Immunotherapy Of AML Research Articles

Abstract 4076: 4-1bb selection augments DC/AML fusion vaccine-educated T cells for adoptive cell therapy

Abstract Introduction: Our group has pioneered a novel vaccine by fusion of patient-derived tumor with autologous dendritic cells (DCs) that presents an array of tumor antigens generating a polyclonal immune response. DC/AML vaccination led to expansion of leukemia-specific T cells with survival benefit in a phase II clinical trial. We postulated that ex vivo generation of vaccine-educated T cells would provide a powerful platform for adoptive immunotherapy with opportunity to augment T-cell functional potency prior to infusion. We report on an ex vivo system in which vaccine-educated T cells are further enriched for activated antigen-specific effector cells via an agonistic 4-1bb antibody. We report phenotypic and functional characteristics of 4-1bb-enriched vaccine-educated T cells. Methods: DC/AML fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc+ AML cells. Splenic T cells were co-cultured with autologous irradiated DC/AML fusions in presence of IL-2/7/15. Selection with biotinylated agonistic 4-1bb (3H3) was performed on vaccine-educated T cells followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for activation (CD25/CD69), immune checkpoints (PD1/LAG3/TIM3), memory (CD44+CD62L-) and enrichment (anti-rat H&L). Cytotoxicity was evaluated by luminescence. Mice were inoculated with C1498 and injected with T cells 7 days later. BLI imaging was performed and 100-day survival measured. Results: Vaccine-educated T cells demonstrated evidence of immune activation and memory phenotype compared to unstimulated naïve T-cell controls (TN) (7.24-fold, CD4+CD25+CD69+; 1.7-fold, CD3+CD44+CD62L-). Vaccine-educated T cells selected based on 4-1bb expression showed enhanced markers of activation (15.3-fold, CD4+CD25+CD69+) and memory phenotype (5-fold, CD3+CD44+CD62L-) compared to TN. Selection enriched for 4-1bb+ vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by tumor lysate induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb+ vaccine-educated T cells showed enhanced cytotoxicity (1.9-fold increase/TN at 10:1 E/T, P<0.0001). Phenotypic and functional analysis support 3-5 days as the optimal duration of time for T-cell vaccine education. In vivo, 60% of mice treated with 4-1bb+ vaccine-educated cells were alive at 60 days vs. 20% treated with unselected vaccine-educated cells. Conclusion: We demonstrate that vaccine-educated T cells subject to selection via an agonist 4-1bb antibody confer enhanced tumor selectivity and potency. Optimal duration for T-cell education was 3-5 days. T-cell stimulation and enrichment by agonistic 4-1bb selection enhanced cytotoxicity and memory phenotype. Thus, 4-1bb selection is a novel approach for antigen-specific T-cell enrichment for superior adoptive immunotherapy in AML. Citation Format: Kathrine S. Rallis*, Jessica Liegel*, Giulia Cheloni, Dina Stroopinsky, Poorva Bindal, Kenel Dufort, Daniela Torres, Isabella Saldarriaga, Samuel Herzlinger, Abigael Morin, Raphael Kesselman, Jeremy Rosenbaum, Georges Chedid, Sophia Adamia, Donald Kufe, Jacalyn Rosenblatt, David Avigan. 4-1bb selection augments DC/AML fusion vaccine-educated T cells for adoptive cell therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4076.

Pan-cancer analysis identifies CD300 molecules as potential immune regulators and promising therapeutic targets in acute myeloid leukemia.

CD300s are a group of proteins playing vital roles in immune responses. However, much is yet to be elucidated regarding the expression patterns and clinical significances of CD300s in cancers. In this study, we comprehensively investigated CD300s in a pan-cancer manner using multi-omic data from The Cancer Genome Atlas. We also studied the relationship between CD300s and the immune landscape of AML. We found that CD300A-CD300LF were generally overexpressed in tumors (especially AML), whereas CD300LG was more often downregulated. In AML, transactivation of CD300A was not mediated by genetic alterations but by histone modification. Survival analyses revealed that high CD300A-CD300LF expression predicted poor outcome in AML patients; the prognostic value of CD300A was validated in seven independent datasets and a meta dataset including 1115 AML patients. Furthermore, we demonstrated that CD300A expression could add prognostic value in refining existing risk models in AML. Importantly, CD300A-CD300LF expression was closely associated with T-cell dysfunction score and could predict response to AML immunotherapy. Also, CD300A was found to be positively associated with HLA genes and critical immune checkpoints in AML, such as VISTA, CD86, CD200R1, Tim-3, and the LILRB family genes. Our study demonstrated CD300s as potential prognostic biomarker and an ideal immunotherapy target in AML, which warrants future functional and clinical studies.

Cellular and Molecular Biomarkers Predictive of Response to Immunotherapy in Acute Myeloid Leukemia.

Immunotherapy has without question revolutionized the treatment of both hematologic and solid malignancies. Over the last several years novel strategies are being developed to incorporate these groundbreaking therapies into the care of patients with AML. Here we present an overview of the recent developments in immunotherapy for AML with a focus on biomarkers of response. Topics reviewed include antibody drug conjugates, BiTEs, DARTs, checkpoint inhibitors, and cellular therapy as well as the development of biomarkers predictive of response in each class.

Novel RNA-based Cancer Vaccine for AML immunotherapy

Abstract Acute Myeloid Leukemia is a rapidly progressing malignancy relying primarily on chemotherapy as the frontline treatment option. Success of personalized neoantigen vaccines in melanoma patients has garnered great attention in the field of immunotherapy. We hypothesize that personalized cancer vaccine, in the form of mRNA, can be a viable treatment strategy for AML patients. Next generation sequencing was performed on both C57BL6Jinv mouse’s and C1498 cell line’s nucleic acid materials. Three different neoepitope prediction pipelines were utilized to identify potential immunogenic neoepitopes. Candidate neoepitopes within intron polyadenlyation sites (iPAS), retained intron (RI) sites and somatic mutation (SM) sites were found. A total of 285 SM-derived neoepitopes were identified. Top 38 highly expressed genes were selected for further analysis. Using Sanger sequencing, we confirmed 37 out of the 38 mutations. A total of 5 iPAS-derived neoepitopes were identified. A total of 26 RI-derived neoepitopes were identified in our first RNAseq analysis and a separate 476 RI-derived neoepitopes were identified in our second RNAseq analysis. With these three categories of candidates, we narrowed down our selection and did a preliminary screen for immunogenic epitopes using a total of 71 synthetic peptides in an ELISpot assay. Conventional ELISpot assay’s results were however non-reproducible, thereby propelling us to adopt Culture ELISpot assay. Subsequently, 10 neoepitope candidates were short-listed for further in-vivo evaluation. mRNA vaccine was synthesized and evaluated in a murine AML model. T cell responses against the 10 neoepitopes were analyzed to provide pre-clinical evidence for patient studies. Supported by NMRC CS-IRG grant

A Polygenic DNA Damage Repair Pharmacogenomics (DDR_PGx8) Score Predicts GO Response in AML

Gemtuzumab Ozogamicin (GO) is a CD33-directed antibody conjugated to calicheamicin used in AML immunotherapy. Children's Oncology Group led pediatric AML phase II trial COG-AAML03P1 (NCT00070174) established that GO could be safely added to the standard chemotherapy regimen consisting of ara-C, daunorubicin and etoposide (ADE+GO) and improved outcome(Cooper et al., 2012). Subsequent COG-AAML0531 phase III trial (NCT00372593) randomized patients to receive either standard chemotherapy alone (ADE) or with addition of two doses of GO (ADE+GO) and showed that addition of GO improved outcome in newly diagnosed AML patients (Gamis et al., 2014). Our group previously established that CD33 genetic variation impacts GO response in AML patients (Lamba et al., 2017). Given that the antileukemic effect of GO is primarily driven by calicheamicin induced DNA damage, in this study we investigated pharmacogenomic impact of SNPs in DNA damage response (DDR) pathway genes on GO treatment response. We genotyped 132 SNPs in 42 genes involved in DNA damage repair pathway in DNA samples from 470 patients treated with standard chemotherapy in AAML0531 trial (ADE arm) and 755 patients treated with addition of GO to standard therapy in AAML03P1 and AAAML0531 trials (ADE+GO arm). Univariate analysis to test for association between OS, EFS, DFS and RR after induction 1 in both ADE+GO and ADE arms identified 20 SNPs in 16 genes that were significantly associated with at least one of the clinical endpoints tested in the only ADE+GO arm but not in ADE arm of the trials. We tested these 20 SNPs in all possible combinations with a maximum of 3 SNP/model using multivariable Cox proportional hazard models for association with EFS and OS in patients treated with ADE+GO. Drastically increased number of models arising from higher SNP combination numbers was a computational challenge thus we restricted our analysis to a maximum of 3 SNP combinations. We performed 1000 permutation tests per model to determine the likelihood of obtaining them falsely. Models were ordered according to their Bayesian Information Criterion (BIC) and weight in favor of each model. Those withleast BIC and a 1000 permutation p≤0.05 were selected for development of DDR pharmacogenomics score. DDR_PGx8 score was defined by adding the genotype scores of 8 SNPs in 7 genes (AKT1, ATR, DDB2, PARP1, PI3KCA, PTEN and RAD51) accounting for mode of inheritance (additive, dominant or recessive) and direction of their association with outcome (positive for beneficial and negative for detrimental association) Fig 1A shows overall study design. The DDR_PGx8 score ranged from -5 to 3 in patients treated with ADE+GO (n=755) or ADE alone (N=470). Based on the distribution, the scores were stratified into high (score ≥0, n=212 in ADE group and n=357 in ADE+GO group) and low score (score <0, n=241 in ADE group and n=329 in ADE+GO group) groups. The distribution of DDR_PGx8 score groups did not differ by risk groups or MRD1 status. However, it differed significantly within race with 81.81% (108/132) of black or African American patients compared to 43.75% (364/832) of white patients in the low DDR_PGx8 score group. Patients with low-DDR-PGx8 score had significantly worse EFS (HR=1.52, 95%CI (1.22-1.90), p<0.001; Fig 1B), OS (HR=1.62, 95%CI(1.24-2.11), p<0.001), DFS (HR=1.88, 95%CI(1.42-2.50), p<0.00001;), and higher RR1 (HR=1.89, 95%CI(1.40-2.40), p<0.00001) compared to high-DDR-PGx8 score patients when treated with GO (ADE+GO cohort). This impact of DDR_PGx8 was not observed in patients treated with standard chemotherapy alone (ADE arm), with no difference between low and high DDR_PGx8 score groups in EFS (Fig 1B), OS, DFS and RR (all p>0.28). In multivariable Cox proportional hazard models for DDR-PGx8 score groups, initial risk group assignment, WBC at diagnosis and age, low-DDR-PGx8 score remained a significant and independent predictor of inferior EFS (HR=1.6, 95%CI=1.21-2.02, p<0.001; Fig 1C) and OS (HR=1.6, 95%CI=1.17-2.22, p=0.003) in ADE+GO arm but not in ADE arm. We establish a DNA damage repair response-based pharmacogenomics score predicting outcome in patients treated with addition of GO to standard chemotherapy. The score was not predictive of outcome in patients treated with standard chemotherapy alone implying its impact is GO-specific. Our results in conjunction with existing CD33 SNPs provide a rationale for use of pharmacogenomics in personalizing GO treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

TIM-3 in Leukemia; Immune Response and Beyond.

T cell immunoglobulin and mucin domain 3 (TIM-3) expression on malignant cells has been reported in some leukemias. In myelodysplastic syndrome (MDS), increased TIM-3 expression on TH1 cells, regulatory T cells, CD8+ T cells, and hematopoietic stem cells (HSCs), which play a role in the proliferation of blasts and induction of immune escape, has been reported. In AML, several studies have reported overexpression of TIM-3 on leukemia stem cells (LSCs) but not on healthy HSCs. Overexpression of TIM-3 on exhausted CD4+ and CD8+ T cells and leukemic cells in CML, ALL, and CLL patients could be a prognostic risk factor for poor therapeutic response and relapse in patients. Currently, several TIM-3 inhibitors are used in clinical trials for leukemias, and some have shown encouraging response rates for MDS and AML treatment. For AML immunotherapy, blockade TIM-3 may have dual effects: directly inhibiting AML cell proliferation and restoring T cell function. However, blockade of PD-1 and TIM-3 fails to restore the function of exhausted CD8+ T cells in the early clinical stages of CLL, indicating that the effects of TIM-3 blockade may be different in AML and other leukemias. Thus, further studies are required to evaluate the efficacy of TIM-3 inhibitors in different types and stages of leukemia. In this review, we summarize the biological functions of TIM-3 and its contribution as it relates to leukemias. We also discuss the effects of TIM-3 blockade in hematological malignancies and clinical trials of TIM-3 for leukemia therapy.

Identification of Novel Targets for Antibody or Chimeric Antigen Receptor (CAR) Based Immunotherapy in AML: Characterizing the Surfaceome from Primary Patient Samples

Background: Despite considerable advances in the field of immunotherapy of acute myeloid leukemia, on-target off-leukemia toxicity remains a challenge. Thus, novel target antigens are sought after. The Cell Surface Capture (CSC) technology enables a discovery-driven approach to detect surface proteins. However, direct processing of primary samples has been limited by the substantial number of viable cells needed for the CSC-workflow. We therefore addressed this issue by improving the technique including the use of our ex vivo culture system. This allowed for an unbiased, direct assessment of primary AML patient samples and thus enabled us to interrogate the AML surfaceome in clinically relevant samples.Methods: Primary cells from patients with newly diagnosed or relapsed AML were cultured in our ex vivo co-culture system as previously described (Krupka et al. 2014) using MS-5 feeder cells. After 3 days, non-adherent cells were harvested and immediately subjected to the CSC-workflow. Glyco-CSC and its variants Cys-Glyco-CSC and Lys-CSC were initially performed as described by Wollscheid and colleagues (Wollscheid et al. 2009 and Bausch-Fluck et al. 2012) and subsequently modified to improve the yield on AML samples. CSC samples were analyzed by tandem mass spectrometry on an Orbitrap Elite instrument and modified peptides were identified using MaxQuant software.Results: To enhance the number of successfully identified surface proteins, we adapted the original protocol. These modifications doubled the yield in identified proteins from AML cell lines from initially 125 to 252 in the modified protocol. More importantly, the modifications increased the specificity of the assay significantly. In the original Glyco-CSC experiments, only 54% of all identified peptides displayed a mass shift (of 0.984 Da) associated with successful N-glycosylation and had a transmembrane domain or a signal peptide annotated in UniProt. After modification of the protocol, 80.4% of all peptides fulfilled these criteria. The modified protocol was therefore used for all primary samples. 5 representative primary patient samples from initial diagnosis and 2 samples from relapsed disease were analyzed. All samples yielded sufficient viable cell numbers after ex vivo culture and could successfully be subjected to the CSC workflow. We identified a total of 719 surface proteins fulfilling all filter rules. 22.9% of these proteins had CD annotations. Next, we only considered proteins that were detected by CSC in at least half of the primary patient samples tested. In addition, proteins were filtered to eliminate targets that are abundantly expressed on normal human hematopoietic stem and progenitor cells as well as relevant healthy tissue using publicly available transcriptome databases. 84 proteins were selected as potential candidates for manual screening. Of note, the expression of several antigens currently under investigation for AML immunotherapy (i.e. CD33, CD123, CD135, CLL-1) were detected by the method. We selected 6 promising novel candidate markers previously not described as relevant targets in AML. These were assessed by FACS analysis in independent patient samples. 3/6 of our novel targets showed uniform expression in all independent primary AML samples tested (defined as MFI ratio >1.5).Conclusion: In conclusion, improvements in the CSC-Workflow combined with our ex vivo culture system allowed for the successful identification of the AML surfaceome from primary patient samples without the necessity of xeno-amplification. We identified 6 novel targets, 3/6 were found to be uniformly expressed in independent primary AML samples. These candidates are now being evaluated further as potential targets for antibody and CAR based immunotherapy in AML. DisclosuresNo relevant conflicts of interest to declare.

Identification of Leukemia Associated Minor Histocompatibility Antigens through Computational Prediction and Targeted Mass Spectrometry

Background and Rationale: T-cell responses to minor histocompatibility antigens (mHA) mediate both anti-tumor immunity (GvL) and graft versus host disease (GvHD) in allogeneic stem cell transplantation. Clinical trials of cellular immunotherapies that target one or more of these mHA are ongoing, however current laboratory and GWAS methods have thus far produced a limited number of confirmed mHA (Oostvogels et al. BMT 2016, PMID: 26501766). Identifying mHA with tight binding affinity to common HLA alleles, narrow tissue restriction, and high population prevalence (GvL mHA) could provide promising immunotherapy targets against leukemia as therapeutic strategies that target these mHA would maximize the number of candidate recipients and may drive a beneficial GvL effect without causing GvHD.Methods: To evaluate a custom GvL mHA prediction pipeline (Figure 1) based on neoantigen prediction software developed in the Vincent lab (Kardos et al. JCI Insight 2016, PMID: 27699256), we predicted mHA for HLA-matched allogeneic stem cell transplant (SCT) donor-recipient pairs (DRPs) from a cohort of 101 patients with myeloid hematologic diseases who had been genotyped at 13,917 non-synonyomous coding polymorphisms (cSNPs) by Illumina NS-12 microarrays (Armistead et al . PLoS One 2011, PMID: 21858034). We computationally predicted GvL mHA by first eliciting in silico all possible 8, 9, 10, 11, 16, 20, and 24-mer peptides arising from DRP genetic variation, then estimating peptide/HLA binding with NetMHCpan and tissue restriction based on Human Protein Atlas and in-house AML sample RNAseq data. We validated our computational approach to mHA prediction by assessing our capacity to predict previously published mHA and by performing two complementary biological validations of a novel GvL mHA we discovered. We purified HLA-restricted peptide epitopes from HLA-A*02:01 expressing U937 cells and used targeted differential ion mobility spectrometry/tandem mass spectrometry (DIMS/MS/MS) (Dharmasiri et al. J. Proteome Res. 2014, PMID: 25184817) to test for HLA-A*02:01 presentation of a predicted novel GvL mHA (UNC-GVL-1). We also tested for T cell responses to UNC-GVL-1 using peptide/HLA tetramers on post-SCT AML patient samples.Results: We identified 18 previously published mHA that were potentially discoverable in our patient cohort because at least one DRP expressed the appropriate HLA and the mHA arose from a cSNP contained in the Illumina NS-12 microarray. Of these 18 possible mHA, we successfully predicted 14 in our analysis (Figure 2A), with the 4 missed mHA resulting from inadequate cohort size (n=2), non-canonical translation of a mHA (n=1), and poor predicted HLA binding (n=1). Of our 101 DRPs, 60 (59%) were predicted to have at least one of these 14 mHA, with T4A the most common, identified in 21 DRPs (21%), and UNC-GVL-1 identified in 13 DRPs (13%). All previously published Class I mHA predicted in our cohort as well as UNC-GVL-1 had similar or tighter predicted binding affinity for the recipient peptide/HLA as compared to the donor (i.e. alternate allele) peptide/HLA (Figure 2B). In addition to predicting previously discovered mHA, we predicted 102 novel public GvL mHA. By optimizing DIMS settings to filter out other peptide epitopes, we confirmed that the novel GvL mHA UNC-GVL-1 was presented on HLA-A*02:01 by HLA-A*02:01 expressing U937 cells. Using UNC-GVL-1/HLA-A*02:01 tetramers, we identified T cell responses in 4 of 9 HLA-A*02:01 expressing post-SCT samples.Conclusions: Computational prediction of GvL mHA with tight predicted binding affinity to common HLA alleles, narrow tissue restriction, and high population prevalence can provide novel public immunotherapy targets. UNC-GVL-1 is a promising novel target for immunotherapy in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Midostaurin Reduces Bite® Mediated Cytotoxicity Against Acute Myeloid Leukemia

T-cell recruiting antibody constructs represent a promising approach to recruit T-cells to tumor cells independent of TCR-specificity. So far, CD33 has been the most commonly targeted antigen in AML. However, its ubiquitous expression pattern within the healthy myeloid hematopoietic system remains a major challenge. To reduce on-target off-leukemia toxicity, alternative AML-associated targets with a more restricted expression pattern are sought after. We evaluated CD135 (FLT3 ; FSM-like tyrosine kinase 3) as a target antigen for T-cell recruiting antibody based immunotherapy in AML. In 159/192 of AML patient samples (83%; MFI ratio of >1.5) a significant expression level was detected by flow cytometry with no differences in expression level at time of initial diagnosis versus relapse (Initial diagnosis: n=161, median MFI ratio 3.0 vs relapse: n=18, median MFI ratio 3.5). Expression level could neither be correlated to FLT3 mutational status (median MFI ratio FLT3-ITD vs FLT3 wild type (WT): 3.7 vs 4.0, n=52 and 173 respectively) nor to FLT3 allelic ratio (median MFI ratio allelic ratio high vs low: 4.1 vs 4.5, n=13 and 19 respectively). Importantly, CD34+/CD38- leukemia initiating cells (LICs) also expressed CD135 at a similar level compared to AML bulk cells (Median MFI ratio LIC: 2.4 vs bulk: 2.6, p=0.1, n=81). Bone marrow progenitor cells (CD45DIM/SSCLOW) cells and CD34+/CD38- expressing hematopoietic precursors from healthy bone marrow expressed CD135 to a significantly lower level compared to AML bulk cells and LICs, respectively (median MFI Ratio: AML bulk vs healthy progenitor cells: 3.2 vs 2.1, p<0.0001; LICs vs HSCs: 3.1 vs 0.7, p=0.0003). Next, we evaluated a CD135xCD3 BiTE® antibody construct (FLT3 BiTE®) for its ability to mediate cytotoxicity against AML cell lines and primary AML cells. Target antigen expression level was relevant for FLT3 BiTE® mediated cytotoxicity as demonstrated by preferential lysis of CD135bright (MM6, MFI ratio 15) vs CD135dim (OCI-AML3, MFI ratio 1.5) AML cell lines. Cytotoxicity against primary AML cells was tested in our long-term culture system using either allogeneic or autologous T cells (Krupka et al, Blood 2014). In the allogeneic set-up, with a fixed E:T ratio of 1:5, the FLT3 BiTE® antibody construct mediated efficient elimination of primary AML cells within 9 days of culture (median % specific lysis 75.9, n=6). This was accompanied by a strong T-cell proliferation (fold change CD2+ T cells: 7.9, n=6). In the autologous system, the E:T ratio was not fixed and influenced by the number of residual T-cells in the respective AML sample. Despite low E:T ratios (1:12-1:74) the FLT3 BiTE® induced high cytotoxicity and T-cell proliferation against primary AML cells within 14 days of culture (median specific lysis: 34.4%; fold change CD2 T cells: 6.6, n=7). We further investigated the influence of Midostaurin on FLT3 BiTE® mediated cytotoxicity against AML cells. Midostaurin (1 µM) drastically decreased FLT3 BiTE® mediated cytotoxicity in 7/8 patient samples (median % cytotoxicity FLT3 BiTE® vs +Midostaurin: 59.0 vs 39.9, p=0.02). This corresponded to a significant decrease in BiTE®- as well as CD3/CD28-mediated T-cell proliferation (median % CD3/CD28 mediated T-cell proliferation FLT3 BiTE® vs +Midostaurin: 56.4 vs 1.46, n=5). It has been reported that Midostaurin inhibits Akt phosphorylation (Kawai, J Bio Sci 2015), a major factor in T-cell proliferation, which might explain the decrease in T-cell proliferation and cytotoxicity. In summary, CD135 was shown to be expressed in the majority of AML patients on AML bulk cells as well as LICs with a restricted expression pattern in the healthy hematopoietic compartment. The FLT3 BiTE® antibody construct mediated strong cytotoxicity against primary AML cells. This correlated to profound T-cell activation and proliferation. The combinatorial approach with Midostaurin abrogated BiTE® mediated cytotoxicity due to a decrease in T-cell proliferation. As T-cell recruiting antibody construct mediated cytotoxicity relies on T-cell function, our data do not support the combinatorial approach with the FLT3 -TKI Midostaurin for targeted therapy of AML. DisclosuresLindl:AMGEN Inc: Research Funding. Krupka:Amgen, Inc: Research Funding; Amgen Research (Munich): Research Funding. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Amgen, Inc.: Research Funding; Amgen Research (Munich): Research Funding.

Impact of HLA-B Dimorphism in the Leader Peptide on Clinical Outcome in AML

NK cells are negatively regulated by inhibitory receptors, such as the family of Killer Immunoglobulin-like Receptors (KIR) and the NKG2A/CD94 heterodimer. These receptors recognize cognate HLA class I molecules, and recent studies suggest that an HLA-B dimorphism in the leader peptide at position -21 dictates whether NK cell regulation mainly relies on the KIRs or the NKG2A/CD94 receptor. Thus, haplotypes with -21M delivers functional peptides to HLA-E, but will rarely harbor genes encoding the KIR ligand HLA-C2. However, little is known regarding the clinical implications of this dimorphism. For this study, we performed HLA typing and an extensive flow cytometry analysis of the NK cell repertoire in 80 AML patients who received immunotherapy with histamine dihydrochloride (HDC) and IL-2 for the prevention of relapse in the post-consolidation phase. Below median levels of HLA class I expression, reflecting low-grade NK cell inhibition, were found to correlate with significantly better outcome in HDC/IL-2-treated patients, indicating that the efficacy of the immunotherapy is NK cell-dependent. In contrast to healthy controls, the majority of NK cells in AML patients in complete remission displayed expression of NKG2A, which was even more prominent in patients after 3 weeks of HDC/IL-2 treatment, or after in vitro culture of NK cells with IL-2. Thirty-eight patients (47.5 %) carried the M/x genotype in HLA B -21 (predicting NKG2A dependence) and 42 patients (52.5 %) carried the T/T genotype (KIR dependence). Despite the high percentage of NKG2A+ NK cells, M/x patients showed significantly improved leukemia-free survival (p=0.04) and overall survival (p=0.007) compared with T/T patients. Only patients with M/x genotype benefited from low expression of HLA class I. The IL-2-induced NKG2A expression was associated with enhanced NKG2A-mediated inhibition as pure populations of NKG2A-expressing NK cells were inhibited by a leukemic cell line transfected to express the NKG2A ligand HLA E, which was reverted by antibody blockade of NKG2A. Our results suggest that the outcome of AML immunotherapy is affected by (i) expression levels of HLA class I, and (ii) the HLA B-21 dimorphism and, hence, whether the KIR or NKG2A-CD94 are the dominant inhibitory pathway for NK cells. Furthermore, the pronounced expression of NKG2A in HDC/IL-2-treated patients suggests that combination strategies, including checkpoint inhibitors targeting NKG2A together with HDC/IL-2, may be feasible for relapse prevention in AML. DisclosuresBrune:Novartis: Other: reimbursement for lecture. Aurelius:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Martner:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Hellstrand:Immune Pharmaceuticals: Patents & Royalties: Filed or pending patents protecting the use of NOX2 inhibitors in cancer. Thoren:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer.

Prolonged Azacitidine Exposure Alters Immunophenotype in Myeloid Leukemia Cell Lines

Hypomethylating agents (HMA), namely azacitidine and decitabine, are used for treatment of MDS and AML. Although HMAs have demonstrated efficacy in a subset of patients, their mechanism of action is not well established. Some studies indicate that HMAs induce expression of specific target proteins, thus enhancing the efficacy of targeted therapies. For example, studies of HMAs used in combination with gemtuzumab ozogamicin (GO) suggest that they can apply additional epigenetic stress to AML cells and increase surface CD33 expression, thereby increasing the efficacy of GO. Despite suggestions of epigenetic modulation of target antigens, data on comprehensive response to HMAs is lacking. We proposed to evaluate the full extent of surface antigen expression in different AML cell lines after prolonged exposure to azacitidine.The dose and duration of azacitidine therapy used for prolonged exposure was established in Kasumi-1 cells (t(8;21), N822K KIT mutation). A daily dose of 250 nM allowed continued cellular proliferation for 18 days. We then treated two cell lines, Kasumi-1 and MV4-11 (t(4;11), FLT3 -ITD+) as above for 14-18 days. Cells were submitted for multi-dimensional flow cytometry (MDF) every 2-4 days.Prolonged azacitidine exposure resulted in broad changes in hematopoietic cell surface antigens, as demonstrated by MDF analysis. CD33, the premier cell surface target for AML immunotherapy, increased significantly in Kasumi-1 cells, approximately doubling by day 14 of treatment (p=0.003, Fig. 1). C -kit, or CD117, which is overexpressed in Kasumi-1 cells and is a potential target of tyrosine-kinase inhibitor (TKI) therapy in t(8;21) AML, was significantly decreased with azacitidine exposure (p <0.0001, Fig. 1). In contrast, treatment of MV4-11 cells resulted in significant decrease in CD33 with a corresponding increase in CD117 (Fig. 1).Kasumi-1 and MV4-11 both exhibited significant increases in surface CD38, CD13, and CD36, and significant decreases in CD64, CD123, and CD11b. Cell marker changes observed in only one cell line include significantly increased CD34 in Kasumi-1 and significantly increased CD56 in MV4-11. Many of the observed changes peaked after day 10 of treatment. No significant changes were seen in either line in CD7, CD14, CD16, CD19, CD45, and HLA-DR. Notably, the shared pattern of surface antigen changes in both Kasumi-1 and MV4-11 indicates that prolonged azacitidine exposure may induce a less mature myeloid phenotype, as typified by increased CD38 and decreased CD11b and CD123 (Fig. 1).The results above were validated in an experiment in which 4 leukemia cell lines were exposed to azacitidine as above for 10 days, with MDF analyses on days 5 and 10. Cell lines included Kasumi-1, MV4-11, K562 (t(9;22)), and the ALL cell line REH (t(12;21)). Similar trends in surface antigens for Kasumi-1 and MV4-11 were again observed. K562 cells exhibited increased CD38, CD36, CD7, CD14, and CD19, and decreased CD33, CD56, CD64, and CD45 (Fig. 1). No change was seen in K562 in CD117, CD13, CD11b, CD16, CD34, CD123, and HLA-DR (Fig. 1). Of these myeloid markers tested, REH cells had only a significant increase in CD13, a marker also seen in very early B-lymphocytes. Thus, HMAs do not appear to promote lymphoid to myeloid phenotype shift in REH.We observed broad changes in cell surface antigen expression in myeloid leukemia cell lines treated with prolonged azacitidine, which may signify changes in gene expression induced by hypomethylation. Among AML cell lines, prolonged azacitidine exposure resulted in a less mature immunophenotype. We observed significant changes in CD33 and CD117, both of which are considered potential therapeutic targets in AML. CD33 expression was increased by azacitidine exposure in Kasumi-1 cells, and reduced in MV4-11 and K562. C -kit was also noted to variably change in response to azacitidine. Downregulation of cell surface antigens is a known resistance mechanism to immunotherapeutic agents, including antibody-drug conjugates such as GO. Resistance to c -kit-targeted agents has been proposed to occur through a variety of mechanisms, including cell surface expression, therefore changes to c -kit levels induced by HMAs may be an important avenue of investigation in averting TKI resistance. The use of prolonged, low-dose hypomethylating agents warrants further investigation as a potential means to modulate surface protein levels for therapeutic benefit. [Display omitted] DisclosuresPardo:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership. Brodersen:Hematologics Inc: Employment.

Dasatinib Increases MHCII Surface Levels and Can Synergize with Anti-PD1 Therapy to Increase the Anti-Tumor Effect in a Pre-Clinical Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Model

Anti-PD1 therapy in hematologic malignancies has shown clinically inferior effects when compared to solid tumors.1 However, immunotherapy has been part of the standard of care in the treatment of acute leukemia for over 40 years. Recently, loss of MHCII was shown to be a mechanism of Immune escape in patients with acute myeloid leukemia after stem cell transplantation.2 Here we demonstrate, in pre-clinical models, that responses to anti-PD1 can be enhanced by increased antigen presentation through induction of MHCII. We used a Philadelphia chromosome positive (Ph+) Acute Lymphoblastic Leukemia (ALL) syngeneic mouse model and treated 4 cohorts of mice with the following regimens: 1) vehicle 2) single agent Dasatinib; 3) single agent anti-PD1; 4) combination anti-PD1 + Dasatinib (Figure 1a). Single agent Dasatinib, was highly active against this model compared to vehicle. Although anti-PD1 therapy showed little or no activity as a single agent, when combined with sub-therapeutic doses of Dasatinib, we observed significantly enhanced survival of mice compared to single agent anti-PD1 or to single agent Dasatinib. (Figure 1b) In our mouse model, Dasatinib increased the MHCII on the surface of the antigen presenting cells in the tumor microenvironment, most notably in dendritic cells (CD11c+ cells) from mouse tumor-infiltrating lymphocytes collected from the mice bone marrow after treatment (Fig 1c). To prove whether Dasatinib-induced MHCII expression can occur independently of a tumor microenvironment, we treated antigen presenting cell lines (KG1) with Dasatinib and were able to confirm an increased MHCII expression by flow cytometry and by western blotting. (Fig 1d) Since Dasatinib is approved for the treatment of Philadelphia chromosome positive ALL, CML, and has been used in a variety of other settings in the treatment of both malignant hematology and solid tumors, this data has immediate translational potential. In fact, Dasatinib in combination with anti-PD1 therapy is currently being tested in a variety of phase I/II trials in various kinds of malignancies (NCT04284202, NCT03516279). Here we show that induction of MHCII by Dasatinib may serve as a biomarker and could predict the potential benefit from the combination treatment. Additionally, targeting anti-PD1 following Dasatinib administration may increase response rates of patients treated with Dasatinib, providing a rationale for sequential treatment design in patients with persistent minimal residual disease. Figure 1. (a) C57BL/6J mice were injected via tail vein injection with a transplantable, immunocompetent BCR-ABL+ B-ALL model and treated with vehicle/Dasatinib/or anti-PD1 as described in the schema. (b) Overall survival of the mice treated in the previous experiment. (c) MHCII cell surface expression on dendritic cells (CD11+ cells) from the bone marrow of treated mice. (d) relative Cell surface expression of MHCII in the KG1 cell line (APC cell line) treated with Dasatinib vs. vehicle. (e) protein expression of MHCII in the KG1 cell line (APC cell line) treated with Dasatinib vs. vehicle. 1. Masarova L, Kantarjian H, Ravandi F, Sharma P, Garcia-Manero G, Daver N. Update on Immunotherapy in AML and MDS: Monoclonal Antibodies and Checkpoint Inhibitors Paving the Road for Clinical Practice. Adv Exp Med Biol 2018;995:97-116. W2. Christopher MJ, Petti AA, Rettig MP, et al. Immune Escape of Relapsed AML Cells after Allogeneic Transplantation. New England Journal of Medicine 2018;379:2330-41. Figure Disclosures Koller: Jazz Pharmaceuticals, Inc.: Consultancy. Konopleva:Stemline Therapeutics: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; Ablynx: Research Funding; Cellectis: Research Funding; Agios: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents &amp; Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AstraZeneca: Research Funding; Amgen: Consultancy; Calithera: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding. OffLabel Disclosure: anti-pd1 therapy in acute lymphoid leukemia

Complete Remission after the First Cycle of Induction Chemotherapy Determines the Clinical Efficacy of Relapse-Preventive Immunotherapy in Acute Myeloid Leukemia

Background: Contemporary studies have identified &amp;gt;1 cycle of induction chemotherapy to induce complete remission (CR) as an independent risk factor for relapse and mortality in younger adults with acute myeloid leukemia (AML; Othus et al., Leukemia 2019). This study aimed at defining the potential impact of previous induction chemotherapy on the clinical benefit of relapse-preventive immunotherapy in AML. Methods: In a previously reported phase 3 trial (Brune et al., Blood 2006; Martner et al., Blood Rev 2013), two hundred and sixty-one patients with AML in first CR (18-84 years, median 55) who were not eligible for allogeneic transplantation were randomly assigned to receive immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) or standard of care (no treatment, control). The HDC component of this regimen aims at countering immunosuppression induced by reactive oxygen species, which are formed by the NOX2 enzyme expressed by normal and malignant myeloid cells. HDC, a NOX2 inhibitor, improves the function and viability of anti-leukemic lymphocytes such as natural killer cells and cytotoxic T cells (Martner et al., J Pathol 2019). The immunotherapy was initiated in CR1 after the completion of chemotherapy with the aim to prevent relapse in the post-consolidation phase. Cycles 1 to 3 comprised 3 weeks on treatment and 3 weeks off treatment. In cycles 4 to 10 the off-treatment periods were extended to 6 weeks. In each cycle, patients in the treatment arm received HDC (Noventia Pharma, Milan, Italy) at 0.5 mg and human recombinant low-dose IL-2 (aldesleukin; 16 400 IU/kg; Chiron Corporation, Emeryville, CA) subcutaneously twice a day. All patients were followed for at least 36 months after random assignment. The median follow-up time was 48 months. Results: The number of previous induction cycles was known in 260 patients, and 203 of these attained CR after 1 induction cycle (78%). Among the 57 remaining patients, forty-seven attained CR after 2 cycles, and 3 (n=7) or 4 (n=3) cycles were required to induce CR in 10 patients. We compared outcomes of patients who had achieved CR1 after 1 vs &amp;gt;1 cycle of induction chemotherapy and observed a non-significant trend towards inferior outcome in control arm patients who had required &amp;gt;1 induction with a similar trend in younger control patients (&amp;lt;60 years old). In the HDC/IL-2 arm, outcome was significantly superior in younger patients who had attained CR1 after 1 vs &amp;gt;1 induction (leukemia-free survival, LFS, P=0.0007, hazard ratio, HR, 1.67-6.77; overall survival, OS, P=0.009, HR 1.30-6.33 in multivariable analyses correcting for potential confounding factors for relapse and death). When comparing outcomes between treatment and control arms within groups of patients who had attained CR1 after 1 cycle of induction chemotherapy, we observed that the LFS of patients in the HDC/IL-2 arm was significantly superior over controls (P=0.01 in multivariable analysis, N=203). In patients who were &amp;lt;60 years old at random assignment (N=130), treatment with HDC/IL-2 entailed significantly improved LFS (P=0.0008 vs control, HR 0.29-0.72) and OS (P=0.01, HR 0.28-0.75). HDC/IL-2 did not improve LFS or OS in patients who had required &amp;gt;1 cycle of induction to attain CR1 (P&amp;gt;0.5 vs control) and was not significantly beneficial in older patients. A test of interaction supported that the effect size of HDC/IL-2 vs control for LFS was more pronounced in younger patients who had attained CR after 1 (HR=0.48) vs &amp;gt;1 (HR=1.33) cycle of induction (P=0.04). Figure 1 shows Kaplan-Meier analyses of outcome, with unadjusted P-values from logrank tests, in the phase 3 trial arms among all patients attaining CR1 after 1 induction (A and B), in corresponding patients &amp;lt;60 years old (C and D) and in all patients who had attained CR1 after &amp;gt;1 cycle of induction (E and F). Conclusions: These findings imply that the efficiency of previous induction chemotherapy independently determines the clinical benefit of relapse-preventive immunotherapy in adult AML patients who are not candidates for allogeneic transplantation. The results may have implications for the selection of patients who are likely to benefit from treatment with HDC/IL-2 and for the design of clinical trials that evaluate relapse-preventive immunotherapy. Figure 1 Disclosures Thorén: Cytovia, New York: Patents &amp; Royalties: Holds patents protecting the use of NOX2 inhibitors in cancer. Martner:Cytovia, New York: Patents &amp; Royalties: Holds patents protecting the use of NOX2 inhibitors in cancer. Hellstrand:Cytovia, New York: Patents &amp; Royalties: Holds patents protecting the use of NOX2 inhibitors in cancer. OffLabel Disclosure: HDC/IL-2 is approved for the prevention of AML relapse within the EU but is not yet approved in the US.

Transgenic Expression of IL15 in CD123-Specific BiTE-Secreting Engager T-Cells Results in Improved Anti-AML Activity

Background: CD123 is frequently expressed on hematologic malignancies including 96-98% of AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) showed a promising approach anti-AML activity, however T-cell persistence was limited. Interleukin-15 (IL15) has emerged as a candidate immunomodulator as it enhances T-cell expansion and persistence, and induces long-lasting memory T-cells. To improve the efficacy and persistence of CD123-ENG T-cells we developed IL15 expressing CD123-ENG T-cells. Here, we report on the characterization and efficacy of IL15-secreting CD123-ENG T cells in vitro and in vivo models of adult AML. Methods/Results: A cDNA encoding IL15 was cloned into retroviral vectors encoding CD123-ENG or CD19-ENG (CD123-ENG.IL15; CD19-ENG.IL15). ENG T-cells were generated from human peripheral blood mononuclear cells (PBMCs) from normal donors or T-cells from AML patients by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells and T-cells expressing CD123 (CD123-ENG T-cells) served as controls. IL15 production of CD19-ENG.IL15 and CD123-ENG.IL15 T cells was confirmed by ELISA (144-159 pg/ml vs 38 and 46 pg/ml of NT and CD123-ENG T cells, p&lt;0.01, n=6). Both CD123-ENG and CD123-ENG.IL15 T-cells recognized CD123+ AML cells as judged by IL2 and interferon γ (IFNγ) production (p&lt;0.01, n=5). In contrast, NT and CD19-ENG.IL15 T-cells did not, confirming specificity. In addition, CD123-ENG.IL15 and CD123-ENG T-cells induced killing of only CD123-positive target cells as well as of primary adult patients' AML blasts in luciferase- or 7AAD-based cytotoxicity assays (p&lt;0.001, n=10). CD123-ENG.IL15 T-cells showed greater cytolytic activity than CD123-ENG T-cells as determined by luciferase activity (p=0.0002, n=3). In a Molm13 AML xenograft model, CD123-ENG.IL15 and CD123-ENG T-cells exhibited potent anti-leukemic activity as judged by bioluminescence imaging. Moreover, CD123-ENG.IL15 T cells had enhanced anti-leukemic activity and greater persistence in BMs, spleens, and livers in comparison to CD123-ENG T cells, resulting in improved anti-AML activity (Figure 1, p&lt;0.01 vs CD123-ENG T-cell group, n=12 per group) and extended survival (Figure 2, p=0.0097 vs CD123-ENG T-cell group). Finally, AML PDX models and ENG T-cells were generated from AML blasts and T-cells from 3 patients with active AML. Infusion of autologous ENG T-cells (1.5x106 cells/mouse, n=7) in AML PDX#6697688 mouse model revealed significant reduction of leukemia burden in the CD123-ENG.IL15 or CD123-ENG T-cells mouse groups but not in the mouse group with NT or CD19-ENG.IL15 T-cells or PBS (p=0.004, n=6-8). We are currently monitoring survival of these PDX models. Conclusion: We here demonstrated that transgenic expression of IL15 in CD123-ENG T-cells results in improved expansion and persistence, and anti-AML activity. These results warrant further exploration of IL15-modified CD123-targeted T-cells as immunotherapy for AML. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents &amp; Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:EMD Serono: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; ASSISI fundation of Memphis: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy. Velasquez:St. Jude: Patents &amp; Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Andreeff:Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstaZeneca: Consultancy; Amgen: Consultancy; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents &amp; Royalties: Patents licensed, royalty bearing, Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Center for Drug Research &amp; Development: Membership on an entity's Board of Directors or advisory committees; CPRIT: Research Funding; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding.

Targeted Marrow Radiation (TMI) Improves Therapeutic Efficacy of STAT3 Decoy Molecules By Augmenting Its Delivery and Immune Modulation in an AML Mouse Model

Introduction Acute myeloid leukemia (AML) is a highly aggressive form of leukemia with poor long-term survival. Our clinical development of a targeted radiation treatment for relapsed/refractory AML disease led to an impressive two-year overall survival (OS) rate of 41% (Stein et al., 2019) in contrast to &lt;10% reported for similar patients (Duval et al., 2019). Unfortunately, a lack of a preclinical model for TMI has impaired further scientific advancement. Ionizing radiation therapy (RT) has been shown to have immune modulatory functions; however, previous studies demonstrated that its efficacy is limited by activation of the radiation-induced tolerogenic TLR9/IL-6/STAT3 signaling axis in myeloid immune cells (Gao et al., 2013; Won et al., 2017). TLR9+ myeloid cells (macrophages, AML, etc) can be efficiently targeted using TLR9 agonists (CpG) conjugated with oligonucleotide (ODN) drugs (e.g., siRNA) to eliminate STAT3 signaling. Such a strategy has been shown to synergize with local tumor RT to prevent tumor recurrence (Gao et al., 2013). A CPG-STAT3-dODN inhibitor (CSI) has been previously reported as an effective immunotherapy for AML (Zhang et al., 2016), but the efficacy of immunotherapeutic agents is drastically reduced at high disease burden (Pai et al., 2019). Therefore, we hypothesized that the immunomodulatory effects of low dose RT can be augmented by blocking the STAT3 signaling in AML cells, enhancing the efficacy of CSI in a high-disease burden model. Methods We first developed a TMI treatment methodology using a Precision X-RAD small animal irradiator. A whole body CT image was acquired, and three-dimensional (3D) dose calculations were performed using a Monte Carlo dose engine-based SmART-Plan treatment system (van Hoof et al, 2013; Downes et al., 2009; Faddegon et al., 2009). Before treatment, mice were further imaged to verify target anatomy prior to delivering precise radiation doses to the entire skeletal system and spleen while sparing vital organs (lungs, liver, gut). We used a Cbfb-MYH11/Mpl (CMM)-induced mice leukemia model. At 5-10% CMM-GFP cells in peripheral blood (~20-30% in bone marrow; high disease burden), the mice were treated with three doses of CSI (2.5mg/kg) on alternate days with or without 4 Gy radiation (TMI or total body irradiation [TBI]). Cy3 labeled CSI uptake studies were carried out 48h post RT by flow cytometry, and immune cell trafficking and activation studies were conducted by harvesting bone marrow and spleen cells on day 8 post RT. Results Dose volume histogram (DVH) and radiation dose painting show that the TMI treatment plan significantly reduced doses to critical organs (lungs, liver, gut) while maintianing the same dose to the skeleton and spleen, unlike TBI (same dose to entire body) (Figure 1A-D). In TMI-treated mice, the CSI-Cy3 uptake was significantly higher in CMM cells than in the CMM cells of mice treated with CSI alone, indicating improved delivery post RT (Figure 1E, F). The trafficking of T helper cells (CD4+) and cytotoxic T lymphocytes (CD8+; CTLs) as well as effector (CD62L-CD44+) and effector memory T cells (CD62L+CD44+) is significantly augmented in the bone marrow of TMI+CSI treated mice compared to levels in mice treated with TBI+CSI or CSI alone (Figure 1G, H). An increased total number of IFNγ secreting CTLs, and a higher CD8:Treg (CD4+CD25+FOXP3+) ratio indicate enhanced anti-tumerogenic activity in TMI+CSI treated mice over that of mice treated with TBI+CSI or CSI alone (Figure 1I, J). Similarly, myeloid cell trafficking and activation was augmented in TMI-treated bone marrow (data not shown). The benefit of augmented immune modulation in TMI+CSI combinatorial therapy is reflected in a significantly increased survival (~39 median survival days) over untreated mice and those treated with CSI or TMI alone ( ~8-9 days median survival) (Figure 1K). Conclusion This is the first report of a novel radio-immunotherapy using a systemic targeted precise RT (TMI) in combination with STAT3 down-regulation (CSI) in AML. As hypothesized, low-dose targeted RT in combination with blocking of STAT3 signaling improved immune cell trafficking and activation, thereby enhancing the efficacy of this combinatorial therapy in high-disease burden. Further, newly developed low-dose TMI shows enhanced immune modulation over conventional TBI, suggesting the benefits of localized targeted RT in hematological malignancies. Disclosures Hui: Janssen Research &amp; Development, LLC: Consultancy, Honoraria.

Characterization and dynamics of specific T cells against nucleophosmin-1 (NPM1)-mutated peptides in patients with NPM1-mutated acute myeloid leukemia

Nucleophosmin(NPM1)-mutated protein, a leukemia-specific antigen, represents an ideal target for AML immunotherapy. We investigated the dynamics of NPM1-mutated-specific T cells on PB and BM samples, collected from 31 adult NPM1-mutated AML patients throughout the disease course, and stimulated with mixtures of 18 short and long peptides (9-18mers), deriving from the complete C-terminal of the NPM1-mutated protein. Two 9-mer peptides, namely LAVEEVSLR and AVEEVSLRK (13.9–14.9), were identified as the most immunogenic epitopes. IFNγ-producing NPM1-mutated-specific T cells were observed by ELISPOT assay after stimulation with peptides 13.9–14.9 in 43/85 (50.6%) PB and 34/80 (42.5%) BM samples. An inverse correlation between MRD kinetics and anti-leukemic specific T cells was observed. Cytokine Secretion Assays allowed to predominantly and respectively identify Effector Memory and Central Memory T cells among IFNγ–producing and IL2–producing T cells. Moreover, NPM1-mutated-specific CTLs against primary leukemic blasts or PHA-blasts pulsed with different peptide pools could be expanded ex vivo from NPM1-mutated AML patients or primed in healthy donors. We describe the spontaneous appearance and persistence of NPM1-mutated-specific T cells, which may contribute to the maintenance of long-lasting remissions. Future studies are warranted to investigate the potential role of both autologous and allogeneic adoptive immunotherapy in NPM1-mutated AML patients.

91P - A dual-targeting fusion protein, human PVR-4-1BBL for immunotherapy in AML

91P - A dual-targeting fusion protein, human PVR-4-1BBL for immunotherapy in AML

The Potential Utility of Immunotherapy for AML

The Potential Utility of Immunotherapy for AML

TLR9 triggering/STAT3 inhibition to reprogram leukemic cells into antigen-presenting cells and trigger T-cell responses.

118 Background: Clinical studies demonstrated potential of cancer immunotherapies while underscoring the need to block immune checkpoints shielding tumors from immune attack. We recently generated a strategy to target STAT3 transcription factor, critical immune checkpoint regulator, using TLR9-targeted delivery of STAT3 decoy inhibitor (CpG-STAT3dODN) targeting TLR9+ immune cells and all subsets of leukemic cells, including leukemic progenitor/stem cells in vitro and in vivo experiments in murine and human AML cells. Methods: Studies were conducted using mouse Cbfb/MYH11/Mpl and C1498 models of AML and acombination of flow cytometry, electron microscopy, and sequencing techniques were used to define changes in the AML cells after CpG-STAT3dODN treatment. Results: Intravenous injections of CpG-STAT3dODN into engrafted mice, led to significant changes in CD11b/CD11c expression, cell morphology, mitochondrial shape and number within the first 5 days of treatment. RNA-sequencing studies indicated increased expression of genes regulating differentiation and antigen presentation such as IRF8, GADD45A, CIITA, IL-12A, IFNγ. These effects associated with increased number of activated T cells in the leukemic microenvironment, thereby resulting in regression of AML in majority of treated mice. Two week CpG-STAT3dODN treatment was sufficient to generate protective antitumor immunity as demonstrated in rechallenge experiments. Conclusions: Our studies suggested that concurrent TLR9-stimulation and STAT3 inhibition trigger potent immunogenicity of AML cells sufficient to drive effector and memory T cell responses. We hypothesize that STAT3-inhibition can unleash immune-stimulation downstream of TLR9 signaling transforming leukemic cells into antigen-presenting cells Our further studies will dissect role of STAT3 and TLR9-induced signaling mediators (IRF, NF-κB) in regulation of the process.This information should support the design of new therapeutic regimens for AML immunotherapy using CpG-STAT3dODN alone or in combination with T cell-based therapies.