NOD-scid IL2Rgammanull Research Articles

Results of a Non-Interventional Clinical Trial of Apograft — a Novel Technology for GvHD Prevention

IntroductionGvHD is a threat to the wellbeing and survival of patients following hematopoietic stem cell transplantation (HSCT). In-vivo and ex-vivo T cell depletion are the most effective methods for GvHD prevention, but attendant risks of infection, graft failure and leukemic recurrence offset the advantages of these manipulations. Less extensive T cell depletion leaves many patients at risk for the ravages of treatment-resistant GvHD. Alternative approaches to eliminate GvHD-causing cells are needed. Fas ligand (FasL), a member of the tumor necrosis factor (TNF) family, can selectively induce apoptosis of mature T cells while sparing stem and progenitor cells. We have developed a novel FasL-mediated selection process which eliminates mature T cell subsets in hematopoietic stem cell grafts and effectively prevents GvHD in pre-clinical models without impairing engraftment and immune system recovery. We report here data from ex-vivo and in-vivo studies supporting the use of FasL-mediated depletion of mature T-cells, a process we have named ApoGraft, as a novel effective method for GvHD prophylaxis in the context of allogeneic HSCT.Methods:Aliquots of mobilized peripheral blood cells (MPBC) were collected from 104 consenting healthy donors and were used ex-vivo for optimization of the ApoGraft manufacturing process. ApoGraft is manufactured under GMP guidelines, and includes two washing steps, a 2 hr incubation of the graft at 37oC with human recombinant different concentrations of FasL (MegaFasL, Adipogen), and 2 additional washing steps to remove unbound FasL. Aliquots from 25 MPBC apheresis that had been subjected to the complete ApoGraft process were analyzed ex-vivo for cell yield, viability and cellular constitution by FACS and for clonogenicity potential by colony forming unit (CFU) assays. Additionally, engraftment and GvHD prevention of ApoGraft were evaluated with 7 aliquots of MPBC apheresis in NOD-SCID IL2-Rgamma-null (NSG) mice (n=349). Here we report results from representative studies were unfractionated ApoGraft (2.5x10^6 cells) or CD34+ purified cells (1.0x10^5 cells) from ApoGraft were transplanted into irradiated (2-2.75Gy) NSG mice. The mice were monitored clinically for GVHD occurrence post transplantation; engraftment and differentiation were evaluated using FACS analysis.Results:Cellular viability was 98% (±1%) in the ApoGraft samples analyzed. Comparison of ApoGraft and Starting Material (SM) showed a mean 19.9% (±4.3%) reduction of total CD3+ T cells; early apoptosis of CD3+ cell increased by 5-fold as compared to Starting Material. Importantly, the percentage of viable CD34+ stem and progenitor cells was not affected by ApoGraft processing. Proliferation and differentiation of hematopoietic progenitors in colony forming unit (CFU) assay was not affected. In NSG mice infused with unfractionated ApoGraft or CD34+ cells purified from it, chimerism level and differentiation were similar to those of mice infused with control MPBC (figure 1A-1B). Most importantly, ApoGraft infused mice had lower GvHD clinical score and prolonged survival as compared to mice infused with control MPBC (maximal score 1.7±0.7 vs. 8.8±0.8) (Figure 1C).Conclusion:ApoGraft is an apoptosis-based selection process that eliminates mature T cells from stem cells grafts, while preserving CD34+ cells quantity, quality and functionality. In our preclinical murine model, ApoGraft reduced GvHD without impairing engraftment. Ongoing animal studies are evaluating the graft vs. leukemic capabilities of ApoGraft and the preservation of anti-viral immunity after ApoGraft processing. Additionally, ApoGraft is currently being evaluated in a Phase I/IIa study (NCT02828878) in subjects with hematological malignancies undergoing matched related allogeneic HSCT. [Display omitted] DisclosuresZuckerman:BioSight Ltd.: Consultancy. Yarkoni:Cellect Biotechnology Ltd.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nechushtan:Cellect Biotechnology Ltd.: Employment. Rodionov:Cellect Biotechnology Ltd.: Employment. Gez:Cellect Biotechnology Ltd.: Employment. Rosenzwaig:Cellect Biotechnology Ltd.: Employment. Rodin:Cellect Biotechnology Ltd.: Employment. Pereg:Cellect Biotechnology Ltd.: Employment.

CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy

BackgroundTumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and...

ABCB5+ dermal mesenchymal stromal cells with favorable skin homing and local immunomodulation for recessive dystrophic epidermolysis bullosa treatment.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, incurable blistering skin disease caused by biallelic mutations in type VII collagen (C7). Advancements in treatment of RDEB have come from harnessing the immunomodulatory potential of mesenchymal stem cells (MSCs). Although human bone marrow-derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. Our work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgammanull (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Furthermore, ABCB5+ DSCs vs BM-MSCs cocultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of major histocompatibility complex class II and Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms among MSC populations could have broader regenerative medicine implications beyond RDEB treatment.

The Cerebroventricular Environment Modifies CAR T Cells for Potent Activity against Both Central Nervous System and Systemic Lymphoma.

Lymphomas with central nervous system (CNS) involvement confer a worse prognosis than those without CNS involvement, and patients currently have limited treatment options. T cells genetically engineered with CD19-targeted chimeric antigen receptors (CAR) are effective against B-cell malignancies and show tremendous potential in the treatment of systemic lymphoma. We aimed to leverage this strategy toward a more effective therapy for patients with lymphoma with CNS disease. NOD-scid IL2Rgammanull (NSG) mice with CNS and/or systemic lymphoma were treated with CD19-CAR T cells via intracerebroventricular (ICV) or intravenous (IV) injection. CAR T cells isolated after treatment were rigorously examined for phenotype, gene expression, and function. We observed that CAR T cells infused ICV, but not IV, completely and durably eradicated both CNS and systemic lymphoma. CAR T cells delivered ICV migrated efficiently to the periphery, homed to systemic tumors, and expanded in vivo, leading to complete elimination of disease and resistance to tumor rechallenge. Mechanistic studies indicated that ICV-delivered CAR T cells are conditioned by exposure to cerebrospinal fluid in the ICV environment for superior antilymphoma activity and memory function compared with IV-delivered CAR T cells. Further analysis suggested that manipulating cellular metabolism or preactivating therapeutic CAR T cells with antigen ex vivo may improve the efficacy of CAR T cells in vivo Our demonstration that ICV-delivered CD19-CAR T cells had activity against CNS and systemic lymphoma could offer a valuable new strategy for treatment of B-cell malignancies with CNS involvement.

Flotetuzumab and Other Cellular Immunotherapies Upregulate MHC Class II Expression on Acute Myeloid Leukemia Cells in Vitro and In Vivo

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative therapy for patients with high-risk and refractory acute myeloid leukemia (AML). Unfortunately, up to 50 percent of patients relapse after allo-HCT.Recent research has shown that 30-50 percent of AML samples from patients relapsing after allo-HCT have downregulation of MHC class II (MHC-II) expression, which may promote immune effector evasion and disease relapse. These studies also report that interferon gamma (IFNγ) can restore MHC-II expression. IFNγ has never been systemically administered after allo-HCT and would likely cause significant and potentially life-threatening toxicities. Reinduction of MHC-II expression may lead to re-engagement of immune effectors, restoration of the graft-versus-malignancy effect, and disease control. We hypothesized that T cell immunotherapies targeting AML cells will lead to T cell activation, localized IFNγ release, and upregulation of MHC-II on AML cells. Methods: For in vitro experiments, THP1 cells (THP1s), which have intermediate MHC-II expression, or primary human AML samples with low MHC-II expression from a patient relapsing after allo-HCT (AML-low cells) were co-cultured with or without T-cell immunotherapy and with or without human MHC-mismatched CD3+ T cells. The following T-cell immunotherapies were tested: flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule; a CD33 x CD3 bispecific molecule (Creative Biolabs, Shirley, NY); and CD123-directed chimeric antigen receptor (CAR) T cells. THP1 IFNγ receptor-1 (IFNγR1) knockout cell lines were generated using CRISPR-Cas9. MHC-II expression was measured by flow cytometry and IFNγ concentrations via Luminex immunoflourescence assay. In order to rescue THP1s from FLZ-induced death and allow for longitudinal evaluation, a transwell plate system was used, placing THP1s, human CD3+ T cells, and FLZ in the top well and THP1s in the bottom well. This allowed for diffusion of IFNγ but not human T cells to the bottom wells, permitting MHC-II upregulation while limiting death. The upper and lower wells were coincubated together for 24 hours prior to harvesting of the THP1s in the lower well for longitudinal studies and mixed-lymphocyte reactions. For in vivo experiments, NOD-scid IL2Rgammanull mice expressing human IL-3, GM-CSF, and SCF (NSG-S) were irradiated with 250 rads and injected with 10e6 primary AML-low cells per mouse. After 5.5 weeks, mice were divided into the following groups: 1) untreated control; 2) FLZ only (2mg/kg); 3) human mismatched T cells only (10e7 T cells per mouse); 4) FLZ and T cells. Results: In vitro co-culture of THP1 or AML-low cells with FLZ and T cells led to significantly increased MHC-II expression at 48 hours when compared with the control, FLZ only, and T cell only groups (Figure 1A-B). Co-culture of THP1s with the CD123 CAR-T cells led to similar results. Although co-incubation with a CD33 x CD3 bispecific led to a similar result, the MHC-II upregulation was not nearly as dramatic as that seen with CD123 targeting agents. Using a transwell system to rescue THP1s from FLZ-mediated toxicity, FLZ-induced MHC-II upregulation on THP1s peaked at 48-72 hours (similar kinetics to what is seen with IFNγ alone). These THP1s with upregulated MHC-II activated third-party donor mismatched human CD4+ T cells to a greater extent than untreated THP1s controls. Co-cultures of THP1s with CD4+ T cells and FLZ induced the secretion of very high concentrations of IFNγ, and blockade of IFNγ signaling through knockout of IFNγR1 led to abrogation of the effect (Figure 1C-D). Finally, in an in vivo model, NSG-S mice injected with AML-low samples and treated with FLZ and T cells showed significant upregulation of MHC-II expression on the AML cells. Single cell RNA-sequencing of AML cells purified from these mice is ongoing. Conclusions: Use of FLZ and other T-cell immunotherapies targeting AML antigens led to both direct AML killing as well as significant upregulation of MHC-II expression on AML cells both in vitro and in vivo. The effect appears to be mediated primarily by IFNγ. T-cell immunotherapies represent a promising treatment approach for AML patients relapsing after allo-HCT and may lead to enhanced immune recognition in the 30-50% of patients who relapse after allo-HCT. Based on these results, a clinical trial treating patients relapsing after allo-HCT with FLZ is planned. Disclosures Christopher: Boulder Bioscience: Patents & Royalties: IP around the use of interferon gamma to treat stem cell transplant. Kim:Tmunity: Patents & Royalties: methods for gene editing in hematopoietic stem cells to enhance the therapeutic efficacy of antigen-specific immunotherapy (Licensed by University of Pennsylvania); Neoimmune Tech: Patents & Royalties: use of long-acting IL-7 analogs to enhance CAR T cells (licensed by Washington University). Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson:MacroGenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

PD-L1 targeting high-affinity NK (t-haNK) cells induce direct antitumor effects and target suppressive MDSC populations

BackgroundAlthough immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor...

Effects of Brain Irradiation in Immune-Competent and Immune-Compromised Mouse Models.

Patient-derived orthotopic xenografts (PDOXs) closely recapitulate primary human glioblastoma (GBM) tumors in terms of histology and genotype. Compared to other mouse strains, NOD-scid IL2Rgammanull (NSG) mice show excellent tumor take rates, which makes them an ideal host for PDOXs. However, NSG mice harbor a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), which renders them relatively radiosensitive. This has been a frequently voiced concern in studies involving ionizing radiation. In this study, we assessed brain toxicity in NSG mice compared to three other different mouse strains frequently used in radiation studies at radiation doses commonly used in experimental combination therapy studies. C3H/Sed/Kam, C57Bl/6, nude and NOD-scid IL2Rgammanull mice received a single dose of 4 Gy to the right brain hemispheres using an image-guided small animal irradiator. Brains were stained using H&E, luxol fast blue, and antibodies against IBA1 and GFAP one, two, four or six months postirradiation. Additional animals of all four strains were exposed to five daily fractions of 2 Gy (5 × 2 Gy), and tissue sections were stained 72 h later against gH2AX, NeuN, GFAP and IBA1. None of the mouse strains displayed radiation-induced toxicity at any of the time points tested. Radiation doses relevant for testing combination therapies can be safely applied to the brains of NSG mice without the occurrence of radiation-induced normal tissue toxicity.

CXCR4 Blockade By BL-8040 in T Cell Acute Lymphoblastic Leukemia Decreases Mitochondrial Mass and Induces Non-Apoptotic Cell Death

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. CXCL12 is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. We previously reported that BL-8040, a potent new CXCR4 antagonist with sustained receptor occupancy, is active as monotherapy against T-ALL in mice. Indeed, a 2-week course of daily BL-8040 resulted in a median reduction in tumor burden of 32.1-fold (range 6.8 to 176) across 5 different T-ALL xenografts. Preliminary data from a clinical trial of BL-8040 plus nelarabine for relapsed T-ALL also suggest therapeutic activity, with a complete remission rate observed in 4/8 patients (50%), which compares favorably to published response rates of approximately 30% with single agent nelarabine. Here, we explore molecular mechanisms by which CXCR4 blockade induces T-ALL death. NOD-scid IL2Rgammanull (NSG) mice were injected with P12-Ichikawa cells, a T-ALL cell line modified to express click beetle red luciferase and GFP. Following T-ALL engraftment, mice were treated with a single dose of BL-8040, and then leukemic cells in the bone marrow harvested 24-48 hours later. Treatment with BL-8040 resulted in a marked suppression of Akt and Erk1/2 phosphorylation, suggesting that signaling through CXCR4 is the major source of PI3 kinase pathway activation in T-ALL cells. Surprisingly, treatment with BL-8040 did not affect cellular proliferation, as measured by Ki67/FxCycle Violet staining or by EdU labeling. Moreover, no increase in apoptosis, as measured by annexin V or activated caspase 3 expression, was observed. These data suggest that CXCR4 blockade induces a non-apoptotic cell death. To explore this possibility further, we performed transcriptome sequencing on T-ALL cells recovered from mice 24 hours after 1 dose of BL-8040. A total of 151 differentially expressed genes (FDR of < 0.05% and ≥ 2-fold change) were identified. Gene set enrichment analysis was strongly positive for alterations in oxidative phosphorylation, ribosome biogenesis, and carbohydrate metabolism. Ribosome function was assessed using O-propargyl-puromycin (OPP), which monitors global protein translation. No difference in global protein synthesis in T-ALL cells was observed after CXCR4 blockade in vivo. T-ALL cells are dependent on glutamine as a source of carbon, and PI3 kinase signaling positively regulates glutaminolysis. Thus, we hypothesized that CXCR4 blockade may induce T-ALL cell death by reducing glutamine metabolism. However, treatment of T-ALL cells in vitro with BL-8040 did not alter the cellular levels of glutamine or glutamate, as measured using a commercial bioluminescent assay. Confirmatory metabolic tracing studies using 13C-labeled glutamine and glucose are in progress. Finally, to explore the reduction in oxidative phosphorylation, we examined mitochondria function using Mitotracker Green. Treatment of T-ALL cells in vitro with BL-8040 for 24-48 hours induced a significant decrease in mitochondria number, suggesting induction of mitophagy. Collectively, these data suggest that T-ALL cells are addicted to CXCR4 signaling in vivo. CXCR4 blockade with BL-8040 induces a non-apoptotic cell death that is characterized by a loss of mitochondria. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership.

Functional Human CD141+ Dendritic Cells in Human Immune System Mice.

For the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established. Human immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors. Our results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines. Establishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo.

647P - Selective Wnt/β-catenin small-molecule inhibitor CWP232228 impairs tumour growth of colon cancer

BackgroundAberrant activation of Wnt/β-catenin signaling is commonly found in colorectal cancers (CRCs) and is the prime event involved in the rapid proliferation of tumor cells in patients with advanced CRC. However, the limited effects of current therapeutics are unsatisfying, which highlights the importance of novel interventions in CRC chemo-treatment. We designed a highly potent small-molecule inhibitor CWP232228, which antagonizes binding of β-catenin to T cell factor in the nucleus. Thus, the present study aimed to explore the possibility of a selective small molecule β-catenin inhibitor, CWP232228, as a potential therapeutic drug in the treatment of CRCs. MethodsWe observed that CWP232228 at a concentration of as low as 1.0μm induced significant cytotoxic effectsand apoptosis in HCT116. Immunoblot analysis showed that CWP232228 treatment increased the release of cytochrome C, which led to the activation of apical (caspase-9) and effector caspases (caspase-7 and -3) resulting in the increased cleavage of PARP. ResultsIn addition, we observed that CWP232228 caused cell cycle arrest via attenuating the expressions of cyclin D2, D3, and survivin. Thus, we validated decreased expressions of Wnt/β-catenin targeted cell cycle progression regulators, aurora kinase A, c-Myc, cyclin D1, and microphthalmia-associated transcription factor. ConclusionsWe confirmed that CWP232228 targets β-catenin via decreased promoter activities, nuclear expressions of β-catenin. Lastly, we found CWP232228 also inhibited growth of xenografted colon cancer cells in NOD-scid IL2Rgammanull mice. Collectively, results in the current study showed a possibility for CWP232228 as a potential therapeutic drug in CRCs. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Human cord blood (hCB)-CD34+ humanized mice fail to reject human acute myeloid leukemia cells.

Since their appearance, humanized mice carrying human immune system seemed promising tools to study the crosstalk between cancer and immunity. The NOD-scidIL2Rgammanull (NSG) mice engrafted with human cord blood (hCB)-CD34+ cells have been proposed to be a valuable tool to reproduce human immune system in mouse. However, the lack of solid evidences on the functionality of their human immune components limits their usage in immune-oncology. We report that (hCB)-CD34+ cells lose their ability to propagate and originate bone marrow-derived human immune cells after two serial transplantations in NSG mice. We demonstrate that transplants of bone marrow patient-derived acute myeloid leukemias (hAMLs) grow very similarly in the humanized (hCB)-CD34+ NSG and parental NSG mice. The similar extent of engraftment and development of leukemias in (hCB)-CD34+ NSG and controls suggests a poor human immune response against not compatible hAMLs. Our findings suggest that (hCB)-CD34+ NSG mice are transient and/or incomplete carriers of the human immune system and, therefore, represent a suboptimal tool to study the interaction between tumor and immune cells.

Pentosan Polysulfate Demonstrates Anti-human T-Cell Leukemia Virus Type 1 Activities In Vitro and In Vivo.

Human T-cell leukemia virus type 1 (HTLV-1) infection causes T-cell leukemia and inflammatory diseases, most notably including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The underlying mechanism for the pathogenesis of HAM/TSP remains unclear. According to a recent clinical trial, a humanized antibody that targets CCR4+ cells ameliorates inflammation by reducing the number of infected cells in the central nervous system; this result suggests that the transmigration of HTLV-1-infected cells plays a crucial role in HAM/TSP. Partly due to the blood-brain barrier, current treatments for HAM/TSP are mostly palliative. Pentosan polysulfate (PPS), a semisynthetic glycosaminoglycan, has recently been used to treat HAM/TSP and was found to alleviate the symptoms. In this study, we investigated the effect of PPS on HTLV-1-infected cells and provide evidence for its efficacy in HAM/TSP. PPS was cytotoxic to certain HTLV-1-infected cells and significantly suppressed HTLV-1 virion production. PPS also efficiently inhibited HTLV-1 cell-cell transmission in T cells. In addition, PPS blocked HTLV-1 infection of primary endothelial cells (human umbilical vascular endothelial cells) and suppressed the subsequent induction of proinflammatory cytokine expression. Furthermore, PPS was found to inhibit the adhesion and transmigration of HTLV-1-infected cells. We also confirmed the anti-HTLV-1 effect of PPS in vivo using two mouse models. PPS blocked HTLV-1 infection in a mouse model with peripheral blood mononuclear cell (PBMC)-humanized NOD-scid IL2Rgammanull (huPBMC NSG) mice. PPS was also found to suppress the development of dermatitis and lung damage in HTLV-1 bZIP factor (HBZ)-transgenic (HBZ-Tg) mice, an HTLV-1 transgenic mouse model in which the mice develop systemic inflammation.IMPORTANCE HTLV-1 is the first human retrovirus to have been identified and is endemic in certain areas worldwide. HTLV-1 infection leads to the development of an inflammatory disease called HAM/TSP, a myelopathy characterized by slowly progressive spastic paraparesis. There have been no effective therapeutics available for HAM/TSP, but recently, a semisynthetic glycosaminoglycan, named pentosan polysulfate (PPS), has been found to alleviate the symptoms of HAM/TSP. Here we conducted a comprehensive study on the effect of PPS both in vitro and in vivo PPS demonstrated anti-HTLV-1 potential in infected cell lines, as shown by its suppressive effects on HTLV-1 replication and transmission and on the transmigration of infected T cells. Moreover, results obtained from two HTLV-1 mouse models demonstrate that PPS inhibits HTLV-1 infection and inflammation development in vivo Our work offers insights into the treatment of HAM/TSP by PPS and also suggests its possible use for treating other HTLV-1-induced inflammatory diseases.

Abstract 2319: Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma

Abstract Introduction: Metastatic uveal melanoma (MUM) is resistant to currently available treatments and overall prognosis is poor. MUM is an “immune cold” tumor and immune checkpoint blockades have only a marginal effect on this tumor. To bring immune cells to the tumor site and change the tumor microenvironment, we developed Chimeric Antigen Receptor (CAR)-NK cells targeting high molecular weight melanoma associated antigen (HMW-MAA/CPSG4). Methods: NK92 cell line derived from NK cell lymphoma was obtained from the American Type Culture Collection (ATCC). NK92 cells were transfected with retroviral pBMN-I vector co-expressing Green Fluorescent Protein (GFP) and anti-HMW-MAA-CD28-CD3z CAR construct. The efficacy of CAR construct transfection was confirmed by expression of His-Tag and F(ab)2 by using BD FACS Celesta flow cytometry. MUM cell line, TJU-UM001, was established from a hepatic metastasis of a uveal melanoma patient in our institution. TJU-UM001 cells have approximately 37,000 HMW-MAA molecules on their surface and are sensitive to NK92 cell killing. Tdtomato-transfected TJU-UM001 cells (106 cells) were intra-hepatically implanted into NOD-scid IL2Rgammanull (NSG) mice. Once establishment of hepatic tumor was confirmed by IVIS 200 imaging system (PerkinElmer) at Red Fluorescent Protein (RFP) excitation 535nm, 3 x 106 CAR-NK92 cells or NK92 cells without CAR constructs were injected into the spleen. Accumulations of GFP+ NK92 cells in the TJU-UM001 tumors in the liver were analyzed under fluorescence microscopy. In a separate experiment, 107 TJU-UM001 cells were subcutaneously implanted into NSG mice. The size of subcutaneous tumor was measured by Caliper. Once tumor became palpable (7-10 mm), 3 x 106 CAR- or non-CAR NK92 cells were directly injected to the MUM tumors. Forty thousand units of human IL-2 were intra-peritoneally injected into mouse once daily for 4 consecutive days after NK92 cell injections. Results: There were expressions of both His-tag and F(ab)2 on CAR-GFP NK92 cells. GFP and CD54 (ICAM-1) were expressed on 95% and 90% of CAR-GFP NK92 cells, respectively. There was no cross-expression of GFP or human CD54 on mouse cells. Therefore, GFP and CD54 were confirmed to be reliable markers for tracking CAR-GFP NK92 cells in mice. Both CAR- and non-CAR NK92 cells exhibited suppression of TJU-UM001 cell proliferation in in vitro assay. Direct injections of both CAR- and non-CAR NK92 cells showed anti-tumor growth effects on subcutaneously implanted TJU-UM001 tumors. More importantly, via splenic injection, CAR-NK92 cells accumulated selectively in the MUM tumors in the liver, while non-CAR NK92 cells were randomly distributed to normal liver tissue and the MUM tumors. These results support the rationale for hepatic arterial infusion of CAR-NK cells as treatment for uveal melanoma patients with hepatic metastasis. Citation Format: Bao Quoc Lam, Takahito Sugase, Mizue Terai, Meggie Danielson, Nadezhda Anikeyeva, Melissa A. Wilson, Yuri Sykulev, Takami Sato. Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2319.

Abstract 3902: Increased sensitivity for detecting cytokine release syndrome with cancer immunotherapy using a PBMC humanized NSG-SGM3 mouse model

Abstract We have described a novel humanized NSG (NOD-scid IL2Rgammanull, NOD-scidIL2Rgnull; JAX stock number 005557) mouse model to measure cytokine release syndrome (CRS) after treatment with immunotherapeutic monoclonal antibodies (mAb). This humanized mouse model is based on PBMC engraftment of NSG mice and provides a rapid, robust and sensitive platform to assess CRS. Exposure of PBMC-engrafted NSG mice to mAb therapeutics was characterized by clinical readouts of cytokine production and decreases in body temperature. Moreover this model enabled the evaluation of differential responses between individual donors and allowed a more comprehensive evaluation of CRS than in vitro assays. However, human immune system engraftment in NSG mice injected with PBMC is dominated by human CD3+ T and NK cell in early time point. To expand the application of our model to additional human immune cell types, we explored the NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF; JAX stock number 013062) strain as a model to assess CRS. The NSG-SGM3 mouse expresses human KITLG, CSF2 and IL-3 as transgenes and supports increased development of human myeloid and Treg populations following engraftment with umbilical cord CD34+ hematopoietic stem cells as compared to NSG mice. We hypothesized that PBMC-engraftment of NSG-SGM3 mice would augment human myeloid and T cell functionality in vivo. We compared NSG and NSG-SGM3 that were humanized with PBMC from identical donors and evaluated both the human immune cell engraftment profile by flow cytometry and CRS induction with single mAb agent and combination therapy. For PBMC engraftment, six weeks old female NSG and SGM3 mice were irradiated and then injected with human PBMC. Five days after PBMC injection, levels of total human cells (CD45+), and human immune cell subsets including CD3+ T cells, CD33+ myeloid cells, CD14+ monocytes, CD19+ B cells, and CD56+ NK cells, were measured by flow cytometry. Overall no significant differences were observed in the levels of human immune cells between PBMC-injected NSG and NSG-SGM3 mice. Six days after PBMC injection, mice were treated with mAb therapeutics including anti-CD28, pembrolizumab, Anti-thymocyte globulin and Lenalidomide as single agents or in combination. While rapid production of human cytokines (IFN-γ, IL-6, IL-10, IL-2, IL-4 and TNF) was detected in both PBMC-injected NSG and NSG-SGM3 mice, the overall cytokine levels were significantly higher in NSG-SGM3 mouse. Moreover, a lower PBMC concentration was compared in NSG and SGM3 mice for measuring CRS. Overall our results indicate that NSG-SGM3 mice humanized with PBMC are a precise and sensitive model to measure human CRS with cancer immune-therapies. Citation Format: Chunting Ye, Michael Brehm, Mingshan Cheng, Leonard Shultz, Dale Greiner, James Keck. Increased sensitivity for detecting cytokine release syndrome with cancer immunotherapy using a PBMC humanized NSG-SGM3 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3902.

Abstract 1121: Role of c-Met in colorectal cancer lung metastasis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; systemic metastasis to the lungs occurs in approximately 10-20% of patients with CRC. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. The present study was designed to determine the role of c-Met in growth and metastasis of CRC cells to the lung. We evaluated patient-derived xenografts (PDX) tumors and CRC cells for c-Met activation (phospho-Met [Tyr1234/1235]), established PDX cell lines with high phospho-Met (Tyr1234/1235) expression from lung or liver metastases, and investigated the role of c-Met expression in CRC lung metastasis. Methods. (1) PDX tumors were implanted into NOD-scid IL2Rgammanull mice; PDX cell lines were established from F3 tumor generation. (2) The protein expression of phospho-Met (Tyr1234/1235) was observed by western blotting and immunohistochemistry. (3). HT29 LungM3 cell line was derived from the human CRC line HT29 following multiple rounds of in vivo selection for lung metastasis in immunodeficient mice. HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene. HT29LungM3 cells were injected into SCID mice subcutaneously to measure tumor growth or intravenously to examine the role of c-Met in CRC lung metastasis. Results. (1) High phospho-Met (Tyr1234/1235) expression was identified in 6 out of 20 CRC PDX models. Two PDX cell lines with high phospho-Met (Tyr1234/1235) expression, 2377LM (from liver metastasis) and 2387 (from lung metastasis), were established. Metastatic potential of the 2377LM cell line to the liver was confirmed after intrasplenic injection of cancer cells into SCID mice. The metastatic potential of 2387 cells to the lung was confirmed after intravenous injection of cancer cells into SCID mice. (2) Western blot analysis demonstrated high phospho-Met (Tyr1234/1235) expression in HT29LungM3, 2387LM and 2387 cell lines, compared to HCT116 (human CRC), LS174T (human CRC), Caco-2 (human CRC), SK-OV-3 (human ovarian cancer) and JAR (placenta choriocarcinoma) cell lines. (3) HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene with c-Met knockdown of at least 90%. Surprisingly, c-Met inhibition in the HT29LungM3 cell line had no effect on subcutaneous tumor growth or lung metastasis. Conclusions. Aberrant expression of c-Met is associated with the progression of multiple human malignancies and has been identified as a novel target for the treatment of malignant tumors. Here, we examined expression of phospho-Met (Ty1234/1235) in CRC PDX cell samples and determined that c-Met knockdown does not decrease CRC lung metastasis. These results are important for understanding the mechanism of CRC lung metastasis and proper strategy selection for prevention and treatment of patients with advanced disease. Citation Format: Piotr G. Rychahou, Nick Roller, Eun Y. Lee, Nicole Rychagov, Matthew Melton, Carrigan Wasilchenko, B. Mark Evers. Role of c-Met in colorectal cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1121.

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4R334X.

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the ex vivo manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by ex vivo expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4. Reduced CXCR4 expression was associated with dampened in vitro NK cell migration toward its cognate ligand stromal-derived factor-1α (SDF-1α). NK cells isolated from patients with WHIM syndrome carry gain-of-function (GOF) mutations in CXCR4 (CXCR4R334X). Compared to healthy donors, we observed that NK cells expanded from WHIM patients have similar surface levels of CXCR4 but have a much stronger propensity to home to BM compartments when adoptively infused into NOD-scid IL2Rgammanull (NSG) mice. Therefore, in order to augment the capacity of adoptively infused NK cells to home to the BM, we genetically engineered ex vivo expanded NK cells to express the naturally occurring GOF CXCR4R334X receptor variant. Transfection of CXCR4R334X-coding mRNA into ex vivo expanded NK cells using a clinically applicable method consistently led to an increase in cell surface CXCR4 without altering NK cell phenotype, cytotoxic function, or compromising NK cell viability. Compared to non-transfected and wild type CXCR4-coding mRNA transfected counterparts, CXCR4R334X-engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro. Importantly, expression of CXCR4R334X on expanded NK cells resulted in significantly greater BM homing following adoptive transfer into NSG mice compared to non-transfected NK cell controls. Collectively, these data suggest up-regulation of cell surface CXCR4R334X on ex vivo expanded NK cells via mRNA transfection represents a novel approach to improve homing and target NK cell-based immunotherapies to BM where hematological malignancies reside.

Mgta-456 Contains Large Numbers of Expanded Cord Blood (CB) CD34+CD90+ Hematopoietic Stem Cells (HSC) Which Confer All Engraftment Activity and Correlate with Accelerated Neutrophil Recovery after Myeloablative Conditioning in Patients with Hematologic Malignancy

Background: Patient access to well-matched CB containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and a risk of graft failure relative to other hematopoietic stem cell (HSC) sources. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. Thus far, 40 patients with hematological malignancy (n=36) and non-malignant diseases (n=4) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (20 to 143 days) respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p<0.01). The aim of the current study was to fully characterize the expanded CD34+ cell fraction of MGTA-456 phenotypically and functionally and identify the cell population that correlates with time to neutrophil recovery. We found that the expanded CD34+CD90+ population of MGTA-456 were the cells responsible for engraftment in NOD-scid IL2Rgammanull (NSG) mice. We hypothesized that the dose of CD34+CD90+ cells/kg would have the strongest correlation with time to neutrophil recovery.Methods: The expansion culture consisted of StemSpan SFEM supplemented with SCF, FLT- 3L, TPO and IL-6 (all at 50 ng/mL) and the AHRa without the addition of antibiotics. After a 10 or 15 day culture, the CD34 expanded product was characterized by cell surface markers (CD34, CD90, CD133, CD41, CD71, CD235a, CD3, CD4, CD8, CD14, CD15, CD16, CD11b, CD33, CD19, CD56, and CD10), as well as colony-forming unit (CFU) capacity and engraftment potential in NOD-scid IL2Rgammanull (NSG) mice in a subset of products.Results: The expanded CD34+ cell fraction of MGTA-456 consisted of CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded fraction contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present (<1%) in the CD34 expanded fraction of MGTA-456. To identify which of these cell populations contain the NSG engraftment activity, we sorted CD34-, CD34+CD90- and CD34+CD90+ cells from MGTA-456 and transplanted the cell fractions into NSG mice. These studies clearly demonstrated that all NSG engraftment activity resided in the CD34+CD90+ cell subpopulation (Figure 2). Based on this result, we correlated the dose of CD34-, CD34+CD90-, CD34+CD90+ cells, and CFU cells/kg infused with time to neutrophil recovery of patients treated with MGTA-456. The dose of TNC (r2=0.49, P<0.05), CD34+CD90- (r2=0.45, p<0.05), and CD34+CD90+ (r2=0.52, p<0.05) cells/kg all significantly correlated with time to neutrophil recovery. The dose of CD34+CD90+ cells/kg had the strongest correlation with time to neutrophil recovery consistent with the NSG engraftment result.Conclusion: In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large doses of CD34+CD90+ HSC and progenitors, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically. [Display omitted] DisclosuresBoitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.

Modeling Sézary Syndrome for Immunophenotyping and Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy

Background: Sézary syndrome (SS) is a highly-morbid T cell leukemic lymphoma with no widely-effective treatments and few preclinical models. We demonstrated effective T cell lymphoma therapy with allogeneic gene-edited anti-CD7 CARTs (Cooper et al, Leukemia, 2018). However, SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo.Methods: To generate SS PDX models, we injected NOD scid IL2Rgammanull (NSG) mice expressing SCF, GM-CSF, and IL-3 (NSG-SGM3) with ~2x106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers (Staser et al, Cytometry A, 2018). To generate UCART2s, we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9, a TRAC-targeted gRNA, and a CD2-targeted gRNA followed by viral transduction with an anti-CD2 scFv 3rd generation CAR. For initial UCART2 testing, we injected NSG mice with 5x105 cells from a human Sézary cell line transduced with click beetle red luciferase (HHCBR-GFP) four days prior to UCART2 treatment. Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART2 infusion.Results: SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p<0.01, SS vs. normal PBMCs), and loss of effector memory CD4 cells (8% vs 34%, p<0.001, SS vs. normal PBMCs). SS cells were CD3+CD4+CD2+CD5+CD8- with variable CD7 loss and PD1 gain. Four of six unique human SS samples injected in NSG-SGM3 mice engrafted within ~6 weeks with no signs of xenogeneic GVHD. Following engraftment, SS cells showed near ubiquitous PD1 expression (>90% vs ~20%, p<0.001, SS vs. normal PBMCs), CD7 loss, and increased CD30 and CD26 expression. Immunohistochemistry further revealed atypical CD3+CD4+CD8-CD7- lymphocytes lining the dermo-epidermal junction. Second generation PDXs showed infiltration of the spleens, blood, and bone marrow with CD2+CD7- human cells and developed alopecia, scaling, and subcutaneous and intraperitoneal masses, with immunophenotyping, sequencing, and UCART treatment studies ongoing.To test UCART2's efficacy in killing SS cells in vivo, we injected NSG mice with HHCBR-GFP+ cells. UCART2-treated HHCBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HHCBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p<0.0001, UCART2 vs. UCART19). Moreover, UCART2-treated HHCBR-GFP mice survived ~40 days as compared to ~21 days in the UCART19 group. Remarkably, UCART2-treated HHCBR-GFP mice receiving NT-I7 showed virtually no tumor burden (maximum 106 photon flux/s vs. 1010 photon flux/s, UCART2+NT-I7 vs. UCART2 only groups) with 100% of UCART2- and NT-I7-treated HHCBR-GFP mice surviving beyond 49 days (Figure 1).Discussion: We describe the generation of physiologically-relevant SS preclinical models, comprehensive immunophenotyping of patient SS samples, clonal SS PDX outgrowth, and the highly effective anti-tumor activity of UCART2- plus NT-I7-mediated killing of SS cells in vivo using an NSG xenograft model. Ongoing studies involve treating primary SS and CTCL PDX models with UCART2 and NT-I7. These preclinical data validate the use of allogeneic “off-the-shelf” adoptive immunotherapy for the treatment of Sézary syndrome, while demonstrating the dramatic enhancement of CART efficacy using a dose-adjustable, clinic-ready long-acting interleukin-7 agonist given in an adjuvant setting. [Display omitted] DisclosuresPark:NeoImmuneTech: Employment. Lee:NeoImmuneTech: Employment. Musiek:Seattle Genetics: Honoraria; Actelion: Other: Scientific Advisory Committee ; Kyowa Kirin: Honoraria.

A Long-Acting Pharmacological Grade Interleukin-7 Molecule Logarithmically Accelerates Ucart Proliferation, Differentiation, and Tumor Killing

Background: Chimeric antigen receptor T cell (CART) therapy is revolutionizing modern cancer therapy, with two anti-CD19 CARTs FDA-approved for relapsed/refractory B cell lymphoma/leukemia and many other CARTs for solid and liquid tumors currently undergoing clinical trials. Our group recently demonstrated multiplexed CRISPR/Cas9 gene-editing of anti-CD7 CARTs to produce CD7 and T cell receptor alpha constant (TRAC)-deleted “off-the-shelf” universal (U)CART7s that effectively kill CD7+ T cell lymphoma in vivo without causing GVHD or fratricide (Cooper et al, Leukemia, 2018). However, in current clinical practice, suboptimal CART persistence and tumor killing permit tumor cell escape and, ultimately, disease relapse. Reasoning that a pro-lymphoid growth factor could promote CART efficacy, we supplemented UCART infusion with subcutaneous injections of the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7) in vivo using a CD19+ lymphoma xenograft model.Methods: To create anti-CD19 universal CARTs (UCART19), we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9 mRNA and a TRAC-targeted gRNA, and virally transduced an anti-CD19 scFv 3rd generation CAR containing a peptidase 2A-cleaved human CD34 construct for both purification and tracking in vivo. Residual TRAC+ cells were depleted using magnetic selection. For xenograft tumor modeling in vivo, we injected NOD-scid IL2Rgammanull (NSG) mice with 5x105 RamosCBR-GFP cells four days prior to UCART19 (2x106 cells). Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART19 infusion.Results:RamosCBR-GFP mice receiving NT-I7 without UCART19 (NT-I7 only group) survived marginally longer (24 day med survival) than mice receiving RamosCBR-GFP cells alone (No tx group) (21 day medium survival, p=0.018, NT-I7 only vs. No Tx). While RamosCBR-GFP mice treated with UCART19 alone (UCART19 group) survived 33 days, 100% of RamosCBR-GFP mice treated with UCART19 and NT-I7 (UCART19+NT-I7 group) were alive at 80 days (Fig 1a), with no mouse showing signs of xenogeneic GVHD (p<0.0001, UCART19+NT-I7 vs. UCART19). At three weeks post UCART19 infusion, bioluminescent imaging (BLI) revealed minimal tumor signal in UCART19+NT-I7 treated mice (108 vs. 1010 photon flux/s, p<0.05, UCART19+NT-I7 vs. UCART19) and near-undetectable photon flux/s at four weeks (107 vs 1011 photon flux/s, p<0.0001, UCART19+NT-I7 vs. UCART19). Quantitative 17-parameter flow cytometric analyses of the blood, bone marrow, and spleens revealed an up to ~8000-fold increase in UCART19 cells in NT-I7-treated mice four weeks post UCART19 infusion (Fig 1a). These UCART19 cells demonstrated a predominantly effector and effector memory phenotype.Discussion: CARTs engineered to express interleukin-7 and CCL19 showed increased migration to and killing of solid tumors (Adachi et al, Nature Biotechnology, 2018). However, genetically engineered potentiation strategies lack “off-switches” and may preclude additional genetic enhancements required for universal “off-the-shelf” CART development. Here, we demonstrate that a pharmacological grade long-acting interleukin-7 agonist can potentiate adoptive cellular therapies. Specifically, NT-I7 can dramatically enhance gene modified T cell proliferation, persistence and tumor killing in vivo, resulting in enhanced survival, providing a tunable clinic-ready adjuvant for reversing suboptimal CART activity in vivo. [Display omitted] DisclosuresCooper:WUGEN: Consultancy, Equity Ownership. Lee:NeoImmuneTech: Employment. Park:NeoImmuneTech: Employment.

Abstract 484: Tumor suppressor miRNA-101 modulates leukemogenesis by targeting the EZH2/Wnt/β-catenin signaling pathways

Abstract The survival rate of acute myeloid leukemia (AML) patients undergoing standard chemotherapy remains poor due to disease relapse. The main cause of relapse is the failure of chemotherapeutic drugs to eradicate self-renewing leukemic stem cells (LSCs) (Cancers 2017;9:74-97). Because of their intrinsic drug resistance, LSCs are extremely difficult to target by single-gene therapies. MicroRNAs (miRNAs), a class of small noncoding RNAs capable of regulating multiple target genes, represent a promising therapeutic target for elimination of LSCs. While miRNA-101 (miR-101) has been identified as a tumor suppressor in several types of cancers including prostate (Science 2008;322:1695-99) and liver cancer (Cancer Res 2009;69:2623-29), its expression and functional role in AML remains unknown. In this study, we uncover that miR-101 plays a crucial role in leukemogenesis through modulation of key epigenetic and signaling pathways activated in LSCs of mixed-lineage leukemia (MLL)-rearranged AML. Our data showed that overexpression of miR-101 in murine pre-LSCs inhibited cell proliferation in vitro and delayed leukemogenesis in vivo, confirming a tumor-suppressor function of miR-101 in MLL-AF9-induced AML, a highly aggressive leukemia. To assess the therapeutic value of miR-101, we next examined the antitumor function of miR-101 in a xenograft model of human MLL-AF9 AML. Briefly, miR-101 was overexpressed in human MLL-AF9 AML cell line, MOLM-13, that was then transplanted into NOD-scid IL2Rgammanull (NSG) mice followed by in vivo bioluminescence imaging to monitor engraftment of human leukemic cells. Our result revealed a significantly lower engraftment in the xenograft mouse model carrying miR-101-overexpressing leukemic cells compared to control mice. This suggests that inhibition of tumor suppressor miR-101 is essential in the initiation of an aggressive form of human AML. To investigate the potential mechanism by which miR-101 impairs leukemogenesis, we examined key oncogenic pathways regulated by miR-101 by performing luciferase assays and Western blotting. Our data showed that miR-101 inhibited leukemogenesis via directly targeting Enhancer of zeste homolog 2 (EZH2), a crucial epigenetic regulator in leukemia maintenance, as well as key components of Wnt/β-catenin signaling, an oncogenic pathway necessary for LSC self-renewal in AML. Furthermore, immunofluorescence analysis revealed a significant reduction in active β-catenin in the nucleus of miR-101-overexpressing leukemic cells, indicating a direct regulation of miR-101 in β-catenin activity. Collectively, these findings reveal a novel role for miR-101 in leukemia progression that may be used to develop a targeted therapy for AML treatment. Citation Format: Estrella Gonzales-Aloy, Dylan G. Grebert-Wade, Jenny Y. Wang. Tumor suppressor miRNA-101 modulates leukemogenesis by targeting the EZH2/Wnt/β-catenin signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 484.