon, known as "concomitant immunity" suggests that the local tumor and its tumor microenvironment (TME) prevent systemic antitumor immunity to become effective. Oncolytic Newcastle disease virus (NDV), an agent with inherent anti-neoplastic and immune stimulatory properties, is capable of breaking therapy resistance and immunosuppression. This review updates latest information about immunosuppression by the TME and discusses mechanisms of how oncolytic viruses, in particular NDV, and cellular immunotherapy can counteract the immunosuppressive effect of the TME. With regard to cellular immunotherapy, the review presents pre-clinical studies of post-operative active-specific immunotherapy and of adoptive T cell-mediated therapy in immunocompetent mice. Memory T cell (MTC) transfer in tumor challenged T cell-deficient nu/nu mice demonstrates longevity and functionality of these cells. Graft-versus-leukemia (GvL) studies in mice demonstrate complete remission of late-stage disease including metastases and cachexia. T cell based immunotherapy studies with human cells in human tumor xenotransplanted NOD/SCID mice demonstrate superiority of bone marrow-derived as compared to blood-derived MTCs. Results from clinical studies presented include vaccination studies using two different types of NDV-modified cancer vaccine and a pilot adoptive T-cell mediated therapy study using re-activated bone marrow-derived cancer-reactive MTCs. As an example for what can be expected from clinical immunotherapy against tumors with an immunosuppressive TME, results from vaccination studies are presented from the aggressive brain tumor glioblastoma multiforme. The last decades of basic research in virology, oncology and immunology can be considered as a success story. Based on discoveries of these research areas, translational research and clinical studies have changed the way of treatment of cancer by introducing and including immunotherapy.

Generalist in allogeneic hematopoietic stem cell transplantation for MDS or AML: Epigenetic therapy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment for patients with myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, relapse and graft-versus-host disease (GvHD) still affect the survival of patients who receive allo-HSCT, and more appropriate therapeutic strategies should be applied at all stages of transplantation to prevent these adverse events. The use of epigenetics agents, such as hypomethylating agents (HMAs), has been explored to decrease the risk of relapse by epigenetic modulation, which is especially effective among AML patients with poor mutations in epigenetic regulators. Furthermore, epigenetic agents have also been regarded as prophylactic methods for GvHD management without abrogating graft versus leukemia (GvL) effects. Therefore, the combination of epigenetic therapy and HSCT may optimize the transplantation process and prevent treatment failure. Existing studies have investigated the feasibility and effectiveness of using HMAs in the pretransplant, transplant and posttransplant stages among MDS and AML patients. This review examines the application of HMAs as a bridge treatment to reduce the tumor burden and the determine appropriate dose during allo-HSCT. Within this review, we also examine the efficacy and safety of HMAs alone or HMA-based strategies in posttransplant settings for MDS and AML. Finally, we provide an overview of other epigenetic candidates, which have been discussed in the nontransplant setting.

AML-142 Patients at High Risk of Relapse Post-Transplant: A Phase 1 Study Design With a Novel Treatment Strategy Using the Estimand Framework

The risk of disease relapse in patients allografted for acute myeloid leukemia (AML) is high and remains the main cause of transplant failure. A potent graft-versus-leukemia (GvL) effect is exerted post-stem cell transplantation (SCT); strategies that may augment a GvL effect have the potential to reduce relapse risk post SCT. Siremadlin/HDM201 is a novel investigational MDM2 inhibitor with single-agent (SA) anti-AML activity. MDM2 inhibitors possess potent immunomodulatory effects (IME) in murine solid tumor and AML models. The post-allogeneic SCT setting is ideal for investigating IME of MDM2 inhibition on the GvL effect. To present a study design assessing the safety and preliminary efficacy of treatment with monotherapy siremadlin and in combination with donor lymphocyte infusions (DLIs) for AML post SCT using the estimand framework (ICH E9 [R1] addendum). Phase Ib/II proof-of-concept. AML patients in CR post SCT at high risk for relapse based on pre-SCT risk factors. The estimand framework mandates definition of experimental intervention according to the clinical question. Due to the risk of GvHD with DLI early post SCT, siremadlin will first be given to eligible patients as priming monotherapy to prevent early relapse; patients who tolerate siremadlin with no evidence of GvHD are eligible to start siremadlin/DLI. Post combination, patients may continue maintenance SA siremadlin, which intends to prolong/enhance the GvL reaction to eradicate residual leukemic blasts. According to their treatment journey, patients may receive only part of the intervention's sequence (e.g., siremadlin priming monotherapy patients not eligible for DLI combination will continue priming). To determine 1) the siremadlin recommended dose (RD) across all parts of the treatment strategy and 2) preliminary efficacy regardless of the treatment parts received. Siremadlin RD will be determined using a Bayesian logistic regression model to guide dose finding of siremadlin during priming/Cycle 1 and a Bayesian time-to-DLT model to assess the siremadlin combination dose. Efficacy will be assessed by the proportion of patients remaining in CR ≥6 months. The estimand framework guided the design of an AML clinical study post SCT to conceptualize the assessment of safety and efficacy of the treatment strategy, accounting for different patient journeys.

Reduced Intensity Conditioning for Allogeneic Stem Cell Transplantation (HSCT) in Acute Myeloid Leukemia

Allogeneic transplantation (HSCT) is an effective curative therapy for high risk acute myeloid leukemia (AML) which account for 38% of the transplants in Europe (1). Prior to HSCT, a conditioning or preparative regimen is administered. The conditioning regimen has 2 components; one target the myeloid system aiming in eradication of the leukemic clones, while the other target the immune/lymphoid system to ensure engraftment and to prevent rejection. Some of the compounds used in the conditioning are more myeloablative in nature for example busulfan or melphalan) 2-4) while others are more lymphodepliting like fludarabine or Cytoxan (5). Traditionally, the pre HSCT conditioning was myeloablative (MAC) and includes total body irradiation (TBI) in combination with cyclophosphamide (CY) (2-3). High-dose busulfan (Bu) is the most commonly used TBI-free-based myeloablative conditioning (2-3). In HSCT from unrelated or mismatched donors the pre transplantation conditioning typically includes serotherapy with anti-thymocyte globulin (ATG) or the CAMPATH monoclonal antibody in order to avoid rejection and ensure engraftment while preventing graft versus host disease (GVHD) (5). However, the MAC is typically associated with significant morbidity and mortality due to the toxicity of the preparative regimen, GVHD, and the immune-deficient state that accompanies the procedure (2,5-6). This is especially true in patients above the age 55-60 years old and in patients with comorbidities which are the majority of AML patients. Extensive research, including pharmacokinetic and pharmacodynamics studies has been directed therefore towards the development of safer and less toxic conditioning regimens for HSCT, optimizing the conditioning allowing its applications to elderly patients and patients with comorbidities (2,5-6). These modern conditioning regimens which are based in part on the immune-mediated graft versus leukemia (GVL) effect are in principle low-dose, less toxic and tolerable conditioning regimens termed reduced intensity (RIC) with different immunosuppressive and myelosuppressive properties (5-7). These regimens combine immunosuppressive agents (such as fludarabine with or without serotherapy or targeted therapy with agents with moderate myelosuppressive effects or novel agents. However, they typically result in higher relapse rate especially in patients undergoing HSCT while not in remission and in patients with high risk leukemia including patients with adverse cytogenetics, high risk mutations and patients with positive measurable residual disease (MRD) at time of transplants. The optimal regimen is thus the one with intensive anti-leukemic activity, but with limited toxicity-the so called reduced toxicity regiments (RTC). These novel regimens are mostly fludarabine based and incorporate drugs like melphalan; thiotepa; treosulfan and clofarabine (8-11). Other protocols are the so called TBF protocol that include two alkylating agents like busulfan and thiotepa(9,11) and the FLAMSA protocol that includes fludarabine, cytarabine, and amsacrine (11).The RIC and RTC regimens enable HSCT in elderly patients and those with comorbidities reducing drastically transplant related mortality and organ toxicities in combination with improved anti leukemic effect. Efficient safe pre transplant conditioning protocols are continuing to be developed. Future protocols will most probably incorporate specific anti leukemic targeted novel compounds as well as monoclonal and radiolabeled antibodies.

FROM ALLOGENIC TRANSPLANTATION TO PRECISION IMMUNE THERAPY

Allogeneic stem cell transplantation (ASCT) represents a model for immune cellular therapy leading to Immune Precision Medicine. The pioneers Georges Mathé in Paris and E. Donall Thomas (Nobel Prize in 1990) in Cooperstown, New York, pioneered ASCCT in the clinical field. In 1958, the first 4 survivors were seen in patients after accidental exposure to lethal or near lethal dose of TBI, in Paris. However, they were subsequently shown to have autologous recovery. Understanding of ABMT immune support begun in 1954 with 1980 Nobel Prize Jean Dausset. The first ABMTs were performed in severe combined immunodeficiencies with the first success observed in 1968 (syngeneic donor), followed in 1973 by unrelated donor ABMT in London. This was also the time of the initiation of registries. Development time in hematological malignancies The first success of ABMT in acute leukemia was observed in 1976 in Seattle with a related donor and in 1976 with an unrelated donor. Thereafter, the evolution will take place within the framework of the risk-benefit balance with reduction of the intensity of the conditioning regimen, the graft versus leukemia (GVL)/graft versus host disease (GVH) balance and the donor extension with umbilical cord blood and more recently the haplo-identical allogeneic ASTC. Autologous SCT was introduced at the beginning of the 80s to amplify reduction in tumor mass, particularly in lymphoid malignancies. Stem cell transplantation as an immune therapy platform Whatever the autologous or allogeneic context, the hematopoietic SCT is an exceptional platform for combining, modulating immunotherapy. In an allogeneic context, by modifying lymphocyte subpopulations, such as the supply of cytotoxic T-cells, the modulation of Tregs, the addition or activation of NK cells have an impact on GVH/GVL balance. The enhancement of anti-tumor cytotoxicity can be brought about using monoclonal antibodies (moAb), the addition of cancer vaccines. In an autologous context, there are some windows of opportunity, in the aplasia period due to the accessibility to stressed cancer cells, and cytokine burst approximately at D15, to add cell-drugs such as NK, γδ T-cells or anti-cancer moAbs, or to associate chimeric antigen receptor (CAR) immune cells such as CAR-NK, as well as immune checkpoint inhibitors depending on the risks. This paves the way for a real dynamic personalized medicine and should cause the methodology for developing these therapeutic strategies to be rethought. Obtaining an optimization of the clinical efficiency which must be preceded by a reflection of biological efficiency can be helped by mathematical models or AI. We have thus developed a mathematical model for the optimization of the use of anti-IL-6. There is a modeling of use of cytotoxic cells. In cellular therapy, the concept of cell-drugs orients towards non-MHC dependent allogenic cells such as NK and γδ T-cells, as well as obtaining them in large batches to reduce production costs. We are entering a new medical era, with new notions such as dynamic, globalized vision, the use of new tools resulting from the digital revolution, new targeted therapies, immunotherapy, the combination of strategies for better efficiency: the Immune Precision Medicine.

Unrelated umbilical cord blood can improve the prognosis of haploidentical hematopoietic stem cell transplantation

BackgroundHaploidentical hematopoietic stem cell transplantation (haplo-HSCT) is widely used as a curative treatment strategy for most types of hematological diseases. However, strategies for enhancing the graft versus leukemia (GVL) effect without aggravating the graft versus host disease (GVHD) effect are still being pursued.MethodsA retrospective cohort study was performed to compare the outcomes between combined unrelated umbilical cord blood (UCB-haplo HSCT) and haplo HSCT.ResultsThe results showed that neither acute GVHD (aGVHD) nor chronic GVHD (cGVHD) was increased in the UCB-haplo HSCT group, and the engraftment and infection rates were similar between the two groups. However, overall survival and progression-free survival were significantly improved, while transplantation-related mortality and relapse were significantly decreased in the UCB-haplo HSCT group by both univariate and multivariate analyses.ConclusionOur results indicated that the addition of a UCB unit could improve the prognosis of haplo-HSCT and enhance the GVL effect without increasing the incidence of GVHD.Trial registrationThe cohort study was retrospectively registered at https://www.chictr.org.cn as ChiCTR2100046681.

Glucocorticoid and glycolysis inhibitors cooperatively abrogate acute graft-versus-host disease.

Although glucorticosteroids (GCs) are the standard first-line therapy for acute graft-versus-host disease (aGvHD), nearly 50% of aGvHD patients have no response to GCs. The role of T cell metabolism in murine aGvHD was recently reported. However, whether GCs and metabolism regulators could cooperatively suppress T cell alloreactivity and ameliorate aGvHD remains to be elucidated. Increased glycolysis, characterized by elevated 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and higher rates of glucose consumption and lactate production were found in T cells from aGvHD patients. Genetic upregulation of PFKFB3 induced T cell proliferation and differentiation into proinflammatory cells. In a humanized mouse model, PFKFB3-overexpressing or PFKFB3-silenced T cells aggravated or prevented aGvHD, respectively. Importantly, our integrated data from patient samples in vitro, in a humanized xenogeneic murine model of aGvHD and graft-versus-leukaemia (GVL) demonstrate that GCs combined with a glycolysis inhibitor could cooperatively reduce the alloreactivity of T cells and ameliorate aGvHD without loss of GVL effects. Together, the current study indicated that glycolysis is critical for T cell activation and induction of human aGvHD. Therefore, the regulation of glycolysis offers a potential pathogenesis-oriented therapeutic strategy for aGvHD patients. GCs combined with glycolysis inhibitors promises to be a novel first-line combination therapy for aGvHD patients.

S1P/S1PR1 signaling differentially regulates the allogeneic response of CD4 and CD8 T cells by modulating mitochondrial fission.

Graft-versus-host disease (GVHD) significantly contributes to patient morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HSCT). Sphingosine-1-phosphate (S1P) signaling is involved in the biogenetic processes of different immune cells. In the current study, we demonstrated that recipient sphingosine kinase 1 (Sphk1), but not Sphk2, was required for optimal S1PR1-dependent donor T-cell allogeneic responses by secreting S1P. Using genetic and pharmacologic approaches, we demonstrated that inhibition of Sphk1 or S1PR1 substantially attenuated acute GVHD (aGVHD) while retaining the graft-versus-leukemia (GVL) effect. At the cellular level, the Sphk1/S1P/S1PR1 pathway differentially modulated the alloreactivity of CD4+ and CD8+ T cells; it facilitated T-cell differentiation into Th1/Th17 cells but not Tregs and promoted CD4+ T-cell infiltration into GVHD target organs but was dispensable for the CTL activity of allogeneic CD8+ T cells. At the molecular level, the Sphk1/S1P/S1PR1 pathway augmented mitochondrial fission and increased mitochondrial mass in allogeneic CD4+ but not CD8+ T cells by activating the AMPK/AKT/mTOR/Drp1 pathway, providing a mechanistic basis for GVL maintenance when S1P signaling was inhibited. For translational purposes, we detected the regulatory efficacy of pharmacologic inhibitors of Sphk1 and S1PR1 in GVHD induced by human T cells in a xenograft model. Our study provides novel mechanistic insight into how the Sphk1/S1P/S1PR1 pathway modulates T-cell alloreactivity and validates Sphk1 or S1PR1 as a therapeutic target for the prevention of GVHD and leukemia relapse. This novel strategy may be readily translated into the clinic to benefit patients with hematologic malignancies and disorders.

The Role of NK Cells and Their Exosomes in Graft Versus Host Disease and Graft Versus Leukemia.

Natural killer (NK) cells are one of the innate immune cells that play an important role in preventing and controlling tumors and viral diseases, but their role in hematopoietic stem cell transplantation (HCT) is not yet fully understood. However, according to some research, these cells can prevent infections and tumor relapse without causing graft versus host disease (GVHD). In addition to NK cells, several studies are about the anti-leukemia effects of NK cell-derived exosomes that can highlight their roles in graft-versus-leukemia (GVL). In this paper, we intend to investigate the results of various articles on the role of NK cells in allogeneic hematopoietic cell transplantation and also their exosomes in GVL. Also, we have discussed the antiviral effects of these cells in post-HCT cytomegalovirus infection.

Natural killer cells 56bright16− have higher counts in the umbilical cord blood than in the adult peripheral blood

Introduction and hypothesisUmbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplantation in the absence of a compatible donor. The UCB transplantation has a lower incidence of chronic graft versus host disease (GvHD), but is associated with slower engraftment and slower immune reconstitution, compared to other sources. Dendritic cells (DCs) and Natural Killer cells (NKs) play a central role in the development of GvHD and the graft versus leukemia (GvL) effect, as well as in the control of infectious complications. MethodWe quantified by multiparametric flow cytometry monocytes, lymphocytes, NK cells, and DCs, including their subsets, in UCB samples from 54 healthy newborns and peripheral blood (PB) from 25 healthy adult volunteers. ResultsIn the UCB samples, there were higher counts of NK cells 56bright16− (median 0.024 × 109/L), compared to the PB samples (0.012 × 109/L, p < 0.0001), NK 56dim16bright (median 0.446 × 109/L vs. 0.259 × 109/L for PB samples, p = 0.001) and plasmacytoid dendritic cells (pDCs, median 0.008 × 109/L for UCB samples vs. 0.006 × 109/L for PB samples, p = 0.03). Moreover, non-classic monocyte counts were lower in UCB than in PB (median 0.024 × 109/L vs. 0.051 × 109/L, respectively, p < 0.0001). ConclusionIn conclusion, there were higher counts of NK cells and pDCs and lower counts of non-classic monocytes in UCB than in PB from healthy individuals. These findings might explain the lower incidence and severity of chronic GvHD, although maintaining the GvL effect, in UCB transplant recipients, compared to other stem cell sources.

Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway.

The immediate precursor to murine type 1 conventional DCs (cDC1s) has recently been established and named "pre-cDC1s". Mature CD8α+ cDC1s are recognized for suppressing graft-versus-host disease (GvHD) while promoting graft-versus-leukemia (GvL), however pre-cDC1s have not previously been investigated in the context of alloreactivity or anti-tumor responses. Characterization of pre-cDC1s, compared to CD8α+ cDC1s, found that a lower percentage of pre-cDC1s express PD-L1, yet express greater PD-L1 by MFI and a greater percent PIR-B, a GvHD-suppressing molecule. Functional assays were performed ex vivo following in vivo depletion of CD8α+ DCs to examine whether pre-cDC1s play a redundant role in alloreactivity. Proliferation assays revealed less allogeneic T-cell proliferation in the absence of CD8α+ cDC1s, with slightly greater CD8+ T-cell proliferation. Further, in the absence of CD8α+ cDC1s, stimulated CD8+ T-cells exhibited significantly less PD-1 expression compared to CD4+ T-cells, and alloreactive T-cell death was significantly lower, driven by reduced CD4+ T-cell death. Tumor-killing assays revealed that T-cells primed with CD8α-depleted DCs ex vivo induce greater killing of A20 B-cell leukemia cells, particularly when antigen (Ag) is limited. Bulk RNA sequencing revealed distinct transcriptional programs of these DCs, with pre-cDC1s exhibiting activated PD-1/PD-L1 signaling compared to CD8α+ cDC1s. These results indicate distinct T-cell-priming capabilities of murine pre-cDC1s compared to CD8α+ cDC1s ex vivo, with potentially clinically relevant implications in suppressing GvHD while promoting GvL responses, highlighting the need for greater investigation of murine pre-cDC1s.

S231: GLUCOCORTICOID AND GLYCOLYSIS INHIBITORS COOPERATIVELY ABROGATE ACUTE GRAFT-VERSUS-HOST DISEASE

Background: Although glucocorticoids(GCs) are the standard first-line therapy for acute graft-versus-host disease(aGvHD), approximately 50% of aGvHD patients have no response to GCs. The role of T cell metabolism in murine aGvHD has been recently reported. Pharmacological inhibition of glycolysis by targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3) ameliorates aGvHD. However, the question of whether glycolysis is required for the function of activated T cells and whether abnormal glycolysis is involved in the occurrence of human aGvHD need to be elucidated. Moreover, the issue as to whether GCs and metabolism regulators can cooperatively suppress T cell alloreactivity and ameliorate aGvHD remains to be elucidated. Aims: The study aimed to analyse the glucose metabolism profiles of activated T cells in aGvHD patients, and to evaluate the roles of PFKFB3-stimulated glycolysis in alloantigen-activated T cells and aGvHD induction. Furthermore, the study was performed to explore the issue of whether GCs could restore activated T cells by regulating glycolysis and the effect of GCs combined with glycolysis inhibitors on activated T cells. Methods: In this prospective nested case–control study, a total of 15 aGvHD patients at diagnosis and 15 matched non-aGvHD patients. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. Subsequently, to assess whether functional T cell activation is caused by the regulation of PFKFB3, we used lentivirus transduction for the genetic regulation of PFKFB3 in vitro and in a humanized xenogeneic aGvHD model. The effect of GCs and glycolysis inhibitors on activated T cells was further explored in vitro and in a humanized murine model of aGvHD and graft versus leukaemia (GVL). In addition, to evaluate the synergistic effect of GCs, 3PO or their combination, combination index (CI) studies were performed by using the Chou-Talalay method for drug interactions. Results: Increased glycolysis, which was characterized by elevated PFKFB3, as well as higher rates of glucose consumption and lactate production in aGvHD T cells. Importantly, in vitro treatment with glycolysis inhibitor 3PO improved the activity of T cells derived from aGvHD patients through down-regulating glycolytic activity of T cells. Genetic upregulation of PFKFB3 induced T cell proliferation and differentiation into proinflammatory T cells. In a humanized mouse model, PFKFB3-overexpressing T cells aggravated aGvHD, as characterized by high aGvHD clinical scores, pathological scores and rapid lethality. Importantly, our integrated data from patient samples in vitro showed that GCs combined with 3PO decreased proinflammatory T cells and T cell proliferation, reduced glucose consumption, lactate production and PFKFB3 expression compared with the single GCs group. Notably, the average CI values of GCs and 3PO were 0.364 for the synergistic inhibition of IFN-γ synthesis and 0.475 for the synergistic inhibition of proliferation, respectively. In a humanized murine model of aGvHD and GVL demonstrated that GCs and 3PO cooperatively reduced the alloreactivity of T cells and ameliorate aGvHD without a loss of GVL effects. Summary/Conclusion: Our data indicated that glycolysis is critical for T cell activation and the induction of human aGvHD. GCs combined with glycolysis inhibitors demonstrated synergistic effects on reducing T cell alloreactivity and ameliorating aGvHD by regulating T cell glycolysis in vitro and in vivo. Thus, GCs combined with glycolysis inhibitors promise to be a novel therapeutic strategy for aGvHD patients.