Graft-versus-leukemia Research Articles

S100A9 upregulated by IFNGR signaling blockade functions as a novel GVHD suppressor without compromising GVL in mice

AbstractAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1−/− and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.

Enrichment of innate immune cells from PBMC followed by triple cytokine activation for adoptive immunotherapy.

Innate immune cells [Natural killer (NK) and gamma-delta (γδ) T-cells] have the advantage of mediating graft versus leukemia (GVL) without graft versus host disease (GVHD). Therefore, the infusion of activated innate immune cells post allogenic hematopoietic stem transplant (AHSCT) is a promising adoptive immunotherapy strategy for relapsed and/or refractory myeloid malignancies. Microbead depletion of T-cells and B-cells has been used as a graft manipulation method to prevent GVHD post haploidentical AHSCT. These grafts are enriched for NK and γδ T-cell receptor (TCR+) cells. Brief ex vivo activation of purified NK cells with interleukin (IL)-12, IL-18, and IL-15 [triple cytokines (TC)] has been shown to produce cells with a memory like function and significantly enhanced leukemia cytotoxicity. In our studies we depleted αβ TCR+ and CD19+ B-cells from healthy donors' peripheral blood mononuclear cells (PBMC) using microbeads; enriching the frequency of NK and γδ TCR+ cells. Following overnight TC incubation, we observed that these innate immune cells were activated based on phenotypic expression of CD69 and CD25. Further, we observed increased cytotoxicity of TC activated innate immune cells against NK sensitive and NK refractory leukemic cell targets. Further, the presence or absence of monocytes did not alter activation marker expression or in vitro cytotoxicity of innate immune cells. Additionally, we observed correlation between target cytotoxicity and mature activated NK phenotypes (CD56dim or CD56dim with co-expression of the activation markers CD69+ and/or CD25+). This approach of depleting T- and B-cells from PBMCs, combined with overnight TC activation, provides a novel cell population for donor lymphocyte infusion (DLI) post AHSCT.

Graft Versus Leukaemia (GvL): Identification & Characterisation of Alloreactive Antigens and Cognate T Cell Responses in Acute Myeloid Leukemia

Allogeneic stem cell transplantation (allo-SCT) is the most established, curative T cell cancer immunotherapy. The curative effect of allo-SCT relies on a graft-versus-leukemia (GvL) effect; but this can be difficult to dissociate from graft-versus-host disease (GvHD). For decades, the key unsolved challenge has been to identify common alloreactive peptide-human leukocyte antigen (HLA) complexes targeted by T cells that mediate GvL but not GvHD. We developed a workflow for unbiased identification of alloreactive antigens and analysis of cognate donor alloreactive T cells and their T cell receptors (TCRs; Fig 1). We studied sequential bone marrow and peripheral blood samples, collected over 15 years, from 12 high-risk AML patients who remain in continuous complete remission following allo-SCT (all >2 years). All patients had relapsed at least once pre-allo-SCT and/or were AML flow cytometry minimal residual disease (MRD)+ pre-allo-SCT. 7 had received donor lymphocyte infusions. No patients developed >1 grade GvHD. We performed whole exome sequencing of DNA from isolated patient T cells (patient germline), AML blasts and donor DNA to identify: (i) AML-specific coding variants; and (ii) germline coding single nucleotide polymorphisms (SNPs), mismatched between patient and donor. 98.7% of the mismatched variants were germline. More variants were identified in unrelated compared with sibling donors (mean: 5744 vs 3253 variants). To narrow the list of variants taken forward for functional studies, we applied two sequential filtering steps: (i) we restricted analysis to variants expressed in the patients' AML cells (determined by RNA-Seq). 28.7% of all mismatched coding variants were expressed in AML cells; and (ii) we used in silico prediction of HLA-binding affinities of peptides spanning variant amino acids to patient-specific HLA-I and -II using NetMHCpan 4.0 and NetMHCIIpan 3.0. To functionally identify alloreactive antigens, we then screened ~200-900 peptides per patient-donor pair (total 5300 peptides) for recognition by T cells present in patient peripheral blood collected 2-7 years after allo-SCT. This enriches for the identification of long-lived anti-AML responses at a time when patients were both infection- and GvHD- free. Response screening, using IFNγ ELISPOT assays, identified T cell responses to 24 germline, mismatched epitopes. In all cases, responses were only observed when the amino acid encoded by the patient SNP, but not the donor allele, was present in the peptide. The phenotype of the epitope-specific T cells was established by intracellular cytokine staining (ICS) and flow cytometry analysis, and the HLA restriction of each T cell response was determined using HLA-I, -DR, -DP and -DQ blocking antibodies. Of the 24 alloreactive peptides,19 elicited CD4+ HLA class II restricted responses and 3 were targeted by CD8+ class I restricted T cells. Two remain to be characterised (Fig 2). The population allele frequency of the germline variant SNPs targeted varies from 0.5% to 72% in the Caucasian population. Likewise, some of the restricting HLA alleles are very common (e.g. a HLA-DP allele presenting 3 epitope peptides is present in ~66% of the Caucasian population). ICS showed that epitope-specific CD4+ T cells produce Th1 cytokines (IFNg, TNFa) and degranulate (express CD107a) in response to peptide stimulation (Fig 2). Next, we performed single cell 5'RNA-seq with TCR-seq of epitope-restricted T cells (isolated via IFN-g catch assay) for all CD4+ epitope-specific T cells. Alloreactive CD4+ T cells have cytokine-producing and cytotoxic effector potential, expressing MIP1a and b, IFNg, granzyme B and FasL (Fig 2). Finally, lentiviral-based gene transfer of one of the candidate alloreactive epitope-specific TCRs into third party primary T cells confers the capacity to generate IFNg and TNFa in response to peptide stimulation, and to recognise primary AML cell-derived antigen in the presence of monocyte-derived dendritic cells (Fig 2). In vitro and in vivo killing assays are in progress. The high proportion of CD4+ T Cell responses identified by our pipeline is concordant with downregulation of HLA II expression on AML blasts at relapse post-allo-SCT, which is a common mechanism of immune evasion, found in one-third of patients. Our approach dissects the biology of alloreactive anti-AML GvL and GvHD responses and to manipulate them for therapeutic purposes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Associations of Minor Histocompatibility Antigens with Clinical Outcomes Following Allogeneic Hematopoietic Cell Transplantation

Introduction Donor T-cell recognition of tumor-presented minor histocompatibility antigens (mHAs) is thought to be a central mediator of the therapeutic graft versus leukemia (GvL) effect following allogeneic hematopoietic cell transplantation (alloHCT). In contrast, graft versus host disease (GvHD) develops when donor T-cells react to mHAs presented by normal recipient cells. While the role of mHAs in outcomes following alloHCT is recognized, it has not been well-characterized. It remains unclear whether GvL and GvHD are mediated by the cumulative effect of total mHAs in a donor-recipient pair (DRP) or individual immunodominant mHAs. We hypothesized that an increased mHA count and individual mHAs would be associated with lower relapse and disease-related mortality (DRM) and/or increased GvHD mortality. To this end, we predicted genome-wide mHA peptides in a large patient population and analyzed the associations between clinical outcomes and 1) the total number of predicted class I and class II mHAs, and (2) individual and groups of mHAs. Methods The study population consisted of 2249 European American DRPs in the DISCOVeRY-BMT dataset treated for Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) with alloHCT. To predict mHAs in a DRP, we translated nonsynonymous SNP alleles present in alloHCT recipients, but absent in their donor, into all possible peptides of varying lengths that would encompass the altered amino acid. To identify mHAs most likely to be immunogenic, peptides were filtered based on (1) source protein gene expression data in GvL and GvHD target tissues, including AML, liver, skin, and colon, from UCSC's Xena Dataset repository and (2) recipient HLA binding affinity from netMHCpan. To test the association of mHA count and individual mHAs with all-cause mortality (OS) and leukemia-free survival (LFS) we used cox proportional hazard models and competing risk models for DRM, treatment-related mortality (TRM), and GvHD mortality. Individual mHA analyses were stratified by HLA type to recognize that T-cell responses depend on HLA-mHA complexes. Models were adjusted for clinical covariates. Bonferroni correction for multiple testing was based on the number of independent SNPs. Results Patients with a class I mHA count greater than the population median had a 39% (HR=1.39, 95%CI 1.005,1.772, P=0.046) increase in hazard of GvHD mortality compared to those with a count less than the median. Patients with a class II mHA count greater than the population median had a decreased overall survival compared to those with a count less than the median (Fig. 1, P=0.029). In recipients with HLA-B*08:01, the class I mHA DLRCKYISL was associated with increased risk of GVHD mortality (HR=2.84, 95%CI 1.52,5.31, P=0.01). Within HLA-B*40:01, WEHGPTSLL was associated with decreased LFS (HR=1.94, 95%CI 1.27,2.95, P=0.044). Within HLA-C*03:04, STSPTTNVL was associated with increased risk of DRM (HR=2.32, 95%CI 1.5,3.6, P=0.008). In recipients with HLA-DRB1*15:01, the class II mHA YQEIAAIPSAGRERQ was associated with increased risk of TRM (HR=3.05, 95%CI 1.75,5.31, P=0.02). Two mHAs, WEHGPTSLL and STSPTTNVL, were found within the HLA haplotype B*40:01-C*03:04. Recipients with both mHAs (versus neither) had a greater hazard of 1-year all-cause mortality (HR=3.58, P=7.9×10-5) and DRM (HR=3.65, P=0.003) (Fig. 2). Patients with either mHA (versus neither) had greater 1-year all-cause mortality (HR=1.69, P=0.008) and increased DRM (HR=2.05, P=0.008) Conclusions The number of class I mHAs in a DRP was associated with increased GvHD mortality, which is consistent with our hypothesis. We also found two mHAs correlated with GvHD mortality and DRM which may represent increased GvH effects driven by these peptides. However, contrary to our hypothesis, an increased number of class II mHAs was correlated with greater risk of all-cause mortality that may be due to decreased LFS. Similarly, we found two mHAs correlated with increased DRM and decreased LFS. These two mHAs are found in an HLA haplotype where they exhibit a dose effect relationship with the risk of DRM and decreased OS. These findings may reflect an unexpectedly attenuated GvL effect that requires further investigation. Our study reports the first large scale investigation of the role of predicted mHA peptides in clinical outcomes following alloHCT. If validated, these results may be used to help guide clinical decision making. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Systematic Identification of Autosomal and Y-Encoded Minor Histocompatibility Antigens Reveals Predictors of Chronic Gvhd and Candidate GVL Targets

At the core of the therapeutic effect of HLA-matched allogeneic hematopoietic cell transplantation (allo-HCT) is T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient disparity at sites of single nucleotide gene polymorphisms (SNPs). Despite their crucial role in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD), only few mHAgs have been characterized to date. To systematically identify autosomal and Y-chromosome encoded HLA-I restricted mHAgs, we devised a computational pipeline based on: (i) comparison of whole exomes (WES) from paired donor-recipient DNA to define recipient-restricted exonic non-synonymous polymorphisms; and (ii) generation of expression filters by incorporating published single cell expression profiles from normal and malignant hematopoietic cells and GvHD target organs. For each GvHD tissue (skin, liver, GI, lung, oral mucosa and lacrimal gland), a large single cell database combining ≥1 independent datasets was created to cluster and annotate tissue-resident cell types. Cell-type specific genes were defined based on their total number of reads (CPM>5 in any cluster), thus creating an expression atlas capturing less frequent albeit biologically relevant cell types underestimated in bulk expression profiles. For GvL predictions, we built an AML-specific filter by defining AML-expressed genes from single cell and bulk data, and by subtracting genes with expression in non-hemopoietic tissues, per transcriptome and proteome GTEx databases. Candidate GvL/GvHD mHAgs prediction was performed by applying the HLAthena binding prediction tool on recipient-restricted SNPs expressed in GvL or GvHD tissues. To validate the pipeline predictive power, we used a panel of 95 HLA class I monoallelic lines generated from B721.221 cells, which we previously profiled by WES, RNASeq and immunopeptidomics. By assigning the B721.221 genome as surrogate 'HCT recipient’ and the reference hg19 genome as surrogate 'donor', we identified recipient-restricted SNPs, whose class I-restricted presentation we confirmed by mass spectrometry for a median of 5 (range 1-16) predicted epitopes/ allele. For 2 HLA-A0201 epitopes encompassing SNPs with an allelic frequency <0.001, we validated immunogenicity through stimulation of PBMCs from HLA-A0201 healthy donors (HDs) with corresponding synthetic peptides. For Y-encoded mHAgs, we predicted a median of 62 (range 24-107) epitopes/HLA-I allele from 9 genes expressed (>1 TPM) in ≥1 non germline tissue. Analysis of 12 male immunopeptidomes confirmed presentation of 3 known Y mHAgs and 26 novel predicted epitopes across 18 alleles. To estimate the fraction of immunogenic predicted binders, we tested all Y epitopes predicted across 3 common alleles (A0201, B1801, C0501). Of 215 Y peptides tested, 53 (~25%) elicited an antigen-specific T cell response in PBMCs from female HDs, as per dextramer-based detection of ≥0.2% CD8 cells. To test the prognostic potential of the mHAg load on cGvHD risk, we performed WES on 220 donor-recipient pairs treated with matched-related-donor allo-HCT for myeloid disease at DFCI between 2013-2020. Our logistic regression analysis used organ-specific NIH moderate/severe cGvHD as outcome; as covariates, we included tissue-specific mHAg load and standard HCT variables (disease type and prognostic risk, conditioning intensity, graft type, GvHD prophylaxis, female into male [F→M] transplant, relapse). The burden of lung-expressed mHAgs was correlated with lung GvHD occurrence (P=0.023). Liver GvHD was associated with F→M transplants (P=0.023). Subgroup analysis further showed liver GvHD to be correlated with the number of epitopes predicted from the RPS14Y gene (P=0.037). For identification of AML-specific mHAgs, a median of 31 (range 13-50) mHAgs/patient were predicted in our cohort from 269 genes preferentially expressed in AML, with a trend for a protective effect of their load against relapse (P=0.058). Of note, 39 SNPs generated putative mHAgs shared by >10 patients and covering 8 common HLA-I alleles in Caucasians, thereby representing potential targets for post-HCT immunotherapy. Overall, we report the generation of a robust analytic pipeline which enables the broadening of the repertoire of mHAgs available for donor selection and for predicting, monitoring or manipulating GvL and GvHD after allo-HCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Breadth of the Human Leukocyte Antigens Class I Immunopeptidome in Allogenic Hematopoietic Stem Cell Transplantation Outcomes for Myeloid Malignancy

Introduction Expected graft-versus-leukemia (GVL) effect, allogeneic hematopoietic stem cell transplantation (allo-HCT) is performed for myeloid malignancies. GVL is modulated by alloreactive T cells that recognize cancer cell antigens, but not all the mechanisms of GVL have been elucidated. Alloreactive T cells can also cause a harmful immune response, graft-versus-host disease (GVHD). Human leukocyte antigens (HLA) are the key molecules presenting antigens to T cells. They are highly polymorphic and vary widely among individuals. HLA allelic diversity causes differences in the immunopeptidome breadth. Recent reports suggest that a smaller immunopeptidome breadth may result in autoimmune disease such as aplastic anemia or type I diabetes, and that larger differences may enhance the antitumor effects of immune checkpoint inhibitors. This suggests that understanding the impact of the HLA immunopeptidome on allo-HCT, which may also be relevant to GVL and GVHD, might improve outcomes. Aim To examine the association between HLA class I immunopeptide diversity and outcomes in patients undergoing allo-HCT. Methods From 2006 to 2021, 145 patients with myeloid malignancies underwent initial allo-HCT at our institution. Of the 109 patients with evaluable allele data, 53 who received 8/8 allele-matched donors (acute myeloid leukemia, n = 40; myelodysplastic syndrome, n = 13) were analyzed. Graft sources were bone marrow (n = 31) and peripheral blood stem cells (n = 23). Donors were related (n = 23) and unrelated (n = 30). The conditioning regimens were myeloablative (n = 31) and non-myeloablative (n = 21). The disease status pre-transplantation was standard risk (n = 30) and advanced stage (n = 23). Differences in the immunopeptidome breadth between individuals were quantified as HLA evolutionary divergence (HED). HED was calculated using Grantham distance, which predicts amino acids from HLA allelic information and attempts to measure evolutionary distance based on the chemistry of the two amino acids. HLA allele data were drawn from clinical records and analyzed in the HLA laboratory (Kyoto, Japan) or SRL, Inc. (Japan). The calculator program was produced by Tobias Lenz et al. (https://hladiv.net/). The HED of each HLA class I: HLA-A, HLA-B, and HLA-C was calculated. Based on HED degree, overall survival (OS), cumulative relapse rate (CIR), acute/chronic GVHD, and non-relapse mortality (NRM) were evaluated. Result The median HED (range) was HLA-A, 10.34 (0.00-13.70); HLA-B, 7.50 (0.00-14.33); and HLA-C 4.66, (0.00-7.60), respectively. Based on their median HED, patients were divided into HED high or HED low groups for HLA-A, HLA-B, and HLA-C. On univariate analysis, patients with HED low for HLA-A had better 2-year OS compared to HED high for HLA-A (80.4%; 95%CI, 55.6%-92.2% vs. 42.0%; 95%CI, 23.9%-59.1%; p = 0.025) and better 2-year CIR (14.3%; 95%CI, 3.4%-46.1% vs. 59.5%, 42.2%-77.5%; p = 0.0027) (Figure 1). HED levels of HLA-B and HLA-C were not related to OS (p = 0.501, and p = 0.972, respectively) or CIR (p = 0.257 and 0.796, respectively). NRM was not associated with HED levels for any of the HLA. Multivariate analysis found HED low for HLA-A to be a favorable risk factor for OS (HR, 0.46; 95%CI, 0.22-0.95; p = 0.035) and CIR (HR, 0.22; 95%CI, 0.07-0.69; p = 0.0092) when adjusted for HLA-B, HLA-C, donor, graft, conditioning, and disease status. Next, the association between the HED level of each HLA class I and GVHD was analyzed. In acute GVHD, grades 2-4, after adjusting for graft, donor, and conditioning, HED high of HLA-B (HR, 4.66; 95%CI, 1.23-17.7; p = 0.024) and HED low of HLA-C (HR, 3.98; 95%CI, 1.16-13.6; p = 0.028) were risk factors but not for HLA-A. In chronic GVHD, multivariate analysis found HED high for HLA-C was a risk factor (HR, 4.77; 95%CI, 1.13-20.2; p = 0.034). Also, HED low for HLA-A tended to increase chronic GVHD ((HR, 2.61; 95%CI, 0.95-7.14; p = 0.063) (Table 1). Conclusion HED low for HLA-A was a favorable risk factor for OS and CIR, possibly because chronic GVHD tended to increase with HED low of HLA-A. HED levels for HLA-B and HLA-C did not contribute to OS and CIR but did influence acute and chronic GVHD. Since immunopeptidome significance may differ for each HLA class I, consideration of HED level during the donor selection may improve the safety and efficacy of allo-HCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1/1b Multicenter Ascending Dose Study to Evaluate the Safety of HA-1 Minor Histocompatibility Antigen-Reactive TCR-Modified T Cells (BSB-1001) in Patients Undergoing HLA-Matched Allogenic Hematopoietic Stem Cell Transplant (alloSCT) for MRD+ AML or ALL or Poor/Very Poor Risk MDS

Background: AlloSCT can cure patients with acute leukemia and high-risk myelodysplastic syndromes (MDS). However, post-transplant relapse remains the greatest single cause of post-transplant death, occurring in up to 80% of high-risk patients. In HLA-matched alloSCT, alloreactive donor T cells target minor histocompatibility antigens (miHAs), which are peptide products of coding polymorphisms that distinguish recipients from donors. These alloreactive T cells can target leukemic cells, mediating the graft-vs-leukemia (GVL) effect; unfortunately, they also can cause graft-vs-host disease (GVHD). Pre-clinical mouse models (Blood, 2011) demonstrate that CD8+ T cells that target miHAs with expression limited to hematopoietic cells can mediate GVL without causing GVHD. To translate this concept for how to safely augment GVL to the clinic, BlueSphere Bio (BSB) used its high throughput T cell receptor (TCR) discovery and characterization platforms to clone a panel of TCRs reactive against the HLA-A*02-restricted miHA, HA-1, which is expressed mostly in hematopoietic cells. These receptors were cloned from a multiparous A*02:01 HA-1- woman who was immunized to HA-1 through pregnancy. A high affinity TCR specific for HA-1 and not reactive against allogeneic HLA molecules was chosen to create a cellular therapeutic designed to mediate GVL and enhance donor engraftment with a lower risk for GVHD, relative to polyclonal donor T cells. The anti-HA-1 T cell product (BSB-1001) is composed of donor CD8+ T cells transduced with a lentivirus vector encoding the anti-HA-1 TCR and the RQR8 tag, which expresses an epitope from CD20, thereby enabling the in vivo killing of BSB-1001 cells with rituximab. CRISPR-cas9 editing of the CD8+ T cells deletes the endogenous TCRα and β chains with >95% efficiency. This increases expression of the TCR and reduces risk of GVHD caused by native TCRs, as well as possible off-target reactivities generated by pairing of endogenous and introduced TCR chains. BSB-1001 cells specifically kill HLA-A*02:01, HA-1+ targets, with no activity against cells that are HLA-A*02:01+ but HA-1-, or cells that are HA-1+ but are HLA-A*02:01-. BSB-1001 is also not reactive against a panel of B lymphoblastic cell lines (LCL) expressing allogeneic HLA molecules. Importantly, efficient target killing is seen at effector:target ratios as low as 0.1, and maintained after several rounds of ex-vivo expansion following target cell challenge, a likely predictor of potency and persistence in vivo. Study Design: The first in human Phase 1/1b study is a multicenter, open-label, single dose study with a 3+3 ascending design with the option to expand accrual at a promising dose. Subjects must have ALL, AML or high-risk MDS with evidence of disease after the last therapy, be HLA-A*02:01 HA-1+ and have a 10/10 HLA-matched HA-1- related or unrelated donor. GCSF-mobilized donors will undergo leukapheresis. From this product, a small fraction of cells will be removed for BSB-1001 manufacturing; the remainder will undergo CD34-selection and cryopreservation until SCT. After BSB-1001 manufacturing is complete, patients will undergo ablative conditioning (choice of three regimens), followed by infusion of the CD34-selected cells and then BSB-1001. Patients will not receive anti-T cell antibodies and will receive no prophylactic pharmacologic immunosuppression. Subjects must otherwise be suitable for ablative conditioning using standard criteria. The primary objective is to establish a recommended dose of BSB-1001 based on its dose-limiting toxicities and the magnitude of post-infusion BSB-1001 expansion. The primary endpoint is determining the incidence of treatment-emergent adverse events, including dose-limiting toxicities. Additional endpoints include rates of engraftment, incidences of cytokine release syndrome (CRS) and acute and chronic GVHD, quantitation of malignant hematopoiesis and lineage-specific donor chimerism. CRS and GVHD, should they occur, will be treated per institutional standards. Rituximab can be administered to kill BSB-1001 cells in case of refractory CRS or GVHD. Enrollment is anticipated to begin in 1Q2023. BSB is also developing panels of TCRs targeting additional relatively hematopoietically-restricted miHAs with prioritization based on the frequencies of the restricting HLAs and the immunogenic/nonimmunogenic miHA alleles.

MO-TRANS: A Phase 2b Placebo-Controlled Study of Mocravimod As Adjunctive and Maintenance Treatment in AML Patients Undergoing Allogeneic Hematopoietic Cell Transplant

Introduction: Mocravimod is a sphingosine 1-phosphate receptor (S1PR) immunomodulator that blocks lymphocyte egress from lymphoid organs via its phosphorylated active metabolite. Allogeneic hematopoietic cell transplant (allo-HCT) is a common treatment in AML, which is indicated as consolidation therapy for intermediate/high risk AML. The therapeutic benefit includes graft-versus-leukemia (GvL) effect of the donor's T cells against the leukemic cells of the patient. One of the major risks of allo-HCT is graft-versus-host disease (GvHD) and is regarded as the off-target effect of the desirable GvL effect, in which the donor T cells respond to and attack normal host cells other than the leukemic cells. The major causes of post-transplant mortality, morbidity and quality of life impairment, remain disease relapse and GvHD. S1PR modulation does not interfere with T cell cytotoxicity, hence function is maintained, and anti-leukemia response remains potent. Since mocravimod is not immunosuppressive, alloreactive T cells can retain anti-leukemia reactivity to boost GvL while being sequestered in the lymphatic organs and thus not promoting peripheral GvHD. Therefore, treatment with mocravimod as an adjunctive and maintenance therapy to allo-HCT may trigger two positive effects: 1) increase the GvL effect through the eradication of residual malignant cells by alloreactive T cells sequestered in the lymphoid tissues such as lymph nodes, the bone marrow, and the spleen white pulp, and 2) reduce the GvHD because alloreactive T cells are retained in the lymphoid tissues and thus do not migrate into peripheral tissues. Both effects will ultimately result in improving overall survival (OS). Based on the encouraging results of a phase Ib study with mocravimod in the allo-HCT setting, a phase 2b trial is planned. Study Design and Methods: In this multicenter, double-blind, phase 2b study, approximately 249 pts will be randomized 1:1:1 to receive mocravimod 1mg or mocravimod 3mg or placebo. Patients will be stratified by the complete remission status (CR1 versus CR2), the treatment used for GvHD prophylaxis cyclosporine A (CsA) versus tacrolimus (TAC), and the measurable residual disease (MRD) status (MRD pos versus MRD neg). Key eligibility criteria include age (> 18 years and < 75 years), diagnosis of AML (excluding acute promyelocytic leukemia) with European LeukemiaNet (ELN) high risk disease in CR1, intermediate risk in CR1 if MRD pos, or AML of any risk in CR2. Patients need to be planned to undergo allo-HCT, with either fully matched related or unrelated donor. The planned use of GvHD prophylaxis needs to be either methotrexate (MTX) plus CsA or MTX plus TAC. Treatment in all arms will consist of 28-day cycles with daily administration of either 1mg mocravimod or 3mg mocravimod or placebo. Treatment will start 2 days before conditioning treatment and will be ongoing for 12 cycles or until relapse, unacceptable toxicity, or consent withdrawal. Patients will be observed for overall survival after end of treatment. Primary efficacy endpoint is relapse free survival (RFS), which is calculated as the time from randomization to relapse or death. It will be assessed once 46 events are reached in one mocravimod arm and the placebo arm and will be observed by an unblinded independent data monitoring committee. The first key secondary objective is to assess the effect of mocravimod relative to placebo on Overall Survival (OS). A second key objective is the comparison of the effect on RFS between the second mocravimod arm and the placebo arm and it will be assessed once the primary endpoint analysis timepoint will be reached. The FDA activated the IND #127312 on 22 Apr 2022, and five European Health Authorities approved the study to date: France, the UK, Spain, Switzerland and Germany. Enrollment is expected to begin in Q3, 2022. The ClinicalTrials.gov identifier is NCT05429632.

Expansion of a CD8+ Temra Population and Activating T-Cell Interactions Characterize the Graft Versus Leukemia Response in Relapsed AML

Graft-versus-leukemia (GvL) activity underlies the curative potential of allogeneic hematopoietic stem cell transplantation. Its effectiveness is well-exemplified by donor lymphocyte infusion (DLI), an established adoptive cellular therapy for treatment of relapsed disease. Whereas ~80% of chronic myelogenous leukemia (CML) patients achieve durable remissions to DLI, responses for acute myelogenous leukemia (AML) are only 15-20%. We hypothesized that systematic characterization of immune cell composition and cellular interactions within the marrow tumor microenvironment in relation to DLI would illuminate the molecular basis of the disease-specific susceptibility to GvL. Given the heterogeneous nature of AML, we suspected AML response to comprise more complex leukemic-immune cell interactions than in CML. To these ends, we generated 5’ single-cell RNA- and TCR-sequencing (scTCRseq) and cell surface marker profiling (scCITE-seq) on 24 bone marrow biopsy samples collected before and after DLI from 9 patients with relapsed AML (5 responders [R], 4 nonresponders [NR]). We merged this dataset with our previously reported CML dataset (14 patients, 8 R, 6 NR [PMID: 34758319]) and batch corrected these data with scVI to obtain a total of 414,000 high-quality single-cell transcriptomes for analysis. Evaluation of the immune cell subset proportions over time revealed 10 populations with distinct dynamics in AML responders to DLI but not CML, while 3 other populations showed similar dynamics across diseases. Both CML and AML DLI response were associated with pre-treatment enrichment of an NK cell population highly expressing XCL1, XCL2, CCL3, CCL4, CCL4L2, and CCL5, consistent with a tumor-infiltrating expression profile. Unique to CML (i.e. not observed in AML), in addition to post-DLI enrichment of the TPEX population previously identified, we found a γδ-like T cell population and a classical dendritic cell population exhibiting post-DLI enrichment associated with response to DLI. In AML, response to DLI was most consistently associated with expansion of a CD8+ T cell population (6.03% of total pre-DLI cells vs 18.31% post-DLI for R [p=0.016] compared to 8.68% pre-DLI vs 9.55% post-DLI for NR [p=0.454]) and a naïve B cell population (4.02% pre-DLI vs 11.68% post-DLI for R [p=0.002] compared to 5.42% pre-DLI vs 5.17% post-DLI for NR [p=0.471]). The CD8+ T cell population was characterized by high expression of cytotoxicity genes (e.g. GZMA, GZMB, GZMH, GZMM, PRF1, KLRG1, TOX, EOMES, TBX21) and was most consistent with a T effector memory re-expressing RA (TEMRA) profile, supported by high CD45RA expression by scCITE-seq. This population also highly expressed ZNF683, which encodes the transcription factor Hobit, known to regulate cytotoxicity in CD8+ T cells. Differential gene expression between AML R and NR TEMRA cells revealed enriched expression of ZNF683, GZMH, NKG7, CCL5, and CXCR4 in R and the exhaustion marker TIGIT and the senescence marker KLRG1 in NR. scTCR-seq of the TEMRA subset demonstrated clonal expansion and differentiation into a more proliferative expression profile post-DLI. Among AML cases, 2-20% of the post-DLI repertoire of responders originated from the DLI product while no TCR from the DLI product was detected in the single NR patient available for analysis. We applied predictive interaction modeling among all 50 cell type pairs using CellPhoneDB (PMID: 32103204), which utilizes a publicly available curated database of receptor-ligand interactions. Predicted interactions unique to R between CD8+ TEMRA and AML leukemia cells included CD226-PVR, CD6-ALCAM, and CD46-JAG1, which have been associated with T cell activation. Interactions unique to NR between CD8+ TEMRA and AML leukemia included the inhibitory interactions TIGIT-NECTIN and PTPRC-MRC1. These interactions were not observed in CML patients in either R or NR between CD8+ TEMRA and CML cells, suggesting disease-specific response and resistance mechanisms. Here we show that response to DLI in AML is associated with largely distinct expansion of immune cell populations compared to CML, including not only T cells but an orchestration of B, NK, and myeloid cell subsets. AML responders were uniquely characterized by expansion of a TEMRA subset marked by high expression of the pro-cytotoxic gene ZNF683. Further investigation is needed to evaluate the role of this population in mediating GvL response.

Transcription Factor Fli-1 Regulates T Cell Immunity and Tolerance in Gvh and GVL Responses

The E26 transformation-specific (ETS) transcription factor (TF) family member Friend Leukemia Virus Integration 1 (Fli-1) is linked to the oncogenesis of virally induced leukemia and Ewing's Sarcoma, as well as the development of autoimmune diseases including systemic lupus erythematosus and systemic sclerosis. A recent report presents compelling evidence that Fli-1 restrains CD8 effector T cell differentiation and protective immunity against cancer. How Fli-1 regulates primary T cell differentiation and function remain largely elusive. In this study, we investigated the role of Fli-1 in T-cell pathogenicity of graft-versus-host disease (GVHD) and validated Fli-1 as a therapeutic target. Using conditional knock-out models featured as homozygous (Fli-1KO) or heterozygous (Fli-1HET) deficiency of Fli-1 in T cells, we found that Fli-1 dynamically regulates different T-cell subsets in allogeneic responses and pathogenicity in the development of acute and chronic GVHD. Compared to Fli-1KO or Fli-1WT T cells, Fli-1HET T cells induced the mildest aGVHD and cGVHD supported by lowest Th1 and Th17 differentiation (Fig. 1 A-B). In addition, Fli-1HET T cells were able to maintain a strong graft-versus-leukemia (GVL) effect while inducing significantly attenuated GVHD. Fli-1-deficient CD8+ T cells expressed comparable levels of cytolytic molecules, reduced levels of exhaustion markers, and differentiated more into memory precursor effector cells compared to WT CD8 T cells after been transferred into allogeneic recipients. To further understand how Fli-1 regulates T-cell gene transcription and heterogeneity, we performed scRNA seq analysis of donor T cells, including Fli-1WT, Fli-1HET and Fli-1KO, isolated from recipient spleens. The scRNA seq analysis on the donor T cells indicated a major difference in the transcriptional regulation by Fli-1 in CD4 versus CD8 T cells and Fli-1 gene dose dependent modification in transcriptional pathways in Fli-1Het vs Fli-1KOT cells. Fli-1 deemed to play a distinct role in regulating gene transcription in CD4 versus CD8 T cells, in that more activation and less exhaustion gene pathways were enriched in CD8 Fli-1 deficient cells, while fewer genes in Th1/Th17 pathogenic pathways were enriched in CD4 Fli-1 deficient cells (Fig. 1 C-D). Overall, Fli-1 deficiency increased gene enrichment in OXPHOS pathway in both CD4 and CD8 T cells, and reduced gene enrichment in glycolysis pathway majorly in CD4 T cells (Fig. 1 E-F). Consistent with the lowest pathogenicity of Fli-1Het T cells for GVHD induction, Fli-1Het CD4 T cells had the lowest Th1/Th17 pathway gene enrichment and TCR pathway downstream gene expression. Camptothecin, topotecan or etoposide exhibited action as potent Fli-1 inhibitors at low-doses. For translational purpose, we tested these drugs and found that each of these drugs significantly attenuated GVHD severity while preserving the GVL effect. The efficacy of camptothecin or etoposide at low doses was dependent of Fli-1 expression on donor T cells. Mechanistically, blocking Fli-1 suppressed activated effector T cells while sparing Tregs and had no side effect on hematopoietic stem cells. Importantly, using the same treatment schedule as post-transplantation cyclophosphamide (PTCy), two doses of camptothecin or etoposide administered on day 3 and 4 were sufficient for the prevention of GVHD and leukemia relapse, in contrast to GVHD mortality in some of those treated with bendamustine and leukemia relapse in some of those treated with PTCy or cyclosporine (Fig. 1 G-I). In addition, the beneficial effect of camptothecin was extended in GVHD induced by human T cells in a xenograft model. In conclusion, we provide the first 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 GVL effect. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haploidentical Hematopoietic Stem Cell Transplant Plus Add-Back NK Cells in Pediatric High-Risk ALL

Introduction: Natural killer (NK) lymphocytes are cells of innate immunity that protect against tumor and infections. NK cells recognize self and non-self HLA class I ligands, are regulated by a balance between activator and inhibitory signals through surface receptors, and can interact with HLA class I molecules on target cells. Killer-cell Immunoglobulin-like Receptors (KIRs) are inhibitory molecules expressed on NK cells and belong to the immunoglobulin superfamily. KIRs function to "educate" NK cells in effective defense against unhealthy self/non-self cells and tolerance against healthy self cells. NKs are thought to play a key role in preventing early relapse of hematologic malignancies, virus infection and graft versus host disease (GvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT).In B-/T-cell alpha/beta depleted haplo-HSCT, donor-versus-recipient allo-reactive NK cells can exert an efficient graft versus leukemia (GvL) effect, mediated by inhibitory KIRs where the HLA ligand is missing on recipient cells. Donor NK cells after haplo-HSCT are "immature" and not effective as the mature NKs. These immature NK cells are CD56bright, are derived from CD34+ stem cells, and mature to a CD56dim phenotype. The mature CD56dim NK cells are characterized by acquisition of KIRs, CD16 and CD57 in mature stages, which have functional significance in targeting and medicating their cytolytic activity. Based on these considerations, we performed haplo-HSCT with donor NK cell add-back to provide mature, functional immunological surveillance against tumor (MRD) and virus reactivation, which can frequently occur in manipulated haplo-HSCT for pediatric acute lymphoblastic leukemia (ALL). Methods: We evaluated the alloreactive effect of donor-derived NK cells in three pediatric high-risk ALL who underwent B-/T-cell alpha/beta depleted haplo-HSCT followed by donor NK infusion. Patients' characteristics are summarized in the Table. All 3 patients were male; median age was 13 months. Patients 1 and 2 had infant ALL, and patient 3 had refractory-relapsed (r/r) ALL. All patients received a median of 3 lines chemotherapy. Patient 1 received a prior allo-HSCT. Patients 1 and 2 received chemo-based conditioning regimens consisting of thyotepa, and fludarabine; In addition, patient1received treosulfan, and patient 2 received busulfan. Treosulfan was given to patient 1, due to less toxicity profile given prior allo-HSCT. Patient 3 received Total Body Irradiation (TBI), thyotepa, and fludarabine. All patients received rituximab and letermovirfor EBV and CMV prophylaxis, respectively, for all patients. Mother was Donor for all patients. Median CD34+, CD19+, CD3+-alpha/beta cell dose was 8.8, 0.05, 0.41 x 106/kg, respectively. NK cell allo-reactivity was tested by evaluating KIR genotyping and KIR/KIR-ligand mismatch in graft-versus-host direction. All three patients present KIR/KIR-ligand mismatch. Results: Engraftment was day +13, +14 and +19 for ANC, and +14, +18 and +17 for PLT (for both >20K and >50K), for patients 1, 2 and 3, respectively. NK cell number were monitored on day +7, +14, +21 and +28 (data pending). Patients received NK cell infusion on day 30 +/- 2 days. No infusion reactions were observed. MedianNK infused were 256x106/kg. None of the patients developed CMV, EBV or ADV reactivation. Patient 2 developed Grade 2 acute GvHD (skin) treated with steroids. No Grade GvHD 3-4 was observed. Median follow up was 58 days. All patients had full donor chimerism at day 30 ±2. All patients are currently in hematological CR; patient 1 converted to positive MRD at nine months from transplant. Conclusion: Alloreactive NK cell add-back infusion after B-/T-cell alpha/beta depleted haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation. These NK cells may improve efficacy by enhancing GvL and could contribute to maintain the "disease control" through immunologic surveillance in high-risk ALL. Indeed, NK killing, which is higher in mature NK cells, depends on acquisition of sequential phenotypic maturation. Thus, infusion of mature donor NK cells may help to maintain control of MRD and virus reactivation through immunologic surveillance. Although our preliminary observations seem to be promising, this hypothesis should be confirmed in clinical trial of a larger number of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Higher Proportions of Monocytic-Myeloid-Derived Suppressive Cells (mMDSC) before Allotransplant Predict Relapse in Acute Leukemia Patients

Introduction: Myeloid-derived suppressive cells (MDSC) are an heterogeneous group of immature myeloid cells with strong immunosuppressive activities which made them of particular interest in the context of allogeneic stem cell transplant (Allo-SCT). In mice models, MDSC modulate the function of alloreactive T cells and prevent graft versus host disease (GVHD) without impairing graft versus leukemia (GVL) effect. Data are limited in humans, but higher proportions of MDSC in the graft seem to protect against both acute and chronic GVHD. However, the influence of circulating MDSC is not fully explored especially regarding their impact on post-transplant GVL effect and survivals. Here we focused on the role of peripheral levels of monocytic-MDSC (mMDSC) in a cohort of adults with acute (both myeloid or lymphoid) leukemia (AML or ALL) having received Allo-SCT. Methods: This prospective monocentric study aimed at documenting the impact of the percentage of mMDSC before and after allo-SCT in terms of incidence of acute and chronic GVHD, overall (OS) and leukemia-free (LFS) survivals, (grade 3/4 acute or extensive chronic) GVHD-free relapse-free survival (GRFS), relapse and non-relapse mortality (NRM) in a cohort of ALL or AML patients (pts). Inclusion was performed after informed consent at diagnosis as part of a more global study (Abstract ASH 2021 # 3465), pts who were ultimately transplanted being included in this sub-group analysis. mMDSC were defined by the minimal CD14+/CD11b+/CD33+/HLA-DR-/low immunophenotypic pattern, at variance from granulocytic MDSC that lack CD14 and express bright CD15. Cells were assessed in a lysis-no-wash flow cytometry technique using peripheral blood collected on EDTA before conditioning then at day+60 post-transplant. Data acquisition was performed on a Navios® flow cytometer (Beckman Coulter, Miami, FL). mMDSC were expressed as a percentage (%) of total nucleated cells defined as CD45+. The study was registered at the French Commission Nationale de l'Informatique et des Libertés as CNIL 2016-038 and approved by the Ethic Review Board of Nantes University Hospital. Results: Among 73 AML and 14 ALL pts included at diagnosis, 47 and 9, respectively, were ultimately allografted between February 2018 and October 2021. Pts characteristics are provided in Table 1A. The median follow-up for alive pts from Day 0 was 24 months (range: 8-48). Considering the whole cohort, 2-year OS, LFS and GRFS were 61.9+6%, 55.9+7% and 32.4+6%, respectively, while 2-year NRM and Relapse incidence were 11+4% and 29.6+6%. In pre-graft, 46 pts were evaluated at a median of 20 days from Day 0 (range: 6-83). The median % of mMDSC was 0.45% (range: 0-11.85) with no differences between AML and ALL (p=0.32). The best cut-off assessed by ROC analyses was 0.47%. According to this value, the incidence of cytologic relapse at 2 years was significantly higher in pts with >0.47% of mMDSC (14.5+8% vs 44.4+11%, p=0.02, Figure 1) while no impact on survivals, NRM or GVHD was observed. Of note, the incidence of relapse was similar between AML and ALL (29% vs 37.5%, p=0.95). In multivariate analysis (Table 1B), higher pre-graft mMDSC % remained associated with higher risk of relapse (HR: 3.71; 95%CI: 1.05-13.09, p=0.04). At day+60, 42 pts were evaluated. The mMDSC median level was 0.52% (range: 0-5.03) with no differences between AML and ALL (p=0.32). No best cut-off was determined. Survivals were similar between pts with ≤ or >0.52% of mMDSC at this time. It was also the case for incidences of relapse, NRM and chronic GVHD. However, the incidence of severe acute GVHD (grade 3/4) was significantly and paradoxically higher in the group of pts with >0.52% mMDSC (33% vs 5%, p=0.04) suggesting a possible amplification of immunosuppressive mMDSC as a reaction to GVHD since the median time of GVHD grade 2-4 occurrence was 30 days (range: 20-123) post-transplant. Of note, the kinetics (increases or decreases of mMDSC %) between pre-graft and D60 had no influence on outcomes. Conclusion: This prospective study demonstrates a negative impact of higher proportions of mMDSC before allotransplant in leukemic pts. This higher proportion is predicting relapse, suggesting a protective role and tolerance of tumoral cells by mMDSC themselves. This paves the way to therapeutic intervention to decrease the incidence of relapse in such pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pilot Trial of IFN-γ and Donor Lymphocyte Infusion to Treat Relapsed AML and MDS after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Allogeneic stem cell transplantation (alloSCT) can cure high-risk myeloid malignancies. While non-relapse mortality has declined over the last several decades, more than half of alloSCT recipients die from recurrent leukemia, and little progress has been made in preventing or treating it. Only 10-20% of subjects with relapsed leukemia post-alloSCT become long-term survivors despite receiving donor lymphocyte infusions (DLI) or a second alloSCT. In mouse models, we identified that optimal CD4- and CD8- mediated graft-versus-leukemia (GVL) responses require interferon-gamma receptor (IFN-γR) expression on myeloblastic leukemias (Matte-Martone and Shlomchik, J Clin Invest. 2017). Other groups reported that IFN-γ restores HLA expression on myeloid blasts from patients who relapsed post-alloSCT. These data support the hypothesis that IFN-γ sensitizes myeloid leukemia cells to alloreactive T cell-mediated killing. We conducted a phase I trial to evaluate whether a standard dose of IFN-γ can safely be administered to patients with relapsed acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) post-alloSCT (NCT04628338). Methods: This investigator-initiated trial aimed to evaluate 1) the feasibility and safety of IFN-γ post alloSCT as monotherapy and combined with DLI; and 2) whether the standard dose IFN-γ is sufficient to upregulate IFN-γ-inducible molecules in myeloblasts in vivo. Major eligibility criteria included: 1) HLA-matched alloSCT recipient with relapsed AML/MDS; 2) measurable bone marrow (BM) myeloblasts (0.5-30%) by flow cytometry, 3) no increases in systemic immunosuppression for 4 weeks; and 4) no history of grade IV graft-versus-host disease (GVHD). The study had two stages: IFN-γ monotherapy (stage 1) with a fixed dose of IFN-γ (ACTIMMUNE®, Horizon Therapeutics) 100 mcg subcutaneously 3 times per week (outpatient self-injection) for 4 weeks and subsequent DLI combined with IFN-γ (stage 2) for 8 more weeks. Clinical responses were evaluated by BM pathology and donor chimerism at 4 weeks (pre-DLI), 12 weeks, and 6 months after the first dose of IFN-γ. BM, peripheral blood mononuclear cells, and plasma were collected before, 48-60 hours after IFN-γ, pre-DLI, and post-DLI. Phosphorylated STAT1 and HLA-ABC/DR, ICAM-1, and PDL-1 expression on BM leukemia blasts were measured by flow cytometry. Results: Four patients with post-transplant relapsed AML/MDS were enrolled (Table 1). All patients tolerated IFN-γ monotherapy well with anticipated injection site reactions, flu-like symptoms, and stable cytopenia. Three patients who received DLI achieved complete molecular and cytogenetics remissions and conversion to full donor chimerism coincident with steroid-sensitive GVHD. One of the three responders with donor T cell chimerism of >90% pre-treatment achieved a CR with IFN-γ monotherapy before DLI (Figure 1). All responders are currently alive and in complete remission without requiring additional anti-leukemia maintenance therapy (median follow-up 283 days; range 164-371). One patient who did not have available DLI and had poor donor T cell chimerism (8%) died from disease progression on day 139. BM blasts from pre-treatment phosphorylated pSTAT1 in response to in-vitro IFN-γ, demonstrating intact upstream IFN-γ R signaling. Blasts from all four patients increased HLA-DR expression 48-60 hours after IFN-γ therapy in vivo. We are performing single-cell RNAseq of BM cells to better understand IFN-γ -induced transcriptome changes across different maturation stages of leukemia blasts and donor-derived normal hematopoietic cells. Conclusion: IFN-g was safe in this pilot cohort of subjects with relapsed AML/MDS post-alloSCT. To our knowledge, this is the first time that IFN-γ has been tested on such subjects. In vivo, IFN-γ induced the predicted changes in myeloblasts. Most importantly, we observed durable CRs and conversion to 100% donor hematopoiesis with the IFN-γ/DLI combination, which is very promising for such poor-risk patients. These data provide a strong rationale for developing a phase II trial of IFN-γ/DLI for post-alloSCT relapse, and we are currently planning such a study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Time-Sequential Change of the Predictive Power of Peripheral Blood WT1mRNA Levels after Allogeneic Stem Cell Transplantation for Myeloid Neoplasm Relapse and Development of a Dynamic Relapse Prediction Model

Introduction:Wilms tumor 1 messenger RNA (WT1mRNA) is detected in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), and is reportedly a useful minimal residual disease (MRD) marker. Allogeneic stem cell transplantation (allo-SCT) is the established cellular immunotherapy with a graft-versus-leukemia (GVL) effect and eliminates residual leukemia cells via the alloimmune response. The GVL effect is generally considered to occur after immune reconstruction. Therefore, the predictive power of WT1mRNA as an MRD marker for relapse may differ between the early and late periods after allo-SCT because the GVL effect is not expected in the early period without enough immune reconstruction. However, no studies have reported whether the predictive power of WT1mRNA for relapse changes in a time-sequential manner after allo-SCT. Hence, we investigated the relationship between WT1mRNA as a time-dependent variable and days after allo-SCT for AML/MDS relapse using a dynamic landmarking approach, and developed a dynamic relapse prediction model using the WT1mRNA level and measurement timepoint after allo-SCT. Methods: We retrospectively analyzed AML and MDS patients who received allo-SCT at Osaka Metropolitan University Hospital, and whose WT1mRNA levels were measured at least once from January 2008 to April 2021. WT1mRNA levels in peripheral blood were measured using an "Otsuka" WT1mRNA assay kit (Otsuka Pharmaceutical Co., Ltd.). Relapse was defined as hematological relapse or salvage chemotherapy intervention and/or donor lymphocyte infusion. To evaluate time-sequential changes in the predictive power of WT1mRNA for relapse and to develop a dynamic relapse prediction model, dynamic landmarking analysis was performed, which was reported by Hans C. van Houwelingen et al. The probabilities of the 1 year cumulative incidence of relapse (CIR) post-landmark timepoint were estimated with the Kaplan-Meier method. The Cox proportional hazard regression model was used to calculate the time-sequential hazard ratio for 1 year CIR not only post-landmark but also post-any timepoints after allo-SCT, and to develop a dynamic relapse prediction model. p < 0.05 was considered significant. Results: We analyzed 265 patients (AML: 197 and MDS: 68) and 2,539 WT1mRNA tests. Median patient age was 46 years, 55% of patients achieved complete remission at the time of allo-SCT, and the 2 year relapse-free survival rate was 46%. First, patients were divided into two groups, namely, those with WT1mRNA levels higher and lower than 200 copies/μg RNA, which is a previously reported cutoff value. Figure 1 shows a summary plot of the 1 year CIR of the two groups post-landmark timepoint. The 1 year CIR decreased over time after allo-SCT in the lower WT1mRNA group but not in the higher WT1mRNA group. The difference in 1 year CIR between the two groups increased over time after allo-SCT. Moreover, the hazard ratio of log WT1mRNA levels for 1 year CIR post landmark timepoints after allo SCT increased over time, and there was a significant interaction between WT1mRNA levels and landmark time (p < 0.001). These findings indicate that the predictive power of WT1mRNA levels for relapse increases over time after allo-SCT. Second, we developed a dynamic relapse prediction model that can predict the 1 year CIR post-any timepoint after allo-SCT based on individual post-transplant WT1mRNA levels and measurement timepoints. Figure 2 shows a time series of predictive values for 1 year relapse post-any timepoints derived from this prediction model, which assumed that WT1mRNA levels remain constant over time after allo-SCT. Conclusions: We demonstrated that the predictive power of peripheral blood WT1mRNA for relapse increases after allo-SCT over time. This finding implies that MRD in the early phase after allo-SCT is more likely to disappear owing to the GVL effect after immune reconstruction compared to one in the late phase. Transplant clinicians should pay attention to the difference in predictive power for relapse depending on the period when they observe elevated WT1mRNA levels. Dynamic landmark analysis also enabled prediction of continuous 1 year CIR post-any timepoint after allo-SCT by inputting the individual WT1mRNA level and measurement timepoint. Dynamic relapse prediction will offer useful information for clinical decision making. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Graft-Versus-Leukemia Effect Is Independent from Graft-Versus-Host Disease Following Ptcy-Based HLA-Haploidentical Transplantation

Background. The potential link between graft-versus-host disease (GVHD) occurrence and acute myeloid leukemia (AML) relapse in patients given human leukocyte antigen (HLA)-haploidentical allogeneic hematopoietic stem cell transplantation (Haplo-HCT) with post-transplant cyclophosphamide (PTCy)-based GVHD prophylaxis has remained debated. Here, we addressed this issue in a large cohort of AML patients with active AML at transplantation, a group of patients who mainly depends on graft-versus-leukemia (GVL) effects for tumor eradication. Methods. Population selection criteria for this study consisted of adults (>= 18 years old), first Haplo-HCT between 2010 and 2020 using PTCy-based GVHD prophylaxis, and primary refractory or relapsed AML (i.e. all patients had active disease at the time of transplant conditioning initiation). Twelve serial Landmark analyses from day 30 to day 365 were generated to assess the impact of GVHD on transplantation outcomes (dynamic landmarking). Results. A total of 803 patients met the population selection criteria. One hundred and thirty-three of them were excluded from the analyses because they died before achieving engraftment (n=90), received a second transplantation for graft failure (n=10) or because missing data in the registry on GVHD (n=31) or missing conditioning intensity (n=2). Therefore, the analyses were carried out in a cohort of 670 patients who were alive and had engrafted. Status at transplantation was primary refractory (55%), first relapse (35%) or second or more advanced relapse (10%). Conditioning was myeloablative in 289 patients. Two-year relapse and nonrelapse mortality incidences were 49% and 19%, respectively. Two-year leukemia-free survival (LFS) was 32%. There were no associations between acute nor chronic GVHD of any grade and lower relapse incidence. In contrast, each grade III-IV acute (HR=3.79, P<0.0001) and extensive chronic (HR=3.23, P<0.0001) GVHD correlated with higher nonrelapse mortality, translating to decreased LFS (HR=1.51, P=0.008 for grade III-IV acute and HR=1.90, P=0.0008, for extensive chronic GVHD, respectively). Conclusions. Our study suggests a dissociation of GvL effects from GVHD in patients with active AML given PTCy-based Haplo-HCT.

Age-Related Immune Cell Dynamics Influences Outcomes after Allogeneic Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) holds a pivotal place in the setting of therapeutic algorithms for hematological disorders. Its therapeutic index is principally related to the immunogenic graft-versus-leukemia (GvL) effect that, together with an efficient immune reconstitution ensures the success of the procedure, contributing to disease control and immune competence. Factors influencing recipient immune recovery have been largely investigated across multiple cohorts issuing heterogenous results. Differences in outcomes in adult and pediatric populations suggest an age-related contribution to post-transplant immune reconstitution, however it is unclear how this parameter may affect the dynamics of single immune cells. Here we conducted a monocentric prospective study questioning on the modifications of immune cell recovery in a cohort of 176 patients (58 children and 118 adults) with hematological disorders receiving a HCT between 2015 and 2020 and followed at our institution. Complete flow-based immune reconstitution data on T-CD4, T-CD8, NK and B cell populations were collected at defined time-points (+1, 3, 6 and 12 months post-HCT). After ascertaining the dynamics in adult and pediatric populations of each cell component, the effect of a set of baseline clinico-biological variables on late (>6 months) reconstitution for each cell population was tested through univariable and multivariable linear or logistic regression models. First, we analyzed the linear relationship existing between recipient and donor age and each cell compartment. Interestingly, while donor age did not impact on any of the parameters in study, CD4- T (Adj R2 0.4, p=5.7x 10-15) and, in minor part, B cell post-transplant increments (Adj R2 0.05, p=0.004) were inversely correlated with recipient age, which instead did not influence CD8-T nor NK cell reconstitution. Results of stepwise multivariable analyses showed that CD8-T cell reconstitution was positively impacted by recipient positive CMV serostatus (p=0.00009) while donor CMV serostatus positively influenced CD4-T cell recovery (p=0.017). This effect was likely dependent from the expansion of CMV related T cell clonotypes in case of previous CMV immunity. The inverse correlation between CD4-T cells and recipient age was confirmed in multivariable analysis (p=0.0148). Donor EBV positive serostatus resulted instead to be a negative predictor of B cell recovery (p=0.0442). Interestingly none of the parameters analyzed influenced NK cell recovery. The impact on age mirrored a different kinetics of immune reconstitution in adult and pediatric patients (<18 years). Therefore, while early after HCT immune cell composition was similar in the two groups, with NK cells representing the largest component, followed by CD8-T and CD4-T, and minimal B cell contribution, in later phases we assisted to a faster expansion of CD4-T arm in children whereas CD8-T remained the major contributor of the immune recovery processes in adults still one year after transplant. B cell reconstitution was also faster in children, with a better immunoglobulin levels recuperation (measured on IgM to avoid bias of prophylactic IgG infusions) despite no differences in post-transplant pre-emptive rituximab utilization. When looking at different outcomes of pediatric and adult patients, both overall and event free survival were significantly higher in children (p=0.00062 and p=0.0062). While no difference in acute grade III-IV GvHD, adults experienced a significantly higher cumulative incidence (CI) of chronic GvHD (p=0.0258) and of specific infections, including EBV (p=0.031) and fungi (p=0.007). Although CI of relapse was not different, children experienced later relapses as compared to adults (median of 294 vs. 139 days, p=0.039), indicating a better disease control at least at early post-transplant stages. Here, starting from immune reconstitution data prospectively collected for patients transplanted at our institution, we dissected the effect of age on post-transplant immune cells. A faster CD4-T and B cell recovery in children may be the driver of a better immune competence underpinning superior outcomes in pediatric populations. If age-related thymus involution may reflect suboptimal CD4-T cell differentiation in adults, younger donor selection does not seem to improve recipient post-transplant immune reconstitution.

Immunotherapy By Targeting CD45 in Mismatched HLA-DP to Treat Patients after Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation (alloSCT) is a curative treatment for patients with hematological malignancies. The anti-tumor or Graft-versus-Leukemia (GvL) effect of alloSCT is mediated by donor T cells targeting patient hematopoietic cells. Donor T cells may, however, also target non-hematopoietic tissues of the patient, thereby inducing Graft-versus-Host Disease (GvHD). Patients without suitable related donors are transplanted with unrelated donors matched for HLA-A, -B, -C, -DRB1, and -DQB1 (10/10 match). HLA-DP alleles are mismatched in most patients transplanted with unrelated donors. HLA class II molecules are mainly expressed on hematopoietic cells, but under inflammatory conditions the expression of HLA class II on non-hematopoietic tissues is upregulated, thereby increasing the risk of GvHD. To reduce this risk and retain the anti-tumor effect, patients transplanted with unrelated donors may be treated with immunotherapy specifically targeting hematopoietic peptides presented by mismatched HLA-DP. CD45 is an attractive source protein for these peptides because of its broad expression in all hematopoietic lineages. Despite CD45 expression, healthy hematopoietic cells after alloSCT are of donor origin and will therefore be spared. To investigate whether CD45 peptides are presented by HLA-DP on hematopoietic cells, peptides were eluted from HLA-DP and measured by tandem mass spectrometry. HLA-DP peptidome data were generated from acute myeloid leukemia (AML) cases as well as hematopoietic (CD45pos) K562 cells separately transduced with 13 different HLA-DP alleles. Peptidome searching revealed one CD45 peptide on K562 cells transduced with HLA-DP2 (DPA1*01:03 & DPB1*02:01) or HLA-DP4 (DPA1*01:03 & DPB1*04:01 or DPB1*04:02) as well as AML cases expressing these HLA-DP alleles. Next, (CD45pos) K562 cells transduced with HLA-DP2 or HLA-DP4 and costimulatory molecules (CD40, CD80, CD86) were used to stimulate CD4 T cells from healthy individuals negative for these HLA-DP alleles. After 14 days of stimulation, CD4 T cells were restimulated and activated CD137+ and CD154+ CD4 T cells were single cell sorted. Growing T cell clones were screened for reactivity against CD45 peptide loaded on non-hematopoietic (CD45neg) HeLa cells transduced with HLA-DP2 or HLA-DP4. Three clones were able to specifically recognize CD45 in HLA-DP2 or HLA-DP4 (Figure 1A). T cell clones M6-5 and M6-6 were specific for CD45 in HLA-DP2, and clone M6-7 recognized the CD45 peptide in HLA-DP4. No cross-reactivity was observed against other HLA-DP alleles. In addition to HLA-DP transduced K562 cells, all T cell clones specifically reacted against HLA-DP2 or HLA-DP4 positive AML cases. We next investigated whether the T cell receptors (TCRs) of the T cell clones could be used to engineer and redirect CD4 T cells to CD45-derived peptides in mismatched HLA-DP. The HLA-DP2 restricted TCRs of M6-5 and M6-6 and HLA-DP4 restricted TCR of M6-7 were sequenced, cloned and transferred to CD4 T cells from three HLA-DP2 and HLA-DP4 negative healthy donors. TCR-T cells showed specific reactivity against K562 transduced with HLA-DP2 or HLA-DP4, whereas HeLa cells with these HLA-DP alleles were not recognized (Figure 1B, example of one donor). In conclusion, we identified three TCRs for a CD45 peptide presented by HLA-DP2 or HLA-DP4 that are able to target hematopoietic cells. The TCRs can be used for CD4 T cell immunotherapy to treat patients with hematological malignancies after HLA-DP mismatched alloSCT. Figure 1. CD45 as a target for specific CD4 T cells. (A) CD4 T cell clones recognizing CD45 in HLA-DP2 or HLA-DP4 on AML. (B) M6-5, M6-6 and M6-7-TCR transduced CD4 T cells recognize HLA-DP2 or -DP4 transduced K562 (CD45pos) but not HeLa (CD45neg) cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a mRNA Lipid Nanoparticle (mRNA-LNP) Cancer Vaccine to Prevent Leukemia Relapse after Stem Cell Transplant

Introduction The most common acute leukemia in adults is acute myeloid leukemia (AML). Approximately 20,050 new cases and 11,540 deaths are expected for 2022 in the US. Allogeneic stem cell transplant (alloSCT) remains the only cure for high-risk AML. AlloSCTs leverage the immune system to kill leukemic cells through a graft-versus-leukemia (GVL) response mediated by donor CD4+ and CD8+ T cells that recognize recipient leukemic minor histocompatibility antigens (mHAs) as foreign. Unfortunately, transplantation failure related to relapse is common. We postulate that proper targeting of mHAs could enhance leukemia-specific immune responses without off-target effects, improving relapse rates. Using computational modeling, we predicted a series of GVL mHAs, confirmed our first GVL mHA (UNC-GRK4-V) through targeted mass spectrometry, and generated a high-affinity T cell receptor (TCR) against it. Using UNC-GRK4-V as our target, we developed a novel mHA mRNA lipid nanoparticle (LNP) vaccine that we hypothesized could induce an antigen-specific immune response. Methods A custom mRNA was ordered from TriLink that encoded the UNC-GRK4-V peptide linked to an MHC Class I trafficking domain (MITD) and was modified with a 5' cap and polyadenylated tail. We created mRNA LNPs by mixing an ethanol lipid solution made of ionizable lipid (SS-OP, SM-102, or MC3)/ DOPE/Cholesterol/DMG-PEG 2000 with an aqueous solution of mRNA (eGFP or UNC-GRK4-V mRNA) dissolved in a citrated buffer solution. We measured mRNA LNP sizes with a Malvern ZS90 and quantified encapsulation rates using a Quant-it RiboGreen RNA assay kit. Immature monocyte-derived dendritic cells (mo-DCs) were differentiated from monocytes collected from fresh blood and transfected with mRNA LNPs. As a control, we measured eGFP mRNA LNP transfection rates of mo-DCs using flow cytometry. CD8+ T cells were transduced with a retroviral supernatant containing a high-affinity UNC-GRK4-V T-cell receptor and stimulated. We co-cultured mRNA LNP transfected mo-DCs with transduced CD8+ T cells. IFN-g was measured from the co-culture supernatant using IFN-g ELISA assays. We used flow cytometry to determine the cytotoxic killing of transfected mo-DCs by transduced CD8+ T cells. Results To identify the most efficient ionizable lipid platform for mRNA delivery and transfection, we tested three commercially available ionizable lipids: SS-OP, SM-102, and MC3. We found that all three ionizable lipids were easily reproducible with SS-OP 75.2nm ± SD 14.8, MC3 97.0nm ± SD 5.4, SM-102 102.1nm ± SD 0.651. The encapsulation rates of mRNA were 75.4% ± SD 6.3% for SS-OP, 70.5% ± SD 2.6% for MC3, and 73.7% ± SD 0.1% for SM-102. The transfection rate of mo-DCs using eGFP mRNA LNPs were 0.48%, 19.3%, and 62.5% for SS-OP, MC3, and SM-102, respectively. Based on poor transfection rates with SS-OP, we used MC3 and SM-102 LNP formulations to study cytokine expression and cytotoxic killing using a co-culture model of transfected mo-DC with transduced CD8+ T cells. We found a 16.8-fold increase in IFN-g release for the SM-102 based LNPs compared to control empty LNPs (p < 0.0001) and a 4.1-fold increase in IFN-g release for the MC3 based LNPs compared to control empty LNPs (p < 0.0001). We then evaluated SM-102 LNPs for cytotoxic killing as they demonstrated the highest IFN-g release. SM-102 demonstrated increased cell death of 13.5% versus 3.5% for control (empty SM-102) after being normalized for background mo-DC death. Conclusions The SM-102 based UNC-GRK4-V mRNA LNP vaccine elicits robust, antigen-specific T-cell responses and could be a potent therapeutic for decreasing relapse rates post-alloSCT. Furthermore, advances in alloSCT have created regimens that eliminate traditional long-term calcineurin inhibitor use in post-alloSCT, allowing therapeutic cancer vaccines to be given much earlier after transplant.

Targeting Molecular Remission with Donor Lymphocyte Infusions Shows Excellent Outcomes for Persistent or Relapsed Myelofibrosis after Stem Cell Transplantation

About 10-30% of myelofibrosis patients who undergo allogeneic hematopoietic cell transplantation will eventually experience relapse within 5 years. While significant improvement has been achieved in stratifying patients according to their risk for posttransplant mortality, preventing and treating relapse remains an unmet clinical need. Notably, no standard of care exists for treatment of relapsed myelofibrosis. On option in the transplant setting (mainly established in CML and AML) is transferring donor T cells to reinforce a graft-versus-leukemia (GVL) effect targeting the recurring malignant cells. Previous small sample studies also indicated a strong graft-versus-myelofibrosis effect of donor lymphocyte infusion (DLI). Additionally, molecular monitoring posttransplant may further help to target outcomes and identify cohorts with distinct risk for and outcome after relapse receiving DLI. Here, we evaluated the largest cohort to date of myelofibrosis patients receiving DLI. The aim of this study was to induce molecular complete remission (CR) and to evaluate associations with overall outcomes. Patients were eligible to receive DLI if they were free of GVHD and immunosuppressive therapy. If no GVHD or response occurred within 6 weeks, another escalating dose (half-log) of DLI was administered. Highly sensitive chimerism and molecular monitoring with developed driver mutation-specific PCRs for JAK2, CALR and MPL was done every month (until day 360 posttranspant) and with routine visites thereafter We present results of 57 genetically annotated myelofibrosis patients who received donor lymphocyte infusion (DLI) for persistent disease (n=20), molecular relapse (n=17), or hematologic relapse (n=20), aiming to achieve molecular complete remission (CR). A total of 143 DLI procedures were given (range, 1-5 cumulative DLIs). Median CD3+ starting dose of first DLI was 1 x106 (range, 1 x105-1 x107), escalated by half-log after at least 6 weeks if no response nor graft-versus-host disease occurred. Overall molecular CR rate was 70% (n=38) and seemed to be higher for patients who received first DLI for molecular relapse (88%, 15 out of 17). For each DLI, molecular CR was achieved in 40% (23 out of 57) after first, in 44% (17 out of 39) after second, in 32% (8 out of 25) after third, in 470% (7 out of 15) after fourth, and in 43% (3 out of 7) after fifth DLI. In terms of acute GVHD, 15 overall cases occurred after a total number of 143 procedures (11%) and appeared to be associated with response. Of 22 patients who achieved initial molecular CR after first DLI, 11 patients (50%) received subsequent DLIs, of whom 4 patients initially received first DLI for hematological relapse, 4 patients for molecular relapse, and 3 patients for disease persistence. Relapses after first DLI were hematological in 8 patients (73%) and molecular in 3 patients (27%), and second DLI was given within a median of 10 weeks (range, 6-323 weeks) from first DLI. Of those, 9 patients could be eventually converted with subsequent DLIs into durable molecular CR (82%) with a median of 7 years after last DLI. Overall outcome was driven by response to first DLI. Five-year overall survival after first DLI was 85% (95% CI, 67-100%) for patients with molecular CR versus 52% (95% CI, 33-71%) for patients with less than molecular CR. Achievement of molecular CR after first DLI was associated with 78% reduced risk of death (P=0.02). All patients who relapsed after initial molecular CR after first DLI (50%, n=11) were alive at last follow-up. Multivariable analysis on overall survival and disease-free survival, adjusting for time-dependent and continuous effects of total number of DLIs, showed significantly reduced risk of death for patients with molecular CR after first DLI (with patients achieving less than molecular CR as reference), with hazard ratio of 0.22 (95% CI, 0.06-0.80; P=0.02). Similar results were retrieved for risk of death or relapse/disease persistence after last DLI, with a hazard ratio of 0.14 (95% CI, 0.05-0.39; P<0.001). No significant effect of number of DLIs or indication for first DLI were identified. Targeting molecular CR after DLI is essential to achieve excellent long-term survival in persistent or relapsed myelofibrosis, and DLI together with molecular monitoring should be standard of care for relapsed myelofibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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