Primary Acute Myeloid Leukemia Blasts Research Articles

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

Abstract 3604: NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts

Abstract Introduction: Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD+) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D CAR and membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD−). In this study, we utilize a flow cytometry-based approach to (i) evaluate the cell surface expression of NKG2D-Ls on normal cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine NKX101 cytotoxic killing of LSC and non-LSC AML blasts. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients (n=20) and healthy donors (n=10) in accordance with approved IRB protocols. AML somatic mutation status was determined using an Illumina TruSight® Myeloid Sequencing Panel. NKX101 cytotoxicity against patient-derived AML blasts was assessed using a flow cytometry-based assay that measured specific cytotoxicity and AML blast/LSC markers. NKG2D-L expression was determined using flow cytometry with antibodies specific to individual NKG2D-Ls. Results: Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on LSCs compared to non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 potently kills patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 had similar cytotoxic potency against both LSC and non-LSC AML blasts. Conclusions: In summary, we show that NKG2D-Ls are expressed on the surface of both LSC and non-LSC AML blasts and at levels above those observed in normal hematopoietic cells. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSCs in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR-NK therapy for the treatment of r/r AML. Citation Format: Kyle Hansen, Cynthia Cho, Nishi Kothari, David Shook, James Trager. NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3604.

Adaptor Anti-P329G CAR T Cells for Modular Targeting of AML

Introduction Chimeric antigen receptor (CAR) T cell therapy has achieved promising results for treatment of B cell and plasma cell malignancies, but success has been limited in the treatment of acute myeloid leukemia (AML). Numerous antigens including CD33, CD123 or CSF1R have been proposed for AML therapy. However, clinical success of CAR T cell therapy in AML is impaired by antigen heterogeneity, therapy-associated toxicities, and antigen escape. Adaptor CAR T cells which allow sequential, transient or simultaneous targeting of multiple antigens have the potential to overcome these limitations. These adaptor CAR T cells were combined with tumor antigen-directed CAR-adaptor molecules for indirect tumor binding. We developed a modular P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human IgG1 antibodies containing P329G L234A/L235A (PGLALA) mutations. These PGLALA mutations render the Fc part inactive for binding to Fc gamma receptors (FcgRs) and to the complement system, and up to now, more than 20 such Fc-silenced therapeutic antibodies including an approved T cell recruiting bispecific antibody have been clinically validated. Compared to other modular CAR T cell platforms this approach does not rely on haptens or artificial tags fused to the targeting antibody and may ease development and testing of the platform. Methods A scFv-based second generation CAR vector system was used in primary human T cells. P329G-targeting CAR T cells were combined with PGLALA-Fc-mutated antibodies targeting CD33, CD123 or CSF1R. Classical, directly binding anti-CD33 and anti-CSF1R CAR T cells have been used as positive controls, while anti-CD19 CAR T cells and untransduced T cells have been used as negative controls. Different AML (MOLM-13, OCI-AML-3, PL-21, Mv4-11, THP-1 and U-937) and an ALL cell line (NALM-6) were used as target cell lines expressing the target antigens in different intensity. Effector function of CAR T cells was also tested against primary human AML blasts derived from patients. Luciferase-based killing assays, ELISA-based and flow-based analysis were used to evaluate CAR effector functions. Results Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, anti-P329G CAR T cells revealed specific effector functions only when combined with antibodies carrying PGLALA-Fc-mutations. Anti-P329G CAR T cells alone revealed no activation. Anti-P329G CAR T cells could be specifically activated by recombinant protein when combined with antibodies targeting CD33, CD123 and CSF1R leading to upregulation of T cell activation markers. Anti-P329G CAR T cells combined with PGLALA-mutated antibodies targeting CD33, CD123 und CSF1R achieved efficient in vitro cytotoxicity against tested AML cell lines and primary human AML blasts, while antigen-negative ALL cell line NALM-6 was not killed. Remarkably, effector functions of anti-P329G CAR T cells were similar to classical CAR T cells particularly when targeting CD33. This led to specific cytokine production when anti-P329G CAR T cells were co-cultured with respective antibodies and tumor cells. P329G-CAR T cells activated by recombinant CD33, CD123 or CSF1R showed no cytokine production in the presence of an antibody which was not recognizing the respective antigen. However, by switching the antibody after 24 h of stimulation towards the tumor antigen-targeting antibody, CAR T cell activation could be again achieved by the recombinant protein. Depleting the tumor antigen-targeting antibody after 24 h of stimulation decreased the cytokine production after 48 h and 72 h despite ongoing presence of the recombinant protein. This confirms the modularity and reversibility of this adaptor CAR T cell platform. Conclusions Taken together, anti-P329G CAR T cells combined with Fc-silenced tumor antigen-targeting IgG1 antibodies carrying the clinically validated PGLALA-mutations in the Fc part achieved profound effector functions against various human AML cell lines and primary AML blasts. The modular platform has the potential to overcome recent limitations of CAR T cell therapy in AML. These results support the rational for clinical translation of this novel and modular CAR T cell platform.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Transfer of mRNA Encoding CEBPA in Lipid Nanoparticles to CEBPA Mutated Leukemia Cell Line KO52 and Primary AML Reduces Cell Growth and Induces Myeloid Differentiation

There are known constitutional mutations associated with bone marrow failure and progression to malignancy, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). For the majority of the defects associated with clonal hematopoiesis and progression to MDS/AML, there are no specific treatments. We sought to determine if messenger RNA (mRNA) transfer can restore expression of the normal proteins, modulate abnormal proliferation and differentiation, and enhance response to chemotherapy and targeted agents. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor involved in myeloid differentiation that is mutated in ~7-20% of patients with AML (Su L et al., Frontier Oncol. 2022). A germline alteration was identified in 4.5% of AML patients with CEBPA mutation (Ho PA et al., Blood 2009). While it was originally believed that biallelic mutations of CEBPA confer favorable prognosis, more recently it was realized that a single basic region leucine zipper (bZIP) mutation in the C terminal region suffices to confer favorable risk, as categorized by ELN2022. There are currently no targeted therapies for mutated CEBPA, and no known specific therapy for patients with constitutional or acquired CEBPA mutations. Although most patients with mutated CEBPA achieve initial CR, there is a moderate relapse rate (30-50%). The objective of this study was to determine if restoration of normal CEBPA mRNA would result in expression of normal CEBPA protein in AML cell lines (e.g.-U937), as well as an AML cell line with mutated CEBPA (KO52), and in primary AML blasts, including those with mutated CEBPA. We tested whether expression of normal CEBPA would lead to phenotypic changes including overcoming differentiation block, induce apoptosis, and eliminate leukemic blasts. There are normally two translation initiation sites, leading to production of the CEBPA-p42 or CEBPA-p30 proteins. U937 has high level expression of normal CEBPA-p42, while the KO52 cell line has two CEBPA mutations, p.E50* and p.L324P, the former leading to decrease in CEBPA-p30. To study the consequences of mRNA transfer, we performed immunofluorescence, multicolor flow cytometry (MFC), and Western blot (WB) analysis for non-transfected control cells, GreenLantern mRNA lipid nanoparticle (LNP) transfected control cells, and cells transfected with two types of CEBPA mRNA LNP (#1 and #2). We observed transfection efficiencies of 90-97% in cell lines and 20-95% in primary cells. Figure 1A shows the mean fluorescence intensity (MFI) of Alexa Fluor® 647-labeled monoclonal antibody to the C-terminus of CEBPA. Wild-type CEBPA-p42 was expressed in U937 and KO52 cell lines at 2-4 times control levels by WB quantitation. With two additions of LNP at 0h and 48h, the induced CEBPA protein maintained stable expression up to 5 days. Immunofluorescence demonstrated staining of CEBPA in the nucleus, co-localizing with nuclear DAPI fluorescence. To evaluate the functional consequences of CEBPA introduction, we analyzed morphology by Wright/Giemsa staining, proliferation by cell counting, apoptosis by Annexin V staining, cell cycle using BrdU labeling assay, and immunophenotype with stem and lineage markers including CD45, CD34, CD38, CD123, CD117, HLA-DR, CD13, CD33, CD16, CD11b, CD64, CD14. Strikingly, the enhanced expression of CEBPA was associated with decrease in proliferation and fraction in S phase, and increase in apoptosis and myeloid differentiation, suggesting induction of differentiation that could have therapeutic implications similar to ATRA in acute promyelocytic leukemia. The myeloid differentiation was evident by MFC showing decreased expression of primitive markers CD34, CD117 and HLA-DR, with increased expression of CD64 (Figure 1B), CD38, and CD11b. With this method we have also transfected primary AML patient blasts including a sample that harbored 4 CEBPA mutations. We observed 95% transfection efficiency and >50% decrease in cell number relative to controls with increased myeloid differentiation. Studies of the combination of CEBPA mRNA LNP transfection with targeted inhibitors to the other mutations present in this sample are in progress. Our findings reveal a novel therapeutic strategy using CEBPA mRNA LNP applicable to inherited or acquired disorders of CEBPA. This approach of restoration of normal mRNA could have potential for therapy of many pre-malignant and malignant disorders.

Cytokine Release Syndrome Results in Reduced AML Killing By CD123 CAR T Cells

Background: Chimeric antigen receptor (CAR) T cells have repeatedly demonstrated capacity to induce high rates of response and durable remissions for B cell cancers. In contrast, CAR T cells for the treatment of acute myeloid leukemia (AML) have not yet achieved this impact. We previously demonstrated that CD123 is expressed on AML blasts at high frequency, thus making it an attractive target antigen for AML-directed CAR T cells. Methods and Results: We conducted a pilot study of CD123-directed CAR T cells (CART-123) in adults with relapsed or refractory AML. The primary objective was safety with a secondary objective of anti-leukemia efficacy. Twenty-two subjects were screened, and 20 were eligible for the trial. Fludarabine and cyclophosphamide were used for lymphodepletion (LD). Twelve subjects were infused with CART-123. There were two treatment-related deaths due to cytokine release syndrome (CRS) and infection. Four subjects (33%) achieved complete response with incomplete count recovery (CRi), three subjects had progressive disease, and five had stable disease. Of the four who achieved CRi, one subject remains alive in remission after a relapse and subsequent consolidative hematopoietic stem cell transplant (HSCT). One other subject is alive after HSCT following CART-123 failure. There was evidence of CAR T cell expansion and ten subjects (83%) experienced clinical CRS ranging from Grade I to Grade V. There was no neurotoxicity. To address the apparent paradox of high rates of CART-123 bioactivity (clinical CRS and in vivo expansion) co-occurring with low rates of response, we hypothesized that cytokines produced during CRS may undermine the anti-AML effect of CART cells. We obtained serum drawn from trial subjects at baseline (prior to LD chemotherapy) or during CRS. Serum drawn during CRS promotes primary AML survival in cell culture, while matched baseline serum from the same subjects does not. Individual cytokines secreted during CRS that signal through receptors expressed on myeloid progenitors (such as GM-CSF and Flt3 ligand) reproduced this survival advantage in primary AML blasts when tested at physiologically relevant levels. In contrast, these cytokines did not produce a pro-survival effect in primary patient B-ALL blasts. Crucially, candidate myeloid-acting cytokines, as well as serum drawn during the peak of clinical CRS, promoted resistance of AML to killing by CART-123 in vitro. Using scRNAseq of bone marrow aspirates from subjects on this clinical trial, we found that GM-CSF and FLT3L were most frequently expressed by CD4+ T cells. Exposure of primary AML samples to CRS cytokines led to upregulation of signaling (e.g. JAK/STAT5) and pro-survival pathways. In previous work in B-ALL, we showed that relative resistance to killing can translate to a functional deficit in CART-19 due to development of exhaustion in the CART cells. We similarly find that that exposure to GM-CSF reduces AML blast sensitivity to CART-123, and that the persistence of antigen leads to exhaustion in CART cells (Figure 1). Specifically, co-incubation of CART-123 cells and cytokine-exposed primary AML blasts leads to increased populations of PD-1+/CD39+/CTLA4+/LAG3+ CAR T cells, consistent with an exhaustion phenotype. Finally, we found that anti-apoptotic effects of cytokines can be prevented, and CART-123 killing of AML can be restored, via ruxolitinib blockade of JAK/STAT signaling in both in vitro and in vivo settings. Significance: Our work reveals CRS as active in undermining CART-123 lethality first by promoting survival of AML, and subsequently by exhausting CAR T cells. This is in stark contrast to its role in lymphoid malignancies and underscores biological differences in myeloid versus lymphoid neoplasms. These results highlight a need to tailor therapy to limit cytokine effects on AML blasts to improve outcomes with AML-directed CAR T cell therapy.

Selective Targeting of TP53-Y220C Mutant AML By PC14586 Results in TP53 Wild-Type Conformation and Synergistical Apoptosis Induction By Concomitant Inhibition of Xpo-1, MDM2, or Bcl-2

Patients with TP53-mutant acute myeloid leukemia (AML) constitute the worst outcome group with a survival of only several months. TP53 mutations frequently become dominant following exposure to chemotherapeutic agents, Bcl-2 or MDM2 inhibitors, and confer resistance to multiple other therapies. Y220C is a recurrent hotspot TP53 mutation observed in solid tumors and hematological malignancies, predominantly in AML and MDS (Gener-Ricoset al., ASH 2022). PC14586 (PMV Pharmaceuticals) is designed to bind to the p53-Y220C mutant and restore wild-type (WT) p53 protein conformation and function. PC14586 has been shown to convert Y220C-p53 mutant into WT-p53 protein conformation, induce the activation of p53 transcriptional pathways resulting in selective inhibition of proliferation ofTP53-Y220C mutant solid tumor cell lines and achieved partial responses and stable disease in an ongoing phase I clinical trial in solid tumors (Dumbrava, ASCO 2022, NCT04585750). However, the activity of PC14586 in TP53-Y220C mutated AML/MDS has not been investigated, either as monotherapy or in mechanism-based drug combinations. We investigated the mechanisms of action of PC14586 in AML using cells with TP53-WT, knockout (KO), and mutations (one allele with Y220C or R175H and the other truncated) generated by CRISPR (Boettcher et al., Science 2019) and found that PC14586 converts Y220C mutant p53 to the active form of p53 and selectively activates p53 signaling proteins including MDM2 and p21 in TP53-Y220C AML cells. PC14586 decreased cell viability but had limited apoptogenic activities in the TP53-Y220C AML cells . In primary AML cells with Y220C mutations, PC14586 induced cell death in bulk AML cells and, importantly, also in stem/progenitor cells in 2/3 samples. We reported previously that sustained nuclear retention of p53 by XPO-1 inhibition greatly enhances the transcriptional activity of activated p53 and that activation of p53 by inhibiting its negative regulator MDM2 effectively induces cell death in TP53-WT AML (Kojima et al., Blood 2013, 2005), and that XPO-1 inhibition vastly increased apoptosis induced by MDM2 inhibition through 20-60-fold increased p53 transcriptional activities. We also reported that combined Bcl-2 inhibition and p53 activation is synthetically lethal in TP53-WT AML (Pan et al., Cancer Cell 2017). We hypothesized that XPO-1 synergizes with PC14586 by retaining PC14586-activated p53 in the nucleus and that inhibition of MDM2 induced by PC14586-mediated p53 activation or inhibition of Bcl-2 synergizes with PC14586 in TP53-Y220C AML cells. Indeed, we found that PC14586-induced p53 target proteins were further upregulated when PC14586 was combined with XPO-1 or MDM2 inhibition. Inhibition of XPO-1, MDM2, or Bcl-2 synergistically induced massive apoptosis and decreased cell viability in PC14586 treated TP53 Y220C mutant AML cells (Fig.1), in primary AML blasts, and in CD34+CD38- stem/progenitor cells but not in normal bone marrow cells or stem/progenitor cells through conformational changes of mutant p53 to p53-WT. While PC14586 had limited activity on proliferation in TP53-Y220C AML cells, the combination with Bcl-2 inhibition largely blocked G1-S transition and enhanced apoptosis. The G1-S transition block and apoptosis induction were even more pronounced when PC14586 was combined with XPO-1 or MDM2 inhibitors as determined by flow cytometry measurement of DNA content, and PARP cleavage. Experiments in TP53-Y220C AML PDX models are ongoing and will be reported. In summary, PC14586 converts Y220C-mutant p53 into WT-p53 conformation and induces p53 target proteins. Mechanism-based combinations with XPO-1, MDM2, and Bcl-2 inhibitors induce massive apoptosis. PC14586 is presently in early clinical trials.

AML Inhibits Lipid Uptake By the Liver through an HGF/c-MET Dependent Mechanism Which Results in an Increased Supply of Free Fatty Acid to the Tumor

Acute myeloid leukemia (AML) requires a high metabolic turnover to allow for constant expansion. We and others have previously shown that primary human AML blasts rely on fatty acid (FA)-oxidation for survival and proliferation. (Shafat MS, et al. 2017, Jones CL, et al. 2018). The liver plays a major role in carbohydrate, protein, amino acid, and lipid metabolism (Hodson L, and Gunn PJ. 2019). Given that AML is a FA dependent tumor, we have studied the interactions between AML and the liver, and specifically how these affect lipid metabolism, the availability of pro-tumoral FFA and ultimately the mechanism by which this supports AML growth. To understand the role of lipid metabolism we used two syngeneic models of AML (HOXA9/Meis1 or MN1). When engrafted with AML cells, mice body weight during the latter stages of disease. After sacrifice, the size of epididymal and inguinal fat pads were significantly reduced in mice with AML compared to controls. Mice with AML also had elevated free fatty acid (FFA) and reduced glucose in the plasma. We and others have shown that AML induces lipolysis of white adipose tissue for uptake by blasts and resulting in tumor expansion (Shafat MS, et al. 2017, Ye H, et al. 2016). Since the liver is the master regulator of available FA in the plasma, we isolated livers from AML engrafted mice. Transcriptomics revealed down-regulation of genes involved in FA uptake and metabolism. RT-qPCR of RNA isolated from liver samples confirmed these results showing down-regulation of FA transport proteins, (FABP1, SLC27A2, SLC27A5, and CD36) and FA metabolism genes (CPT1, ACADM, and HMGCS2). Furthermore, primary hepatocytes isolated and cultured with AML conditioned media showed a down-regulation of FA uptake and metabolism genes. These data support the hypothesis that AML secretes a factor that alters lipid metabolism in the liver. To discover the factor that AML secretes, and which regulates FA uptake and metabolism in the liver, a proteome profile of the serum from AML engrafted mice was compared to conditioned media from AML cultured cells. Pathway analysis revealed hepatocyte growth factor (HGF), IL-1B and Granulocyte colony stimulating factor (G-CSF) as possible key factors involved in liver FA metabolism (Jing Y, et al. 2019, Kaibori M, et al. 2002). In vitro gene expressions analysis of primary hepatocytes revealed that addition of HGF to the medium, but not Il-1b or G-CSF, inhibited expression of FA uptake genes. In addition, using Seahorse mitochondrial stress kit we confirmed that primary hepatocytes decreased FA oxidation when cultured with AML conditioned media or HGF. Next, we confirmed that AML derived HGF was responsible for inhibition of hepatocyte FA genes by using a neutralizing antibody to HGF which blocked the transcriptional changes previously observed in hepatocytes cultured in AML conditioned media. This showed that AML derived HGF downregulates hepatocyte FA uptake and metabolism. Finally, to determine the role of AML in the uptake of FA by hepatocytes, cultures of primary murine hepatocytes were pretreated with AML conditioned media or HGF with and without neutralizing antibody. A BODIPY conjugated long chain FA was then added to the culture. Images analysis showed hepatocytes had reduced lipid uptake in the presence of AML condition media or HGF, but this was restored with the addition of HGF neutralizing antibody. Here we report that AML inhibition of lipid uptake in the liver, through an HGF dependent mechanism, increases the availability of plasma FA and forms a fundamental part of the AML disease. Ongoing work to develop our understanding of how AML increases access to FA to aid proliferation, may aid in the development of new therapeutics to target liver metabolism in combination with other AML therapies.

STI-8591, a Novel Tyrosine Kinase Inhibitor, Shows Excellent Antileukemic Activity in FLT3-Mutated (mut) Acute Myeloid Leukemia (AML)

FMS-related tyrosine kinase 3 ( FLT3) mutations, including internal tandem duplications ( FLT3-ITD) and tyrosine kinase domain ( FLT3-TKD), are found in approximately 30% of AML patients. FLT3 has emerged as a promising molecular target in therapy of AML. Herein, we report on the activity of STI-8591, a novel kinase inhibitor developed by Hangzhou ACEA Pharmaceutical Research Co, in AML models carrying FLT3 mut, including those associated with TKI resistance [e.g., FLT3-ITD(F691L)]. We first determined the in vitro antileukemic effects of STI-8591 in FLT3 mut human AML cell lines (MV4-11, MOLM-13), a murine cell line Ba/F3 transduced with FLT3-TKD(D835Y)[Ba/F3 FLT3-TKD(D835Y)] or FLT3-ITD-TKD(D835Y) [Ba/F3 FLT3-ITD-TKD(D835Y)] or FLT3-ITD/F691L [Ba/F3 FLT3-ITD(F691L), which has been associated with drug-resistant, and primary blasts from FLT3 mut AML patients. STI-8591 effectively inhibited cell proliferation and induced apoptosis in FLT3-ITD/TKD mut cell lines (IC 50: 0.33 to 9.19nM at 24 hours). Of note, STI-8591 demonstrates 4-7-fold higher antileukemic activity on otherwise resistant Ba/F3 FLT3-ITD(F691L ) cells than the currently available FLT3 inhibitors, Gilteritinib and Quizartinib. Similar antileukemic activity of STI-8591 was observed on primary FLT3 mut AML blasts (median IC 50: 13.67nM, n=5). The antileukemic mechanism of STI-8591 is associated with inhibition of the phosphorylation of the mutated FLT3 protein and its downstream targets (i.e., STAT5, AKT, and ERK) as shown by western blot analysis. To test the in vivo antileukemic activity of STI-8591, we utilized 4 FLT3 mut AML murine and human models, obtained by transplanting: 1. BM mononuclear cells (MNCs) from CD45.2 MLL PTD/WT FLT3 ITDITD AML mice congenic CD45.1 B6 mice; 2. murine cell lines [i.e. Ba/F3 FLT3-TKD(D835Y) , Ba/F3 FLT3-ITD-TKD(D835Y), or Ba/F3 FLT3-ITD(F691L), 3. Luciferase-expressing FLT3 mut MOLM13 cells (MOLM13 Luci) into NCG mice; 4. primary blasts from FLT3 mut AML patients into NSGS mice. We treated these mice for 3-4 weeks [3 to 30mg/kg/day(d), oral gavage]. For the MLL PTD/WT FLT3 ITDITD model (n=10 mice/group), compared with Vehicle(V)-treated mice [median survival: 44 d], we observed significantly lower engraftment and longer overall survival (OS) in mice treated with STI-8591 10mg/kg (52 d, p<0.0001) or 30mg/kg (57 d, p<0.0001) or Gilteritinib 10mg/kg (49 d, p=0.0029). At 10mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0195). Treatment of STI-8591 also demonstrated significant benefits to mice transplanted with FLT3 mut Ba/F3 cells. For the TKI-resistant Ba/F3 FLT3-ITD(F691L) model (n=10/group), STI-8591 (median:19 and 37 d for 10 and 30 mg/kg) and Gilteritinib (17 and 24 d for 10 and 30 mg/kg) -treated mice had longer OS than V-treated (12 d) mice. At equivalent dosage (10 and 30mg/kg), STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0013 and p<0.0001). For the MOLM13 Luci+ model (n=5/group), mice receiving STI-8591 or Gilteritinib treatment demonstrated a significant reduction in disease burden as observed by bioluminescence imaging (BLI), and prolonged OS. Compared with V-treated mice (median: 23 d), we observed longer OS in Gilteritinib (10mg/kg, 33 d, p=0.0023) and STI-8591 (3mg/kg, 27 d, p=0.0179; 10mg/kg, 34 d, p=0.0023; and 30mg/kg, 35 d, p=0.0023) -treated mice. At 10 mg/kg dosage, STI-8591- and Gilteritinib- treated mice showed no significant difference in OS (p=0.2411). Similar experiments are ongoing in FLT3 mut primary AML patient-derived xenografts (PDXs) and will be reported at the ASH meeting. To evaluate antileukemic activity of STI-8591 in mice bearing disease in central nervous system (CNS), we implanted 5×10 5 /3 µL MOLM13 cells intracranially into SCID mice to construct the CNS leukemia model (n=9/group). Compared with V-treated mice (median: 11 d), we observed a longer OS in mice treated with Gilteritinib (10 mg/kg, 18 d, p=0.0004) or STI-8591 (10 mg/kg, 23 d, p<0.0001; and 30 mg/kg, 26 d, p<0.0001). At 10 mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.005). In conclusion, STI-8591, a novel TKI exhibited a potent antileukemic activity in various FLT3 mut AML models, including those carrying FLT3 mut associated with drug resistance. These results supported further clinical development of STI-8591 for treatment of AML.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.

IL1RAP-Specific T Cell Engager (TCE) Antibody Efficiently Depletes Acute Myeloid Leukemia (AML) Leukemic Stem Cells (LSCs)

LSCs are self-renewal primitive cells that initiate and maintain AML. Because they are highly therapy resistant, they are often responsible for treatment failures. Due to the lack of a specific antigen (Ag) and their quiescent nature, LSCs have been challenging to target and eliminate. IL1RAP is highly expressed on most human AML cell lines and primary blasts, including LSC-enriched CD34+CD38- cells, but not on normal hematopoietic stem cells (HSCs). To leverage this observation, we generated over 50 unique mouse IL1RAP monoclonal (m) antibodies (Abs), grouped them based on sequence, and selected representative members from the clades to generate murine-human chimeric mAbs. One of the mAbs, termed #24, had the strongest antibody-dependent cell-mediated cytotoxicity (ADCC) properties and the greatest in vivo anti-AML activity. Thus, it was advanced to generate a bispecific (Bs) anti-IL1RAP/CD3 TCE Ab termed BiF002, which incorporates a human IgG1 Fc mutated to lack FcR binding. Incubation of BIF002 with healthy donor T cells and AML cell lines or primary AML blasts induced IL1RAP dependent T cell activation, and Ab-dependent T cell lysis of leukemic cells in a dose-, time- and effector to target (E: T) ratio-dependent manner. The IC 50 against MOLM13 and THP-1 was subnanomolar (0.047nM, 95%CI:0.036-0.060nM and 0.025nM, 95%CI:0.14-0.40nM, respectively), at E:T ratio 5:1 at 48 hours using resting T cells. The IC 50 of IL1RAP pos AML bulk and CD34+ blasts (n=6) was also subnanomolar (0.45nM; 95%CI: 0.28-1.96nM). Co-cultures of normal CD34+ cells with BIF002 and T cells did not activate T cells or eliminate the normal CD34+ cells. To test the antileukemic activity of BIF002 in vivo, we engrafted luciferase (luc)-expressing MOLM13 cells into NSG-SGM3 (NSGS) mice. On day (D) 3, in vitro expanded healthy donors' T cells (3x10 6/mouse, weekly), with either BIF002 or a control IgG (10µg,) were administered intravenously (IV) every 3 days x 4-weeks. Mice treated with T cells + BIF002 exhibited a 100-fold greater tumor reduction on D33 than other groups, as measured by bioluminescence imaging (BLI), and had a significantly longer overall survival (OS; median 54.5 D) compared to IgG (27 D, p=0.0045), BIF002 alone (36 D, p=0.0049), or T cells + IgG (33 D, p=0.0062) treated controls. In initial dose finding studies using a similar schema, lower BIF002 doses (0.1 and 1 µg) were tested. Mice treated with T cells and 1 µg of BIF002 had a 100-fold tumor reduction on D29 and longer OS (median 57 D) compared to T cells + 0.1µg of BIF002 (31 D, p=0.0018) or T cells + vehicle (34 D, p=0.0019). We also engrafted luc-expressing primary blasts from a relapsed/refractory complex karyotype AML patient into NSGS mice that treated for 3 weeks using the above described schema (10µg). In this experiment, we used as control BIF026, a bispecific Ab (BsAb) carrying point mutations in both Fab arms to abrogate target binding to IL1RAP and CD3. Mice treated with T cells + BIF002 exhibited a >1000-fold reduction in disease burden and longer OS than BIF026 or BIF002 alone or T-cells+ BIF026-treated mice (median 46 D vs 26, 25 or 30 D, respectively, all comparisons, p=0.0003). In a second patient-derived xenograft (PDX) model, blasts from a relapsed complex karyotype AML patient were engrafted into NSG mice. Treatment was initiated on D10, with T cells given weekly along with 10µg of BIF002 or BIF026 for 3 weeks. After 70 days, all mice treated with T cells + BIF002 were alive, with no detectable blasts in peripheral blood or bone marrow (BM), while all vehicle (median 38 D, p=0.0002) and T cells + BIF026 (38 D, p=0.0001) treated controls had died of leukemia. To determine if T cells + BIF002 eradicated LSCs, we randomly selected 3 female mice from each group on D 34 and transplanted BM mononuclear cells (MNC) into secondary NSGS recipients (n=7). At D 90, no recipients of BM from donors treated with T cells + BIF002 had detectable blasts and remained alive, while all vehicle (median 26 D, p=0.0004) and T cells + BIF026 (median 26 D, p=0.0002) treated controls succumbed to AML. Importantly, no obvious hematopoietic or non-hematologic side effects were observed after BIF002 administration alone or with activated T cells in normal and leukemic mice. In summary, we provide evidence of the subnanomolar potency and efficacy of BIF002, a novel anti-IL1RAP/CD3 TCE, in eliminating AML blasts, including LSCs. BIF002 is currently undergoing IND-enabling studies.

Immunologic Targets in AML.

In this issue of Blood Cancer Discovery, Nelde and colleagues used a sensitive mass spectrometry-based immunopeptidomics approach to characterize the antigenic landscape of acute myeloid leukemia (AML) and were able to identify immunogenic peptides presented by both leukemia stem cells (LSC) and bulk primary AML blasts. These immunogenic peptides elicit primarily CD4 T-cell responses and the diversity of the HLA class II immunopeptidome and presence of CD4 memory T-cell responses were both associated with improved clinical outcome. See related article by Nelde et al., p. 468 (1) .

Preclinical development of ARD103, a CLL-1 CAR-T produced by a 3-day DashCAR technology, for acute myeloid leukemia.

119 Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of the AML patients experienced primary refractory or relapse after current treatments, such as chemotherapy, hematopoietic stem cell transplantation, and targeted therapy. AML patients are in urgent need of novel therapeutic strategies. Leukemic stem cells (LSCs), which cause the treatment failure and leukemia relapsed. Human C-type lectin-like molecule-1 (CLL-1) is a surface antigen expressed on both AML malignant blast cells and LSCs, but not on normal hematopoietic stem cells, suggesting that CLL-1 is a compelling target with low off-tumor toxicity for novel AML treatment. Methods: ARD103 is a second-generation chimeric antigen receptor (CAR)-T cell, expressing a novel extracellular anti-CLL-1 scFv fused to a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ activation domain. ARD103 was generated using lentiviral vector in an optimized 3-day DashCAR manufacturing process and demonstrated high percentage of CAR population (>80%), high percentages of memory T cells (Tscm and Tcm are >70%) and profound effector cytokine production (IFN-γ, granzyme A, perforin and granulysin). Results: ARD103, CLL-1 CAR-T exhibited specific killing on various CLL-1-expressing AML cell lines, such as U937, THP-1, and HL60, correlated to the levels of CLL-1 expression. In the AML U937-Luc xenograft mouse model, the CLL-1 CAR-T cells prolonged the survival time significantly (p < 0.0001), with superior CAR-T cell persistence, to the end point of the study at 48 days compared to the median survival time at 23 days in the untreated or control groups. Furthermore, the xenograft mice receiving as minimum as 3 x105 CAR-T cells per mouse were able to elicit effective recall responses after tumor rechallenge and survived for more than 60 days. The off-target binding interactions of ARD103 CAR-T cells using a human cell microarray showed a specific and strong interaction with CLL-1 but not with other human membrane/extracellular proteins. In the aspect of on-target off-tumor toxicity, the hematotoxicity assessment on ARD103 exerted modest (5%-23%) or no effect on colony formation inhibition of CD34+ cells derived from human normal bone marrow or peripheral blood, respectively. Furthermore, autologous ARD103 cells were successfully prepared from relapsed/refractoryAML patients and demonstrated to mediate specific cytotoxicity to primary AML blasts and LSCs derived from the same patients. Conclusions: Taken together, the ARD103 CLL-1 CAR-T candidate has demonstrated excellent anti-AML potency and efficacy with minimal safety concerns, and warrant to be further developed preclinically and clinically for the treatment of relapsed/refractory AML patients. Keywords: Acute myeloid leukemia; CLL-1; Chimeric antigen receptor-T cell; Immunotherapy.

Abstract 4418: Heterogeneity of IFN-γ responsiveness in myeloid malignancies- implication for the possible impact on IFN-γ immunotherapy

Abstract Introduction: Allogeneic stem cell transplantation (alloSCT) can potentially cure hematologic malignancies through graft-versus-leukemia effect (GVL). Several studies have suggested that IFN-γ pathway is critical for GVL sensitivity or resistance in leukemia cells. In mouse model, acute myeloid leukemia (AML) generated in IFN-γ receptor-deficient mice acquired resistance to GVL (Matte-Martone, Shlomchik WD, J Clin Invest. 2017). Other groups have reported that IFN-γ restores HLA expression on myeloid blasts from patients who relapsed AMLs after alloSCT. These data support the clinical application of IFN-γ to augment GVL in subjects with post-transplant relapse. However, AML is molecularly heterogeneous, and the IFN-γ responsiveness may vary by AML subtypes. We analyzed IFN-γ responsiveness of primary AML cells and investigated the role of IFN-γ on cell death and T-cell recognition of AML cell lines. Methods: Primary AML cells collected at diagnosis (n=60), at relapse after chemotherapy (n=4), and at post-transplant relapse (n=8) were stimulated with IFN-γ at 1 ng/ml or 50 ng/mL in vitro, and IFN-γ responsiveness was analyzed by measuring phosphorylation of STAT1 (pSTAT) after 15 minutes and upregulation of HLA-DR after 48 hours by flow cytometry. U937 and THP-1 cell lines were used to investigate IFN-γ induced cell death and the impact of IFN-γ priming of leukemia cells on the activation of alloreactive T cells. For this, Jurkat cells transduced with anti-HA1 T cell receptor (αHA1-TCR-T) were used, and the activation was analyzed through CD69 upregulation by flow cytometry. Results: IFN-γ responsiveness was heterogeneous in both pSTAT1 and HLA-DR upregulation of primary AML blasts. IFN-γ responsiveness also varied within the subpopulation of leukemic blasts within the sample at diagnosis and relapse, implying clonal evolution. Compared to THP-1, U937 cells fail to upregulate HLA-DR with IFN-γ despite appropriate pSTAT1 response to IFN-γ, suggesting silencing of downstream IFN-γ activation site (GAS) and transcription of IFN-stimulated genes (ISGs). IFN-γ directly induced cell death in THP-1 in vitro, while U937 was resistant to IFN-γ induced cell death. Priming of THP-1 with IFN-γ significantly improved the affinity of αHA1-TCR-T to peptide-stimulated THP-1. Conclusion: IFN-γ responsiveness of AML blasts was heterogeneous, and cell line data suggest an impact of IFN-γ sensitivity on IFN-γ mediated cell death and alloreactive T cell recognition. Future studies aim to understand the mechanisms underlying IFN-γ sensitivity or resistance in AML, which would help to design clinical trials by selecting the patients who would obtain maximal benefit from IFN-γ therapy in post-transplant relapse (NCT04628338). Citation Format: Kedwin Ventura, Iuliia Kovalenko, Jui-En Ray Lee, Biswas Neupane, Daniel Stapor, Sawa Ito. Heterogeneity of IFN-γ responsiveness in myeloid malignancies- implication for the possible impact on IFN-γ immunotherapy. [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 4418.

Therapeutic targeting of PRAME with mTCRCAR T cells in acute myeloid leukemia.

Preferentially Expressed Antigen in Melanoma (PRAME), a cancer-testis antigen, provides an ideal target for immunotherapy in acute myeloid leukemia (AML). We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a chimeric antigen receptor (CAR) construct encoding a targeting domain based on T-cell receptor (TCR) mimic antibodies that target the peptide-HLA complex. We used the antibody sequence from a previously designed TCR mimic (mTCR) antibody, Pr20, that recognizes the PRAME ALY peptide in complex with HLA-A∗02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary samples from patients with AML and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to appropriate controls, PRAME mTCRCAR T cells demonstrate target-specific and HLA-mediated invitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. Invivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared with unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with interferon gamma, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.

Lycorine induces apoptosis of acute myeloid leukemia cells and inhibits triglyceride production via binding and targeting FABP5.

Acute myeloid leukemia (AML) is the most common hematopoietic malignancy with abnormal lipid metabolism. However, currently available information on the involvement of the alterations in lipid metabolism in AML development is limited. In this study, we demonstrate that FABP5 expression facilitates AML cell viability, protects AML cells from apoptosis, and maintains triglyceride production. Our bioinformatics analysis revealed that FABP5 expression was upregulated and correlated with unfavorable overall survival of AML patients. FABP5 expression may be used to distinguish normal and AML with high accuracy. FABP5-based risk score was an independent risk factor for AML patients. AML patients with highly expressed FABP5 predicted resistance to drugs. In vitro study showed that FABP5 expression was remarkably elevated in primary AML blasts and an AML cell line. Silencing FABP5 expression attenuated AML cell viability, reduced triglyceride production and lipid droplet accumulation, and induced apoptosis. We utilized AutoDock online tool to identify lycorine as an FABP5 inhibitor by binding FABP5 at amino acid residues Ile54, Thr56, Thr63, and Arg109. Lycorine treatment downregulated the expression levels of FABP5 and its target PPARγ, impaired AML cell viability, triggered apoptosis, and reduced triglyceride production in AML cells. These results demonstrate that FABP5 is critical for AML cell survival and highlight a novel metabolic vulnerability for AML.

Control of acute myeloid leukemia by a trifunctional NKp46-CD16a-NK cell engager targeting CD123

CD123, the alpha chain of the IL-3 receptor, is an attractive target for acute myeloid leukemia (AML) treatment. However, cytotoxic antibodies or T cell engagers targeting CD123 had insufficient efficacy or safety in clinical trials. We show that expression of CD64, the high-affinity receptor for human IgG, on AML blasts confers resistance to anti-CD123 antibody-dependent cell cytotoxicity (ADCC) in vitro. We engineer a trifunctional natural killer cell engager (NKCE) that targets CD123 on AML blasts and NKp46 and CD16a on NK cells (CD123-NKCE). CD123-NKCE has potent antitumor activity against primary AML blasts regardless of CD64 expression and induces NK cell activation and cytokine secretion only in the presence of AML cells. Its antitumor activity in a mouse CD123+ tumor model exceeds that of the benchmark ADCC-enhanced antibody. In nonhuman primates, it had prolonged pharmacodynamic effects, depleting CD123+ cells for more than 10 days with no signs of toxicity and very low inflammatory cytokine induction over a large dose range. These results support clinical development of CD123-NKCE.

A Phase 1, Open-Label, Safety, Tolerability, and Efficacy Study of NC525 in Subjects with Advanced Myeloid Neoplasms

Background With the exception of certain select subtypes, Acute Myeloid Leukemia (AML) remains incurable for a majority of patients with current therapies. Despite recent drug approvals targeting actionable molecular mutations and BCL-2, relapse remains inevitable for most patients with AML due to leukemia-initiating cells and progenitors {i.e., leukemic stem cells (LSCs)} which are resistant to conventional cytotoxic drugs. Novel agents that preferentially target LSCs to lessen or eliminate the risk for relapse while avoiding significant myelosuppression by sparing normal hematopoietic stem cells (HSCs), are highly sought-after anti-leukemia therapeutics. Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR-1) is an immune inhibitory receptor that is present on most immune cell subsets, including T cells, NK cells, monocytes, macrophages, dendritic cells, and granulocytes. LAIR-1, a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motifs (ITIM), binds to endogenous collagen ligands and plays an important role in inducing an immunosuppressive microenvironment. In AML, the engagement of LAIR-1 inhibits intracellular downstream survival signals, leading to decreased GM-CSF-mediated survival and proliferation of AML blasts. LAIR-1 is expressed on AML blasts and LSCs regardless of FAB subtypes, cytogenetics or molecular mutations. Recent work demonstrates that myeloid leukemia cell lines, primary AML blasts, monoblasts and monocytes, and MDS-EB-2 blasts all express LAIR-1. Critically, while high expression of LAIR-1 is characteristic of LSCs, this receptor is minimally detectable on normal HSCs, making it an ideal anti-leukemic target. NC525 is a humanized immunoglobulin gamma 1, kappa (IgG1κ) monoclonal antibody that targets LAIR-1-expressing AML blasts and LSCs. Extensive IND-enabling preclinical studies have shown the following features of NC525: (i) Inhibition of colony formation of AML CD34+ bone marrow cells; (ii) sparing of colony formation and differentiation ability of healthy CD34+ HSCs; (iii) direct ex vivo killing of AML blasts without killing healthy leukocytes; (iv) suppression of AML in both MV4-11 and THP-1 CDX models; (v) restriction of MV4-11 in a humanized murine model, without depletion or suppression of human T cells; (vi) restriction of MV4-11 tumor growth in murine blood, spleen, and bone marrow; and (vii) reduction of AML disease burden in primary patient-derived AML xenograft (PDX) models. Finally, NC525 completely inhibits leukemia propagation in secondary transplant models, suggesting that NC525 is capable of eradicating LSCs. Furthermore, NC525 destroys leukemic cells by ligation-mediated apoptosis of leukemic blasts and stem cells without evidence of an immunomodulatory effect on other immune subsets. Methods This is an open-label, non-randomized, Phase 1 dose escalation study to determine the safety and tolerability of NC525 in adult patients with advanced myeloid neoplasms. This study will also assess the clinical benefit of NC525. Eligible patients must have relapsed or refractory AML, myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML) that has progressed after standard therapy. Participants will receive NC525 by intravenous infusion on Days 1 and 15 of each 28-day cycle. During Phase 1, the safety and tolerability of NC525 will be evaluated by exploring escalating dose levels of NC525. In addition, the pharmacologically active dose (PAD), defined as the dose that provides a maximal biologic effect, such as achieving a complete remission (CR), CR with partial hematological recovery (CRh) or CR with incomplete hematological recovery (CRi), and the maximum tolerated dose (MTD) of NC525 will be determined. Safety and tolerability will be further assessed through the expansion of a few dose levels to determine an optimal recommended Phase 2 Dose (RP2D) and administration schedule of NC525, based on the PK/PD profiles. Taken together, preclinical data suggest that targeting LAIR-1 in AML and other myeloid malignancies may be an effective therapy for these diseases with the advantage of specifically eradicating leukemic stem cells and sparing normal HSCs.

Senti-202, a Selective, Off-the-Shelf, Preclinical CAR-NK Cell Therapy with CD33 and/or FLT3 Activating CAR, Healthy Cell Protection from Endomucin (EMCN) Inhibitory CAR and Calibrated Release IL-15 for Hematologic Malignancies Including AML

Background: Patients with CD33 and/or FLT3 malignancies, which encompass most myeloid malignancies such as acute myeloid leukemia (AML), have a grim prognosis and a high unmet need. There are no approved cell therapies for patients with AML due to paucity of targets and an immunosuppressive bone marrow milieu. SENTI-202, a preclinical CAR-NK cell is engineered with a logic-gated gene circuit to overcome these challenges. Methods: SENTI-202 is engineered with a FLT3 OR CD33 NOT EMCN logic-gated gene circuit and a proprietary calibrated release IL-15 (crIL-15). These 3 chimeric proteins are engineered onto a natural killer (NK) cell (healthy adult peripheral blood-derived) to create an off-the-shelf chimeric antigen receptor (CAR) NK cell therapy. The bivalent CD33 OR FLT33 (OR GATE) activating CAR (aCAR) enables concurrent targeting of CD33+ and/or FLT3+ AML cells which kills both AML blasts and leukemic stem cells (LSCs) in vitro and in vivo. This concurrent targeting of two tumor antigens could potentially provide longer remission and less chance of relapse. The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) protects healthy EMCN+ hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) from off-tumor toxicity to potentially aid in post-treatment reconstitution of a healthy hematopoietic system. The crIL-15 provides both autocrine and paracrine IL-15 stimulation to the CAR-NK cells (and surrounding immune cells) to promote cell expansion, persistence, and tumor killing. While previous presentations (Garrison et al., 2021) focused on the performance of individual SENTI-202 components in a cell, this presentation focuses on functional data from the complete logic-gated gene circuit and the crIL-15 together in a cell (i.e., the final CAR-NK with all 3 genetic elements). Results: We demonstrated delivery of all circuit components using a single retroviral vector. We then investigated activity of the resulting CAR-NK cells using a traditional in vitro cytotoxicity assay (E:T=1:2; 20hrs) in which SENTI-202 showed >90% killing of leukemia cells (e.g. SEM; P≤0.001), maintained significant serial killing potential (3 rounds; P≤0.0001; P≤0.001; P≤0.001), as well as robust killing against primary AML blasts and LSC-enriched target cell populations. Importantly, SENTI-202 also exhibited significant tumor cell killing and improved survival (P≤0.001) in an MV4-11 xenograft AML mouse model. We investigated the ability of SENTI-202 to reduce off-tumor toxicity toward EMCN+ healthy cells, including primary HSCs. Within in vitro assays, SENTI-202 provided >50% protection to FLT3+ CD33+ model healthy cells that also expressed the EMCN safety antigen, and similar significant protection was maintained through serial protection assays (3 rounds; P≤0.0001; P≤0.001; P≤0.0001). As further evidence of the selectivity imparted using the OR/NOT logic gating approach, in vitro assays with SENTI-202 resulted in killing of leukemia cells but not primary healthy human HSCs and HPCs (~42% protection) (P≤0.01). Finally, in the presence of SENTI-202 in vivo, the NOT GATE protected and enabled significant (P≤0.0001) expansion of EMCN+ FLT3+ CD33+ model healthy cells. Conclusions: In summary, preclinical evaluation of SENTI-202 demonstrated selective killing of CD33 and/or FLT3 expressing AML cell lines while protecting healthy HSCs and HPCs. This selectivity is imparted by the novel logic-gated gene circuit engineered into healthy adult NK cells. Clinical evaluation of SENTI-202 is planned to evaluate the safety and efficacy in patients with hematologic malignancies with high unmet need, including AML.