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Purpose:Trials of adoptive natural killer (NK)-cell immunotherapy for hematologic malignancies have thus far shown only marginal effects, despite the potent in vitro antitumor activity of these cells. Homing of infused cells to tumor microenvironments is critical for efficacy, but has not been well characterized. We established a novel method to track and quantify the distribution of adoptively transferred NK cells using rhesus macaques (RM) as a clinically relevant preclinical model.Experimental Design:RM NK cells were expanded ex vivo for 14–21 days, labeled with 89Zr-oxine complex, and assessed for phenotype, function, and survival. Trafficking of 89Zr-labeled ex vivo–expanded NK cells infused into RMs was monitored and quantitated by serial positron emission tomography (PET)/CT (n = 3, 2.05 ± 0.72 MBq, 23.5 ± 2.0 × 106 NK cells/kg) and compared with that of 89Zr-labeled nonexpanded NK cells, apoptotic NK cells, and hematopoietic stem and progenitor cells (HSPC).Results:NK cells retained sufficient levels of 89Zr for accurate in vivo tracking for 7 days. 89Zr labeling did not alter cellular phenotype, viability, or function. PET/CT showed NK cells initially localized in the lungs, followed by their migration to the liver, spleen, and, at low levels, bone marrow. One day following transfer, only 3.4% of infused NK cells localized to the BM versus 22.1% of HSPCs. No clinical side effects were observed, and dosimetry analysis indicated low organ radioexposures of 6.24 mSv/MBq (spleen) or lower.Conclusions:These data support translation of this technique to humans to track the distribution of adoptively infused cells and to develop novel techniques to improve immune cell homing to tumor microenvironments.

Supplementary Methods

Fig. S1. Zr-89 oxine labeling does not alter NK cell phenotype. Fig. S2. PET/CT reveals low bone marrow homing of NK cells. Fig. S3. CD34+ HSPCs home to the bone marrow.

Zr-89 oxine labeled cell doses infused to rhesus macaques.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow. Here, we compare engraftment of CD34+ cells transplanted in a myeloablative rhesus macaque (RM) model using the OIB method compared with IV delivery. RM CD34+ cells obtained by apheresis were split equally for transduction with lentiviral vectors encoding either green fluorescent protein or yellow fluorescent protein reporters. Following conditioning, one marked autologous population of CD34+ cells was injected directly IB using the OIB method and the other was injected via slow IV push into the same animal (n = 3). Daily flow cytometry of blood quantified the proportion of engrafting cells deriving from each source. Marrow retention was examined using positron emission tomography/computed tomography imaging of 89Zirconium (89Zr)-oxine–labeled CD34+ cells. CD34+ cells injected via the OIB method were retained in the marrow and engrafted in all 3 animals. However, OIB-transplanted progenitor cells did not engraft any faster than those delivered IV and contributed significantly less to hematopoiesis than IV-delivered cells at all time points. Rigorous testing of our OIB delivery system in a competitive RM myeloablative transplant model showed no engraftment advantage over conventional IV infusion. Given the increased complexity and potential risks of IB vs IV approaches, our data do not support IB transplantation as a strategy to improve hematopoietic engraftment.

Summary Background Margetuximab, a novel, investigational, Fc-engineered, anti-HER2 monoclonal antibody, is designed to more effectively potentiate innate immunity than trastuzumab. We aimed to evaluate the safety, tolerability, and antitumour activity of margetuximab plus pembrolizumab (an anti-PD-1 monoclonal antibody) in previously treated patients with HER2-positive gastro-oesophageal adenocarcinoma. Methods CP-MGAH22–05 was a single-arm, open-label, phase 1b–2 dose-escalation and cohort expansion study done at 11 academic centres in the USA and Canada and 15 centres in southeast Asia (Korea, Taiwan, and Singapore) that enrolled men and women aged 18 years or older with histologically proven, unresectable, locally advanced or metastatic, HER2-positive, PD-L1-unselected gastro-oesophageal adenocarcinoma, with an Eastern Cooperative Oncology Group performance status of 0 or 1, who had progressed after at least one previous line of therapy with trastuzumab plus chemotherapy in the locally advanced unresectable or metastatic setting. In the dose-escalation phase, nine patients were treated: three received margetuximab 10 mg/kg intravenously plus pembrolizumab 200 mg intravenously every 3 weeks and six received the recommended phase 2 dose of margetuximab 15 mg/kg plus pembrolizumab 200 mg intravenously every 3 weeks. An additional 86 patients were enrolled in the phase 2 cohort expansion and received the recommended phase 2 dose. The primary endpoints were safety and tolerability, assessed in the safety population (patients who received at least one dose of either margetuximab or pembrolizumab) and the objective response rate as assessed by the investigator according to both Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, in the response-evaluable population (patients with measurable disease at baseline and who received the recommended phase 2 dose of margetuximab and pembrolizumab). This trial is registered with ClinicalTrials.gov , NCT02689284 . Recruitment for the trial has completed and follow-up is ongoing. Findings Between Feb 11, 2016, and Oct 2, 2018, 95 patients were enrolled. Median follow-up was 19·9 months (IQR 10·7–23·1). The combination therapy showed acceptable safety and tolerability; there were no dose-limiting toxicities in the dose-escalation phase. The most common grade 3–4 treatment-related adverse events were anaemia (four [4%]) and infusion-related reactions (three [3%]). Serious treatment-related adverse events were reported in nine (9%) patients. No treatment-related deaths were reported. Objective responses were observed in 17 (18·48%; 95% CI 11·15–27·93) of 92 evaluable patients. Interpretation These findings serve as proof of concept of synergistic antitumour activity with the combination of an Fc-optimised anti-HER2 agent (margetuximab) along with anti-PD-1 checkpoint blockade (pembrolizumab). Funding MacroGenics.

Purpose: Trials of adoptive natural killer (NK)-cell immunotherapy for hematologic malignancies have thus far shown only marginal effects, despite the potent in vitro antitumor activity of these cells. Homing of infused cells to tumor microenvironments is critical for efficacy, but has not been well characterized. We established a novel method to track and quantify the distribution of adoptively transferred NK cells using rhesus macaques (RM) as a clinically relevant preclinical model. Experimental Design: RM NK cells were expanded ex vivo for 14–21 days, labeled with 89Zr-oxine complex, and assessed for phenotype, function, and survival. Trafficking of 89Zr-labeled ex vivo–expanded NK cells infused into RMs was monitored and quantitated by serial positron emission tomography (PET)/CT (n = 3, 2.05 ± 0.72 MBq, 23.5 ± 2.0 × 106 NK cells/kg) and compared with that of 89Zr-labeled nonexpanded NK cells, apoptotic NK cells, and hematopoietic stem and progenitor cells (HSPC). Results: NK cells retained sufficient levels of 89Zr for accurate in vivo tracking for 7 days. 89Zr labeling did not alter cellular phenotype, viability, or function. PET/CT showed NK cells initially localized in the lungs, followed by their migration to the liver, spleen, and, at low levels, bone marrow. One day following transfer, only 3.4% of infused NK cells localized to the BM versus 22.1% of HSPCs. No clinical side effects were observed, and dosimetry analysis indicated low organ radioexposures of 6.24 mSv/MBq (spleen) or lower. Conclusions: These data support translation of this technique to humans to track the distribution of adoptively infused cells and to develop novel techniques to improve immune cell homing to tumor microenvironments.

ICB therapy has transformed cancer treatment; durable responses in difficult-to-treat cancers have been observed. Despite this most patients (pts) don’t respond to ICB (primary resistance), and many pts who initially respond eventually relapse (acquired resistance). Primary resistance is associated with tumor cell extrinsic factors, e.g., the immunosuppressive nature of the tumor microenvironment (TME), while acquired resistance is associated with tumor intrinsic factors, e.g., downregulation of MHC1, preventing T cell recognition. Treatments to overcome ICB resistance are necessary. WU-NK-101 is a PBMC-derived, cytokine-reprogrammed, expanded, and cryopreserved off-the-shelf memory NK cell product. WU-NK-101 cells capture the memory-like NK cell biology of cytokine-induced memory-like (CIML) NK cells, exhibiting enhanced cytotoxicity, metabolic fitness/flexibility, and resistance to TME immunosuppression (Muth et al. EHA 2022; Rutella et al. ESMO 2022). Bone marrow biopsies, collected from R/R AML pts who received CIML-NK cells (NCT01898793), were interrogated using immune gene expression (GE) profiling and spatially resolved proteomics (IO360® panel, n = 740 genes, and GeoMx® DSP; NanoString Technologies). Higher T-cell infiltration was noted post CIML-NK. CIBERSORT deconvoluted GE data inferred higher abundance of macrophages, γδT cells and activated dendritic cells on day 28 post-treatment. GE signatures showed downregulation of NFIL3 and FAM30A (log2 fold-change Citation Format: Tom A. Leedom, Barbara Muz, Jaykumar Vadakekolathu, John J. Muth, Xiao-Hua Li, Gregory Watson, Kristaan Magee, Ryan P. Sullivan, Melissa Berrien-Elliott, Todd Fenigher, Sergio Rutella, Matthew L. Cooper, Ayman Kabakibi, Jan K. Davidson-Moncada. xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB) [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 6418.

Introduction. Approximately 40% of patients (pts) with newly diagnosed AML either fail to achieve complete remission with intensive induction therapy or experience disease recurrence after a short remission (CR1 6 months), the probability of response for PIF/ER pts is particularly poor (~12%) with median expected overall survival of ~3.5 month and no approved therapy for this specific population. We have recently shown that increased immune infiltration of the tumor microenvironment (TME) is associated with induction failure and poor prognosis; conversely, an infiltrated TME predisposes for immunotherapy response1. We provide an update of the first-in-human study of flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule currently in clinical development for PIF/ER AML pts. Methods. In this phase of the study, PIF is defined as being refractory to induction with: ≥1 high-intensity cytarabine-based chemotherapy (CTx) cycles, or ≥2 but ≤4 Bcl-2 inhibitor-based combinations, or gemtuzumab ozogamicin only. ER is defined as relapse following CR1 < 6 months. Pts who receive up to one prior salvage attempt are included. Pts whose AML recurred following HSCT are excluded. The recommended Phase 2 dose (RP2D) of FLZ is 500 ng/kg/day administered as a continuous infusion in 28-day cycles following a step-up ('priming') lead-in dose during Cycle 1 Week 1. Disease status is assessed by modified IWG criteria. Duration of response is measured from initial response to relapse or death. Results. As of July 1, 2020, 38 PIF/ER (as defined above) AML patients have been treated at the RP2D (median age 63yrs [range 28-81]; 31.6% [12] pts female). Most pts (63.2%, 24/38) were PIF and the large majority (94.7%, 36/38) had non-favorable risk by ELN 2017 criteria (25 pts adverse, 11 pts intermediate); 34.2% (13/38) had secondary AML. For ER pts, median duration of CR1 was 2.9 months (range: 0.7-4.0 months). Cytokine release syndrome (CRS) was the most frequently reported treatment related adverse event (TRAE), with all pts experiencing mild-to-moderate (grade ≤ 2) CRS. No grade ≥ 3 CRS events have been reported in this cohort. Most CRS events (51.5%) occurred in the first week of treatment during step-up dosing. The incidence of CRS progressively decreased during dosing at RP2D (34.8% in week 2, 4.5% in week 3, and 6.1% in week 4), allowing outpatient treatment in most cases. Neurologic AEs have been infrequent, with the most prominent event being grade 1 or grade 2 headache in 23.7% (9/38) treated at the RP2D. Two pts experienced grade 3 confusion of short duration (1-2 days) that was fully reversible. Over half (57.9%) of pts had evidence of antileukemic activity (reduction in blast count) with a median decrease of 92.7% in BM blasts (Fig. 1). The overall complete response rate (CRR, Conclusion: FLZ demonstrated encouraging activity in pts with PIF/ER AML, a population with poor prognosis and high unmet medical need, with 42.1% achieving CRR and over half of those receiving a stem cell transplant. Treatment is tolerable with a minimum 8 day inpatient treatment. The study is currently enrolling patients [NCT02152956] 1 Vadakekolathu J, Minden MD, Hood T, Church SE, Reeder S, Altmann H et al. Immune landscapes predict chemotherapy resistance and immunotherapy response in acute myeloid leukemia. Sci Trans Med 2020. Disclosures Aldoss: abbvie: Consultancy, Research Funding; agios: Honoraria; kite: Consultancy; autolus limited: Consultancy; JAZZ: Honoraria, Speakers Bureau; Amgen: Consultancy; Agios: Consultancy. Uy:Genentech: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria. Emadi:Amgen: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; Servier: Membership on an entity's Board of Directors or advisory committees. Walter:Aptevo Therapeutics: Research Funding. Foster:Daiichi Sankyo: Consultancy; Bellicum Pharmaceuticals: Research Funding; Macrogenics: Consultancy, Research Funding. Arellano:Hanmi: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cephalon Oncology: Research Funding. Wieduwilt:Amgen: Research Funding; Macrogeneics: Research Funding; Leadiant: Research Funding; Merck: Research Funding; Shire: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Michaelis:Jazz Pharmaceuticals: Research Funding. Stiff:Kite, a Gilead Company: Research Funding; Gamida Cell: Research Funding; Atara: Research Funding; Unum: Research Funding; Delta-Fly: Research Funding; Macrogenics: Research Funding; Amgen: Research Funding. Advani:Novartis: Consultancy, Other: advisory board; Pfizer: Honoraria, Research Funding; Takeda: Research Funding; OBI: Research Funding; Kite: Other: Advisory board/ honoraria; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Wermke:MacroGenics: Honoraria. Erba:AbbVie, Daiichi Sankyo, Forma, ImmunoGen, Jazz Pharmaceuticals, MacroGenics, Novartis, PTC: Research Funding; Glycomimetics: Other: member of Scientific Steering Committee; Celgene: Other: chair of the Scientific Steering Committee; Covance (AbbVie): Other: chair of the Independent Review Committee; AbbVie, Agios, Celgene, Incyte, Jazz Pharmaceuticals, and Novartis: Speakers Bureau; AbbVie, Agios, Amgen, Astellas, Celgene, Daiichi Sankyo, Glycomimetics, ImmunoGen, Incyte, Jazz Pharmaceuticals, MacroGenics, Novartis, and Pfizer: Consultancy. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Ravandi:Abbvie: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; Celgene: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Orsenix: Consultancy, Honoraria, Research Funding. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Collins:IQVIA: Other: I have worked as a contractor for IQVIA in the past, within the past 24 months.; MacroGenics: Current equity holder in publicly-traded company, Other: I currently work as a contractor for MacroGenics. Guo:Macrogenics: Current Employment. Tran:MacroGenics: Current Employment. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Patel:MacroGenics: Current Employment. Bakkacha:MacroGenics: Current Employment. Jacobs:MacroGenics: Current Employment. Seiler:MacroGenics: Current Employment. Rutella:Kura Oncology: Research Funding; MacroGenics Inc.: Research Funding; NanoString Technologies Inc.: Research Funding. Bonvini:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson-Moncada:Macrogenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

We have recently shown that bone marrow (BM) RNA profiles stratify patients with acute myeloid leukemia (AML) into immune-infiltrated and immune-depleted subtypes and that type I/II interferon (IFN)-related gene signatures associate with complete response to flotetuzumab (FLZ), an investigational CD123×CD3 bispecific DART molecule. Within the AML tumor microenvironment CD8+ T cells exhibit features of immune exhaustion and senescence (IES). IES are dysfunctional states driven by metabolic alterations in the tumor microenvironment (TME) and emerging targets for cancer immunotherapy. The aim of the current study was to determine whether IES predicts response of relapsed-refractory (R/R) AML to FLZ in the CP-MGD006-01 clinical trial. Based on prior knowledge and gene set enrichment analysis, we derived a 61-gene IES signature score from RNA-sequencing datasets (TCGA and Beat-AML Master Trial; 162 and 281 patients, respectively). The immunotherapy cohort included 139 BM samples from 71 patients with R/R AML treated with FLZ at the RP2D of 500 ng/kg/day (NCT02152956). BM samples were collected at time of study entry (n=71; n=66 with response data) and longitudinally post-cycle (PC)1 (n=40), PC2 (n=18), PC3 and 4 (n=4) and end of treatment (n=6). AML status at study entry was classified as primary induction failure (PIF, defined as lack of response to at least 2 induction treatment cycles), and early (ER) or late relapse (LR), defined as complete remission (CR) of 50% decrease or decrease to 5-25% BM blasts. RNAs were profiled on the PanCancer IO 360™ gene expression panel on the nCounter® platform. Formalin-fixed paraffin embedded BM biopsies were profiled using the human IO protein and RNA panels on the GeoMx® digital spatial profiler (DSP). The 61 genes in the IES signature included T/NK-cell markers (granzymes, CD8A, KLRD1, KLRK1), immune checkpoints (ICOS, CTLA4, EOMES), IFNG and IFN-stimulated genes (CXCR6, IFIH1, IL10RA, GBP1), and were enriched in KEGG pathways related to Th1/Th2 differentiation, TCR signaling, cytokine-cytokine receptor interaction, NK-mediated cytotoxicity and CD28 costimulation (false discovery rate In conclusion, features of IES were associated with response to FLZ. T-cell functional rejuvenation by FLZ could benefit patients with R/R AML by counteracting pre-existing immune dysfunction. Figure Disclosures Church: NanoString Technologies, Inc.: Current Employment. Uy:Pfizer: Consultancy; Agios: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Astellas Pharma: Honoraria; Daiichi Sankyo: Consultancy. Emadi:Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; KinaRx: Other: co-founder and scientific advisor; Servier: Membership on an entity's Board of Directors or advisory committees. Walter:Aptevo Therapeutics: Research Funding. Foster:Bellicum Pharmaceuticals: Research Funding; Macrogenics: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. Arellano:Cephalon Oncology: Research Funding; Hanmi: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wieduwilt:Amgen: Research Funding; Leadiant: Research Funding; Merck: Research Funding; Shire: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Macrogeneics: Research Funding. Michaelis:Jazz Pharmaceuticals: Research Funding. Stiff:Kite, a Gilead Company: Research Funding; Gamida Cell: Research Funding; Atara: Research Funding; Unum: Research Funding; Delta-Fly: Research Funding; Macrogenics: Research Funding; Amgen: Research Funding. Advani:Takeda: Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Other: advisory board; OBI: Research Funding. Wermke:MacroGenics: Honoraria. Erba:AbbVie, Daiichi Sankyo, Forma, ImmunoGen, Jazz Pharmaceuticals, MacroGenics, Novartis, PTC: Research Funding; AbbVie, Agios, Celgene, Incyte, Jazz Pharmaceuticals, and Novartis: Speakers Bureau; AbbVie, Agios, Amgen, Astellas, Celgene, Daiichi Sankyo, Glycomimetics, ImmunoGen, Incyte, Jazz Pharmaceuticals, MacroGenics, Novartis, and Pfizer: Consultancy; Glycomimetics: Other: member of Scientific Steering Committee; Celgene: Other: chair of the Scientific Steering Committee; Covance (AbbVie): Other: chair of the Independent Review Committee. Ravandi:Orsenix: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Macrogenics: Research Funding; Xencor: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Topp:Amgen, KITE, Novartis, Regeneron, Roche: Consultancy; Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Gojo:Amgen: Research Funding; Merck: Research Funding; Amphivena: Research Funding; Genentech: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees. Luznik:AbbVie: Consultancy; WindMil Therapeutics: Patents & Royalties: Patent holder; Merck: Research Funding, Speakers Bureau; Genentech: Research Funding. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:Macrogenics: Current Employment. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding; Kura Oncology: Research Funding.

Introduction: CRS is a potentially life-threatening toxicity observed following T cell-redirecting therapies. CRS is associated with elevated cytokines, including IL6, IFNγ, TNFα, IL2 and GM-CSF. Glucocorticosteroids (GC) and the IL6 receptor blocking antibody tocilizumab (TCZ) can reduce CRS severity; however, CRS may still occur and limit the therapeutic window of novel immunotherapeutic agents. Disruption of cytokine signaling via Janus kinase (JAK) pathway interference may represent a complementary approach to blocking CRS. Ruxolitinib (RUX), an oral JAK1/2 inhibitor approved for the treatment of myelofibrosis and polycythemia vera, interferes with signaling of several cytokines, including IFNγ and IL6, via blockade of the JAK/STAT pathway. We hypothesized that RUX may reduce the frequency and severity of CRS in R/R AML patients (pts) undergoing treatment with flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule. Methods: Relapse/refractory (including primary induction failure, early relapse and late relapse) AML pts were included in this study. RUX pts were treated at a single site, Washington University, St. Louis, MO. RUX was dosed at 10 mg or 20mg BID days -1 through 14. Comparator (non-RUX) pts (n=23) were treated at other clinical sites. FLZ was administered at 500 ng/kg/day continuously in 28-day cycles following multi-step lead-in dosing in week 1 of cycle 1. CRS was graded per Lee criteria1. Results: As of July 1st, 2020, 10 R/R AML pts, median age 65 (range 40-82) years, have been enrolled and treated in the RUX cohort (6 at 10mg, 4 at 20 mg of RUX). All pts had non-favorable risk by ELN 2017 criteria (8 adverse and 2 intermediate); 1 (10.0%) pt had secondary AML; pt characteristics in the RUX and non-RUX cohorts were balanced, except for median baseline BM blasts which was higher in non-RUX pts: 15% (range 5-72) vs (40% (range 7-84), RUX and non-RUX pts respectively. Cytokine analysis showed statistically significant (p Conclusion: Prophylactic RUX produced a clear difference in cytokine profiles but no discernable improvement in clinical CRS or response rates in FLZ treated patients. A larger study may be required to determine the prophylactic role of RUX in CRS. References: 1. Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 2014; 124(2): 188-195. doi: 10.1182/blood-2014-05-552729 Disclosures Uy: Pfizer: Consultancy; Agios: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria. Aldoss:abbvie: Consultancy, Research Funding; kite: Consultancy; agios: Honoraria; autolus limited: Consultancy; JAZZ: Honoraria, Speakers Bureau; Amgen: Consultancy; Agios: Consultancy. Arellano:Cephalon Oncology: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Hanmi: Research Funding. Foster:Daiichi Sankyo: Consultancy; Bellicum Pharmaceuticals: Research Funding; Macrogenics: Consultancy, Research Funding. Ravandi:Celgene: Consultancy, Honoraria; Xencor: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding. Advani:Takeda: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Immunogen: Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Other: advisory board; OBI: Research Funding. Wieduwilt:Macrogeneics: Research Funding; Amgen: Research Funding; Leadiant: Research Funding; Merck: Research Funding; Shire: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Emadi:Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; KinaRx: Other: co-founder and scientific advisor; Servier: Membership on an entity's Board of Directors or advisory committees. Michaelis:Jazz Pharmaceuticals: Research Funding. Stiff:Macrogenics: Research Funding; Kite, a Gilead Company: Research Funding; Delta-Fly: Research Funding; Unum: Research Funding; Atara: Research Funding; Gamida Cell: Research Funding; Amgen: Research Funding. Wermke:MacroGenics: Honoraria. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Collins:MacroGenics: Current equity holder in publicly-traded company, Other: I currently work as a contractor for MacroGenics; IQVIA: Other: I have worked as a contractor for IQVIA in the past, within the past 24 months.. Guo:Macrogenics: Current Employment. Tran:MacroGenics: Current Employment. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Patel:MacroGenics: Current Employment. Bakkacha:MacroGenics: Current Employment. Jacobs:MacroGenics: Current Employment. Seiler:MacroGenics: Current Employment. Rutella:MacroGenics Inc.: Research Funding; Kura Oncology: Research Funding; NanoString Technologies Inc.: Research Funding. Walter:Aptevo Therapeutics: Research Funding. Bonvini:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson-Moncada:Macrogenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 37% to 46% of acute myeloid leukemia (AML) with adverse-risk cytogenetics and correlate with primary induction failure, high risk of relapse, and dismal prognosis. Herein, we aimed to characterize the immune landscape of TP53-mutated AML and determine whether TP53 abnormalities identify a patient subgroup that may benefit from immunotherapy with flotetuzumab, an investigational CD123 × CD3 bispecific dual-affinity retargeting antibody (DART) molecule. The NanoString PanCancer IO360 assay was used to profile 64 diagnostic bone marrow (BM) samples from patients with TP53-mutated (n = 42) and TP53-wild-type (TP53-WT) AML (n = 22) and 45 BM samples from patients who received flotetuzumab for relapsed/refractory (R/R) AML (15 cases with TP53 mutations and/or 17p deletion). The comparison between TP53-mutated and TP53-WT primary BM samples showed higher expression of IFNG, FOXP3, immune checkpoints, markers of immune senescence, and phosphatidylinositol 3-kinase-Akt and NF-κB signaling intermediates in the former cohort and allowed the discovery of a 34-gene immune classifier prognostic for survival in independent validation series. Finally, 7 out of 15 patients (47%) with R/R AML and TP53 abnormalities showed complete responses to flotetuzumab (

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Importantly, interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Background T-ALL/LBL represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 85% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage regimens induce remissions in only ~20-30% of cases, and survival is dismal. T-ALL/LBL is a genetically diverse group, but with universal overexpression of CD7, making this a suitable target for immunotherapy. Despite the success of CAR-T cells in B-cell malignancies, CAR-T cell development in T-cell malignancies has proven challenging due to fratricide and high risk of contamination of the genetically modified CAR-T product with the patient's malignant T cells. WU-CART-007 is a CD7-directed, genetically modified, allogeneic, 'off the shelf', fratricide-resistant chimeric antigen receptor (CAR) T-cell product for the treatment of CD7+ve hematologic malignancies. Methods This multicenter, open-label, dose-escalation, Phase 1/2 study (NCT#04984356) of WU-CART-007 in patients ≥ 12 years old, with relapsed or refractory T-ALL/LBL is designed to characterize the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose (MTD)/maximum administered dose (MAD; if no MTD defined) (Phase 1), and to investigate the preliminary anti-tumor activity, as measured by objective response rate (ORR) and duration of response (DOR) (Phase 2). Phase 1 is comprised of a dose escalation segment and will proceed according to a standard 3+3 design testing up to 4 dose levels from 1 to 9 x 10 8 cells. Adolescent patients, ages 12-17, will be eligible for enrollment in Phase 1 Dose Escalation beginning at Dose level 3 and 4, and during Phase 2 Cohort Expansion. Upon reaching the MTD and/or RP2D, the Phase 2 portion comprised of the cohort expansion segment will be launched. A Simon's optimal two-stage design will be implemented to enroll patients (an interim analysis for futility in the first stage and the final analysis in the second stage) for Phase 2 dose expansion cohort to confirm safety and explore preliminary efficacy. All patients will receive a single infusion of WU-CART-007 cells on day 1 following a lymphodepleting conditioning therapy consisting of fludarabine and cyclophosphamide on days -5 to -3. Patients will be hospitalized for a minimum of 7 days following WU-CART-007 administration. Response will be assessed on Cycle 1 Day 28 (± 1 days), and at Months 3, 6, 12, and 24, by bone marrow aspirate and biopsy and PET/CT if indicated. Response will be defined as per modified NCCN Guidelines Version 2.2020. Disclosures Ghobadi: Atara: Consultancy; Amgen: Consultancy, Research Funding; Wugen: Consultancy; Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding. Locke: Janssen: Consultancy, Other: Scientific Advisory Role; Kite, a Gilead Company: Consultancy, Other: Scientific Advisory Role, Research Funding; Iovance Biotherapeutics: Consultancy, Other: Scientific Advisory Role; Legend Biotech: Consultancy, Other; Novartis: Consultancy, Other, Research Funding; Takeda: Consultancy, Other; Wugen: Consultancy, Other; Cowen: Consultancy; Umoja: Consultancy, Other; Bluebird Bio: Consultancy, Other: Scientific Advisory Role; Calibr: Consultancy, Other: Scientific Advisory Role; BMS/Celgene: Consultancy, Other: Scientific Advisory Role; GammaDelta Therapeutics: Consultancy, Other: Scientific Advisory Role; Cellular Biomedicine Group: Consultancy, Other: Scientific Advisory Role; Amgen: Consultancy, Other: Scientific Advisory Role; Allogene Therapeutics: Consultancy, Other: Scientific Advisory Role, Research Funding; EcoR1: Consultancy; Emerging Therapy Solutions: Consultancy; Gerson Lehrman Group: Consultancy; Moffitt Cancer Center: Patents & Royalties: field of cellular immunotherapy. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. Davidson-Moncada: Wugen: Current Employment. Cooper: Wugen: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties; NeoImmune Tech: Patents & Royalties; RiverVest: Consultancy.

Introduction: Somatic TP53 mutations and deletions of 17p, to which TP53 is mapped, (TP53mut) occur in 8-10% of de novo Acute myeloid leukemia (AML) and in up to 37-46% of patients (pts) with adverse-risk cytogenetics and treatment-related myeloid neoplasms and confer a poor prognosis. In addition to its well-characterized function as a tumor suppressor, emerging evidence implicates mutant TP53 in activating genes involved in immune response and inflammation such as chemokines, cytokines and extracellular matrix modulators. An analysis of The Cancer Genome Atlas (TCGA) transcriptomic data showed that TP53 mutations, in 30 diverse cancer types, correlated with increased leukocyte infiltration into tumors with higher proportions of PD-L1-expressing CD8+ T cells and increased expression of T-cell effector genes and interferon (IFN)-γ-related genes. We recently characterized tumor microenvironmental (TME) immune gene sets that capture elements of both type I- and IFN-γ-driven biology and stratify AML into immune-infiltrated and immune-depleted subtypes. Our immune classifier predicted survival in patients receiving cytarabine-based induction and immunotherapy with flotetuzumab (FLZ), an investigational CD123×CD3 bispecific DART® molecule. We hypothesized that TP53-mutated AML represents immune-infiltrated AML that would be particularly responsive to FLZ. Methods: Fifteen TP53mut AML pts have been treated with FLZ on clinical trial CP-MGD006-01 (NCT#02152956). Disease status was assessed by modified International Working Group (IWG) criteria. Specifically, overall response rate (ORR), collectively complete response, defined as 50% decrease or decrease to 5-25% BM blasts. Microenvironmental RNAs were profiled using the PanCancer IO 360™ gene expression panel on the nCounter® platform. Baseline formalin-fixed paraffin embedded BM samples were evaluated for PD-L1, FoxP3, CD8 and CD3 expression by immunohistochemistry (IHC). Slides were stained using a Leica BondRx autostainer. Fluorescence was imaged using a Polaris Vectra 3 and analyzed using inForm software. A density-based clustering algorithm developed and run in QuPath was used to quantify T-cell 'hotspots". Results: Baseline (BL) BM samples for immune gene expression profiling were available in 13 pts with TP53mut (median age 61yrs [range 27-81]; 46.7% [7] pts female); among these, 77% (10/13) had high or intermediate immune infiltration in the TME compared with pts with 33% (10/30) TP53-WT AML (pt characteristics in the TP53-WT AML cohort were balanced) (Fig. 1A). IHC analysis confirmed high CD8+ T-cell, regulatory T cell (Treg) and PD-L1+ cell infiltration in TP53mut BL BM samples (Fig. 1B). ORR was 60% (9/15), with 47% (7/15) achieving complete response. In the TP53mut subgroup, the reduction of BM blasts relative to baseline averaged 51.2% (Fig. 1C). Time on treatment and time to death and/or censoring are summarized in Fig. 1D, including three pts who proceeded to receive allogeneic hematopoietic stem cell transplantation (HSCT). In pts who achieved a complete remission (CR, CRi), median OS was 10.3 months. Furthermore, the tumor inflammation signature (TIS), inflammatory chemokine, Treg and IFN-γ gene expression scores were significantly higher at baseline in pts with complete remission compared with non-responders (Fig. 1E), highlighting the association between response to T-cell engagers and a T cell-infiltrated TME. Conclusion: TP53 mutated AML is associated with immune infiltration in the TME and FLZ immunotherapy demonstrated activity in pts with TP53 alterations. This suggests that FLZ immunotherapy may alleviate the negative prognostic immunological impact of TP53 mutation. Figure 1 Disclosures Lai: Abbvie: Consultancy; Agios: Consultancy; Macrogenics: Consultancy; Astellas: Speakers Bureau; Jazz: Speakers Bureau. Church:NanoString Technologies, Inc.: Current Employment. Advani:Novartis: Consultancy, Other: advisory board; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; OBI: Research Funding; Takeda: Research Funding. Wieduwilt:Macrogeneics: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Merck: Research Funding; Leadiant: Research Funding; Amgen: Research Funding. Arellano:Hanmi: Research Funding; Cephalon Oncology: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Pfizer: Consultancy; Agios: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria. Ravandi:Macrogenics: Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Orsenix: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Foster:Bellicum Pharmaceuticals: Research Funding; Daiichi Sankyo: Consultancy; Macrogenics: Consultancy, Research Funding. Stiff:Atara: Research Funding; Delta-Fly: Research Funding; Kite, a Gilead Company: Research Funding; Amgen: Research Funding; Unum: Research Funding; Gamida Cell: Research Funding; Macrogenics: Research Funding. Emadi:NewLink Genetics: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; Jazz Pharmaceuticals: Research Funding. Walter:Aptevo Therapeutics: Research Funding. Tran:MacroGenics: Current Employment. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Guo:Macrogenics: Current Employment. Gojo:Genentech: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Amphivena: Research Funding; Amgen: Research Funding; Merck: Research Funding. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:Macrogenics: Current Employment. Rutella:MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding; Kura Oncology: Research Funding.

PurposeSomatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 37-46% of acute myeloid leukemia (AML) cases with adverse risk cytogenetics and are associated with primary induction failure (PIF), high risk of relapse and dismal prognosis. Herein, we aimed to characterize the immune landscape of TP53 mutated AML and to determine whether TP53 abnormalities identify a patient subgroup that may benefit from T-cell targeting immunotherapy approaches.Experimental DesignThe NanoString Pan-Cancer IO 360™ assay was used for the immune transcriptomic analysis of 64 diagnostic bone marrow (BM) samples from adults with TP53 mutated AML (n=42) or TP53 wild type AML (n=22), and 35 BM samples from heavily pretreated patients with relapsed/refractory (R/R) AML (11 cases with TP53 mutations and/or 17p deletion with genomic loss of TP53) who received immunotherapy with flotetuzumab, an investigational CD123×CD3 bispecific DART® molecule (NCT02152956). In silico data series included The Cancer Genome Atlas (TCGA) cohort and a Dutch–Belgian Cooperative Trial Group for Hematology–Oncology (HOVON) cohort.ResultsAll TCGA cases with TP53 mutations (n=13) expressed higher levels of negative immune checkpoints, inflammatory chemokines, interferon (IFN)-γ-inducible molecules, and had a higher tumor inflammation signature (TIS) score, compared with TCGA cases with other risk-defining molecular lesions. The comparison between TP53 mutated and TP53 wild type primary BM samples showed higher expression of IFNG, FoxP3, immune checkpoints and markers of exhaustion and senescence in the former cohort and allowed the computation of a 34-gene immune classifier prognostic for overall survival. In vitro modeling experiments with AML cell lines showed heightened expression of IFN-γ and inflammation pathway genes in KG-1 cells (loss-of-function mutation of TP53) compared with Kasumi-1 cells (gain-of-function mutation of TP53). Finally, 5 out of 11 (45.5%) patients with R/R AML and TP53 abnormalities showed evidence of anti-leukemic activity of flotetuzumab immunotherapy and had higher TIS, FoxP3, CD8 T-cell abundance, inflammatory chemokine and PD1 gene expression scores at baseline compared with non-responders.ConclusionsThis study provides evidence for a correlation between IFN-γ-dominant immune subtypes and TP53 abnormalities. The anti-leukemic activity with flotetuzumab encourages further study of this immunotherapeutic approach in this patient subgroup.

Approximately 50% of acute myeloid leukemia (AML) patients do not respond to induction therapy (primary induction failure [PIF]) or relapse after

This study dissected the complexity of the immune architecture of acute myeloid leukemia (AML) at high resolution and assessed its influence on therapeutic response. Using 387 primary bone marrow samples from three discovery cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease subtypes and unraveled critical differences in immune gene expression across age groups and disease stages. Importantly, interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles sheds novel insights into the immuno-biology of AML and will inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated myeloid precursors, resulting in impaired hematopoiesis and often bone marrow (BM) failure. The general therapeutic approach in patients with AML has not changed substantially in more than 30 years. Investigation of new strategies, including immunotherapy, remains a priority. Tumor phenotypes are dictated not only by major oncogene drivers, but also by the tumor immunologic microenvironment (TIME) which is inherently immunosuppressive, is equipped to hamper effector T-cell function and includes immune and inflammatory cells, soluble mediators such as interferon (IFN)-gamma and extracellular matrix components. Herein, we profiled the TIME of AML to identify gene signatures that are reflective of general immune status and predictive of antileukemia immune potential. Methods: We used the hybridization-based nCounter® system (NanoString Technologies®, Seattle, WA) and the RNA Pan-Cancer Immune Profiling PanelTM to analyze BM aspirates from 290 adults and 40 children with newly diagnosed, nonpromyelocytic AML. Data were normalized to a set of reference genes and log2-transformed for bioinformatics analysis. The clinical relevance of the association between immune gene signatures and patient outcome was further validated in silico using publicly available cancer transcriptomic datasets (The Cancer Genome Atlas; TCGA). Results: We identified distinct sets of co-expressed genes corresponding to innate immunity, adaptive immunity and IFN-gamma signaling. BM samples with IFN-gamma-dominant gene expression profiles showed up-regulation of actionable immune checkpoints, such as B7-H3, PD-L1 and CTLA-4. A set of 4 differentially expressed immune genes between children and adults with AML (FDR Conclusions: Our study identified distinct immune gene programs in the TIME of patients with newly diagnosed AML. From a translational standpoint, immune-enriched and IFN-gamma-dominant AMLs may benefit from immune checkpoint blockade and from new immunotherapy approaches, including dual-affinity T-cell redirecting antibodies targeting CD123. Grant support: John and Lucille van Geest Foundation, UK; Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). Citation Format: Sergio Rutella, Jayakumar Vadakekolathu, Tressa Hood, Stephen Reeder, Sarah E. Warren, Patrick Danaher, Jan Davidson-Moncada, Alessandra Cesano, Joseph M. Beechem, Sarah K. Tasian, Mark D. Minden. Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B63.

Natural killer (NK) cells recognize and eliminate infected and malignant cells. Their life histories are poorly understood, particularly in humans, due to lack of informative models and endogenous clonal markers. Here we apply transplantation of barcoded rhesus macaque hematopoietic cells to interrogate the landscape of NK cell production, expansion and life histories at a clonal level long-term and following proliferative challenge. We identify oligoclonal populations of rhesus CD56(−)CD16(+) NK cells that are characterized by marked expansions and contractions over time, yet remained long-term clonally uncoupled from other hematopoietic lineages, including CD56(+)CD16(−) NK cells. Individual or groups of CD56(−)CD16(+) expanded clones segregated with surface expression of specific killer immunoglobulin-like receptors (KIRs). These clonally distinct NK cell subpopulation patterns persisted for over 4 years, including following transient in vivo anti-CD16 mediated depletion and subsequent regeneration. Profound and sustained IL-15-mediated depletion was required to generate new oligoclonal CD56(−)CD16(+) NK cells. Together, our results indicate that linear NK cell production from multipotent hematopoietic progenitors or less mature CD56(+)CD16(−) cells is negligible during homeostasis and moderate proliferative stress. In such settings, peripheral compartmentalized self-renewal can maintain the composition of distinct, differentiated NK cell subpopulations.

Background Despite improvements in treatments, the 5-year survival of GEA patients (pts) is disappointing. Individual molecular subtypes display preferential responses to PD-1 blockade. M, an investigational Fc-optimized anti-HER2 monoclonal antibody, is being tested in combination with P in HER2+ GEA post trastuzumab. We present gene expression data from archival FFPE biopsies. Methods 55 pt samples were assessed by NanoString PanCancer IO360™ assay. Immune signature scores are presented as fold changes (FC) and analyzed by unpaired t-test. Associations examined include baseline IHC PD-L1 (pos vs neg) and HER2 expression (IHC3+ vs 2+), ERBB2 mRNA, inflamed tumor microenvironment (TME), radiographic response, and GC vs GEJ. Results ERBB2 mRNA was increased in pts with HER2 IHC3+ vs 2 + (5.6 FC, p Conclusions Our study documents for the first time ERBB2 expression and inflamed TME in GEA, which can help differentiate immunologically between GC and GEJ tumors. Clinical trial identification NCT02689284. Legal entity responsible for the study MacroGenics, Inc. Funding MacroGenics, Inc. Disclosure S. Rutella: Leadership role: Society for Immunotherapy of Cancer; Research grant / Funding (institution): John and Lucille van Geest Foundation. S.E. Church: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. A.H. Sullivan: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. S. Warren: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. J. Baughman: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. J. Muth: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. H. Park: Research grant / Funding (institution): Amgen; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): BeiGene; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): EMD Serono; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Medivation; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Millennium; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Puma Biotechnology; Research grant / Funding (institution): Regeneron; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Vertex. H. Uronis: Shareholder / Stockholder / Stock options, Full / Part-time employment: GeneCentric; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Research grant / Funding (institution): Advaxis; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): Macrogenics. Y. Kang: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (self): DAE HWA Pharmaceutical; Advisory / Consultancy: Lilly/ImClone; Advisory / Consultancy: Merck Serono; Advisory / Consultancy: Ono Pharmaceutical; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: Taiho Pharmaceutical; Research grant / Funding (self): LSK Biopharma. M.C.H. Ng: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD Oncology; Honoraria (self), Travel / Accommodation / Expenses: Taiho Pharmaceutical; Advisory / Consultancy: Bristol-Myers Squibb; Research grant / Funding (institution): ASLAN Pharmaceuticals. P. Enzinger: Advisory / Consultancy: Astellas Pharma; Advisory / Consultancy: Five Prime Therapeutics; Advisory / Consultancy: Merck; Advisory / Consultancy: Taiho Pharmaceutical. K.W. Lee: Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): ASLAN Pharmaceuticals; Research grant / Funding (institution): AstraZeneca/MedImmune; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Green Cross Corp; Research grant / Funding (institution): LSK BioPharma; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Merck KGaA; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Pharmacyclics. K. Huber: Full / Part-time employment: Macrogenics, Inc. A. Wynter-Horton: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. D. Li: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. Y. Bang: Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca/MedImmune; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): BeiGene; Advisory / Consultancy, Research grant / Funding (institution): Green Cross; Advisory / Consultancy: Hanmi; Advisory / Consultancy, Research grant / Funding (institution): Merck Serono; Advisory / Consultancy, Research grant / Funding (institution): MSD Oncology; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Samyang; Advisory / Consultancy, Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Astellas Pharma; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): CKD; Research grant / Funding (institution): Curis; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Takeda. J. Davidson-Moncada: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. D.V. Catenacci: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Bristol-Myers Squibb; Honoraria (self): Five Prime Therapeutics; Honoraria (self), Speaker Bureau / Expert testimony: Foundation Medicine; Honoraria (self), Advisory / Consultancy: Genentech/Roche; Honoraria (self): Genmab; Honoraria (self): Gritstone Oncology; Honoraria (self), Speaker Bureau / Expert testimony: Guardant Health; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy: Merck; Honoraria (self): NantOmics; Honoraria (self): OncoPlex Diagnostics; Honoraria (self), Advisory / Consultancy: Taiho Pharmaceuticsal; Advisory / Consultancy: Astellas Pharma. All other authors have declared no conflicts of interest.

Approximately 40% of patients (pts) with newly diagnosed (AML) either fail to achieve complete remission with intensive induction therapy or experience disease recurrence after a short remission duration (< 6 months). These pts, herein considered to have primary refractory disease, are an extremely challenging population to treat, with only 14% achieving remission following conventional chemotherapy and with subsequent salvage attempts being nearly universally ineffective (1). Increased immune infiltration of the tumor microenvironment (TME) and high CD123 expression on AML blasts have been associated with primary induction failure and poor prognosis (2, 3). Flotetuzumab (FLZ), a CD123 x CD3 bispecific DART molecule, is currently being tested in a phase 1/2 study in pts with either relapsed or refractory (R/R) AML. We have previously reported FLZ activity in primary refractory AML (4); herein, we provide additional scientific rationale supporting the investigation of FLZ in this patient population. The recommended Phase 2 dose (RP2D) of FLZ identified in an ongoing Phase 1/2 study is 500 ng/kg/day administered as a 7 -day/week continuous infusion. Pts receive a lead-in dose during week (W) 1, followed by 500 ng/kg/day during W2-4 of Cycle 1, and a 4-day on/3-day off schedule for Cycle 2 and beyond. Disease status was assessed by modified IWG criteria; bone marrow (BM) samples were collected to investigate biomarkers, including CD123 receptor density (RD), and gene expression profiling using the NanoString PanCancer IO 360™ panel, which measures the expression of 770 genes, including 14 immune cell types and 32 immuno-oncology biological signatures. Gene expression comparisons are presented at fold change (FC) and a t-test was used for statistical analysis. Fifty pts with R/R AML received FLZ at the RP2D. Thirty (60%) pts had primary refractory AML: 24 failed ≥2 induction attempts and 6 recurred after remission of 30% decrease in BM blasts) was associated with increased baseline immune gene signatures, including significantly higher IFNγ scores (1.8 FC, p=0.0212; AUROC=0.74), and an increased tumor inflammation signature (TIS) score (1.658 FC, p=0.00827, AUROC=0.847 compared to non-responders. FLZ was well tolerated, with no increased cytokine release syndrome events in primary refractory pts (30% G1, 67% G2, 3% G3) compared to relapse pts (26% G1, 58% G2, 16% G3), notwithstanding the increased CD123 RD in the former. In conclusion, we show that FLZ elicits clinical response in heavily treated patients with poor response rates to primary therapy. We also show that increased IFNγ signaling gene expression scores in baseline BM appears to associate with response to FLZ therapy. Enrollment has been expanded to further define FLZ's activity specifically in pts with primary refractory AML, and candidate biomarkers to enable identification of pts more likely to respond to FLZ. Estey E, et al. Blood (1996), 88 (2) 756Vadakekolathu J, et al. Blood (2017) 130:3942Vergez F, et al. Haematologica (2011), 96(12):1792-8Uy GL, et al. Blood (2018) 132:764Duong V, et al. Leukemia (2013),13(6):711-5 Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Aldoss:Agios: Consultancy, Honoraria; AUTO1: Consultancy; Jazz Pharmaceuticals: Honoraria, Other: travel/accommodation/expenses, Speakers Bureau; Helocyte: Consultancy, Honoraria, Other: travel/accommodation/expenses. Foster:Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Sallman:Celgene: Research Funding, Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Jazz: Research Funding; Novartis: Speakers Bureau; Abbvie: Speakers Bureau. Sweet:Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Stemline: Consultancy; Pfizer: Consultancy; Jazz: Speakers Bureau; Incyte: Research Funding. Rizzieri:AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; Stemline: Research Funding. Advani:Amgen: Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Emadi:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Genentech: Consultancy, Honoraria; KinaRx: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and Scientific Advisor, Patents & Royalties; Jazz Pharmaceuticals: Research Funding. Wieduwilt:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen, Leadiant, Merck, Servier: Research Funding; Reata Pharmaceuticals: Equity Ownership. Vey:Novartis: Consultancy, Honoraria; Janssen: Honoraria. Church:NanoString Technologies, Inc.: Employment, Equity Ownership. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Arellano:Gilead: Consultancy. Löwenberg:Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees. Ravandi:Selvita: Research Funding; Xencor: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Macrogenix: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel LTD: Research Funding. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Tran:MacroGenics: Employment. Timmeny:MacroGenics, Inc.: Employment, Other: Stock Ownership. Topp:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Guo:Macrogenics: Employment. Zhao:MacroGenics, Inc.: Employment. Wigginton:MacroGenics, Inc.: Employment, Equity Ownership; Western Oncolytics: Other: clinical advisory board. Bonvini:MacroGenics, Inc.: Employment, Equity Ownership. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding. Walter:Seattle Genetics: Research Funding; Race Oncology: Consultancy; Pfizer: Consultancy, Research Funding; New Link Genetics: Consultancy; Kite Pharma: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. DiPersio:Amphivena Therapeutics: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; Magenta Therapeutics: Equity Ownership; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Consultancy; Incyte: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Karyopharm Therapeutics: Consultancy.

Introduction The infiltration of immune cells into tumors has been associated with therapeutic effects in preclinical models and patients with cancer. In AML, we have previously reported that immune infiltrated TME is predictive of failure to cytotoxic chemotherapy, but associated with response to immunotherapy, specifically FLZ (Uy ASH 2018, Rutella ASH 2018). Furthermore, FLZ also affects immune infiltration in the TME (Rutella ASH 2018). NK cells play an important role in AML control (Ruggieri Science 2012). FLZ (MGD006/S80880) is a humanized DART® molecule that bridges CD123 on AML with CD3 on T cells and mediates anticancer activity via T-cell activation and cytolytic activity against the bound cancer cell. While this is well described in vitro, little evidence of this interaction is available in vivo. Methods Patients (pts) were treated on the recommended phase 2 dose (RP2D) of FLZ (multi-step lead-in dose followed by 500ng/kg/day, in 28-day cycles). We studied the bone marrow (BM) tissue samples for 6 primary refractory pts at baseline and after treatment. Response assessment was performed at day 25±3 days of each cycle. Serial BM samples were evaluated using 2 different staining panels (PD-L1, FoxP3, CD8, CD3, CD103 / CD123, CD3, CD57, CD16) on consecutive slides. Slides were stained using a Leica BondRx autostainer and fluorescence imaged using a Polaris Vectra 3 and analyzed using inForm software. A density-based clustering algorithm developed and run in QuPath was used to quantify CD3+ T cell clusters. Results Six pts with primary refractory AML were included in this report. Pts were heavily pretreated (median prior lines of therapy was 3, range 2-9), and had adverse cytogenetic risk (ELN 2017). Three pts had a complete remission (CR) after 1 cycle of therapy (CR, CRh, CRi), two went on the receive allogeneic stem cell transplant (HSCT). In baseline BM samples, CD3 and CD8 cell infiltrates were higher in CR vs non-responders (CD3+ 18.3% ±6.9 vs 9.3% ±1.8; CD8+ 9.4% ±3.5 vs 4.8% ±1.2; mean±SEM). Two of the three CR patients, who underwent HSCT, developed clusters (Figure 1) in their on-treatment biopsies with 65 and 22 clusters of an average of 34 and 17 T cells per cluster, respectively. All clusters in CR pts were found on or adjacent to CD123+ cells. The BM biopsy of the CR pt with no detected clusters had no unequivocal evidence of residual/recurrent leukemic blasts. This pt had their dose interrupted early due to non-treatment related AE (infectious complication) and did not receive a full cycle of treatment; the response was transient and the pt relapsed shortly thereafter. NK cells (CD57+CD16+) were increased in post treatment biopsies of CR vs non-responders (0.93 ±0.31 vs 0.27 ±0.13; mean±SEM) with the largest fold increase in CR (28 vs 9). Lastly, post treatment biopsy PD-L1 expression was higher in non-responders than CR (23% vs 16%) with non-responders exhibiting the largest fold change in total PD-L1+ cells (10.9 vs 2.2). Summary Consistent with its proposed mechanism of action, these data highlight for the first time, the dynamic induction of an increase in T-cell infiltration, and clustering around CD123 AML cells in the bone marrow microenvironment of two AML patients that responded to FLZ. In pts with resistance to FLZ (non-responders) PD-L1 induction was significantly higher indicating that in some pts treatment with sequential check point inhibitor could obviate this mechanism of resistance A trial combining FLZ with sequential administration of a PD-1 inhibitor (MGA012) is currently recruiting pts. Figure 1. Baseline and on-treatment IHC of BM biopsies of a FLZ-treated CR pt showing cluster formation following treatment. Disclosures Bifulco: Ventana: Other: advisory board; PrimeVax: Equity Ownership, Other: ScientificBoard; BMS: Other: Advisory Board; Providnece: Patents & Royalties: Imaging processing; Halio Dx: Other: advisory board. Wigginton:macrogenics: Employment, Equity Ownership; western oncolytics: Consultancy, Other: consultancy. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. Fox:Akoya: Research Funding; Bristol Myers Squibb: Research Funding; Definiens: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Research Funding; Ultivue: Membership on an entity's Board of Directors or advisory committees.

Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Chemotherapy resistance is common, and relapse is the major cause of treatment failure. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. Methods Immune gene expression profiling for the high-dimensional analysis of the immunological landscape of bone marrow (BM) samples from patients with newly-diagnosed (de novo) non-promyelocytic AML (n=387) was employed to analyze the tumor microenvironment (TME). We derived immune scores from mRNA expression levels and devised an RNA-based, quantitative metric of immune infiltration, as previously published (Danaher P, et al. JITC 2017 and 2018). The PanCancer IO360™ gene expression assay was used to profile BM samples collected prior to and during flotetuzumab (FLZ) treatment from 30 AML patients treated at the RP2D (500 ng/kg/day) in the CP-MGD006-01 clinical trial (NCT#02152956), primary refractory, n=23 or relapsed, n=7. IO360 score comparisons are presented as mean ± SD and significance was assessed by the Mann Whitney U test. Results Analysis of pre-treatment BM samples from de novo AML revealed distinct immune signature modules, reflecting the co-expression of genes associated with 1) an IFNγ-dominant TME, 2) adaptive immune responses, and 3) myeloid cell abundance. When considered in aggregate, the relative intensity of gene expression in the immune modules stratified BM samples into two subgroups, which will be herein termed immune-infiltrated and immune-depleted. When AML patients were dichotomized based on median immune scores, high versus low, a higher percentage of primary refractory patients was observed within the IFNγ high module (65.4% versus 34.6%; p=0.0022). In multivariate logistic regression, an IFNγ high profile (derived from the IFNγ-dominant module) was predictive of therapeutic resistance to induction chemotherapy, even more than ELN risk categories (AUROC = 0.815 versus 0.702 with ELN risk only). In a validation cohort, Beat AML series, the IFNγ high profile when compared to other clinically established prognostic indicators in AML, i.e. disease type (primary versus secondary), WBC count, patient age at diagnosis, and ELN risk categories was significantly more predictive of therapeutic resistance to induction chemotherapy (AUROC=0.921 versus 0.709 with ELN cytogenetic risk alone; two-tailed p value=0.002673). Similarly, a higher percentage of patients with an IFNγ high profile AML in the HOVON series failed to achieve CR in response to induction chemotherapy when compared to AML cases with an IFNγ low profile (27.2% versus 15.2%; p=0.0004). We hypothesized that higher expression of IFNγ inducible genes, while underpinning chemotherapy resistance, might identify AML patients who derive benefit from immunotherapy with FLZ. BM samples from 92% of patients with evidence of FLZ anti-leukemic activity (ALA), which was defined as either CR, CRh, CRi, PR or overall benefit (>30% reduction in BM blasts), had an immune infiltrated TME relative to non-responders (SD or PD). Interestingly, the IFNγ-signaling score was significantly higher in patients with chemotherapy-refractory AML compared with relapsed AML at time of FLZ treatment, and in individuals with evidence of ALA compared to non-responders (p Conclusions Our findings to date suggest that microenvironmental immune gene profiles could be used to inform the delivery of personalized immunotherapies to patients with IFNγ-dominant AML subtypes, and identify patients less likely to respond to cytotoxic chemotherapy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Hood:NanoString Technologies, Inc.: Employment. Church:NanoString Technologies, Inc.: Employment, Equity Ownership. Sullivan:NanoString Technologies, Inc.: Employment. Viboch:NanoString Technologies, Inc.: Employment. Warren:NanoString Technologies, Inc.: Employment. Liang:NanoString Technologies, Inc.: Employment. Cesano:NanoString Technologies, Inc.: Employment. Löwenberg:Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. DiPersio:Celgene: Consultancy; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Bioline Rx: Research Funding, Speakers Bureau. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding.

Background Acute myeloid leukemia (AML) blasts and leukemic stem and progenitor cells typically express higher levels of CD123 than their normal hematopoietic stem cell counterparts, making CD123 an attractive target. Leukemic CD123 expression is associated with poor prognosis, high-risk disease, and increased risk of induction failure (Vergez et al 2011). Single-agent flotetuzumab, an investigational CD123 x CD3 bispecific DART protein, has shown evidence of clinical activity in a Phase 1 study of relapsed/refractory (R/R) acute myeloid leukemia (AML). In this study, flotetuzumab led to T-cell activation which in turn was associated with PD-1 induction on T lymphocytes, enhanced IFNɣ secretion, and upregulation of PD-L1 expression by AML blasts (ASH 2018 Rutella, ASH 2018 Uy). In vitro studies have shown synergistic T-cell mediated cytotoxicity of an AML cell line (KG1A) with flotetuzumab in the presence of PD-1/PD-L1 axis blockade compared to flotetuzumab alone. MGA012, also known as INCMGA00012, is an investigational anti-PD-1 antibody that has shown clinical activity in a Phase 1 study (SITC 2018 Mehnert). We hypothesize that combined checkpoint inhibition with MGA012 together with redirected T‐cell killing of CD123+ cells with flotetuzumab may show enhanced activity over flotetuzumab alone. Methods This is a Phase 1 dose escalation study designed to characterize the safety, tolerability, dose-limiting toxicities, maximum tolerated dose (MTD) or maximum administered dose (if no MTD is defined), pharmacokinetics, and preliminary anti-leukemic activity of flotetuzumab in combination with MGA012, each administered intravenously (IV) in patients with R/R AML. Response evaluation will be determined by modified ELN 2017 criteria. Activity is measured by complete response (CR) (complete remission [CR], or CR with partial hematologic recovery [CRh], or CR with incomplete hematological recovery [CRi], or morphologic leukemia-free state [MLFS]) rate, relapse-free survival, or mortality rate at 1 and 3 months. The impact of flotetuzumab/MGA012 combination on overall survival and event-free survival will be explored. Eligible patients will consist of adults with relapsed or refractory AML (any subtype except acute promyelocytic leukemia) who have exhausted standard of care options. In Induction Cycle 1, patients will be treated with a step-up lead in dose of flotetuzumab, followed by continuous infusion flotetuzumab, starting at week 2 of Cycle 1 and continuing through each 28-day cycle. MGA012 will be administered every two weeks. Depending on response, patients will transition to either consolidation or second induction. Eligible patients who achieve CR/CRh/CRi can receive maintenance MGA012 alone for up to 12 months. Disclosures Wei: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. Fong:Novartis: Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Astellas: Consultancy. Montesinos:Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Research support; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau. Gil:Jazz Pharmaceuticals: Honoraria; Gilead: Honoraria; Daiichi-Sankyo: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Abbvie: Honoraria. Perez De Oteyza:Celgene: Speakers Bureau. Rowe:BioSight: Consultancy. Wolach:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaker; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaker. Sun:MacroGenics, Inc.: Employment, Equity Ownership. Baughman:MacroGenics, Inc.: Employment, Equity Ownership. McNulty:MacroGenics, Inc.: Employment, Equity Ownership. Bonvini:MacroGenics, Inc.: Employment, Equity Ownership. Wigginton:Western Oncolytics: Other: clinical advisory board; MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership.

Background T + chemo is standard 1st line therapy for HER2+ gastroesophageal adenocarcinoma; however, patients (pts) tend to progress in 6-8 months. Up to 40% show loss of HER2 expression post T, likely underlying the lack of efficacy of anti-HER2 agents in 2nd line therapy. We report here a clinical update and biomarker analysis of an ongoing study in pts receiving M, an anti-HER2 Fc-optimized mAb, plus P in HER2+ GEA pts in post T, 2nd line, chemotherapy-free treatment. Methods Endpoints described herein are safety, objective response rate (ORR), disease control rate (DCR), archival HER2 IHC level and/or ERBB2 amplification (amp) status pre-M+P in cell free DNA (cfDNA) by NGS (Guardant360), PD-L1 CPS by IHC (22C3 pharmDx), anti-HER2 T- cell immunity by ELISPOT on PBMCs, and NanoString PanCancer IO360™ assay on archival FFPE biopsies. Results 92 pts (66% GC, 34% GEJ) received 15mg/kg M+200mg P Q3W. 99% were MSS and 43% were PD-L1+(>1%). Related adverse events ≥grade 3 were 18.5%. Confirmed ORR was 19% with 54% DCR; interestingly, higher response rates occurred in GC vs GEJ (25% vs 7% ORR, p=0.047 and 64% vs 33% DCR, p=0.008). A higher proportion of GC vs GEJ cancers was HER2 3+ on archival IHC (90% vs 53%, p=0.0075), furthermore, a 5.3-fold greater fraction of GC vs GEJ pts showed upregulated tumor ERBB2 RNA expression (p 6-fold higher in GC vs GEJ (34.7% vs 5.5%, p=0.03) and associated with 53% confirmed ORR and 82% DCR in GC. GC pts with higher baseline T-cell immunity toward HER2 (p59 class II peptide) had greater probability of ORR (p=0.005). Conclusions In this study, M+P demonstrated tolerability and preliminary evidence of anti-tumor activity as a chemotherapy-free regimen in 2nd line HER2+ GC; biomarker analysis suggests an association with higher expression and retention of HER2 and PD-L1 together with pre-existing anti-HER2 immunity. Clinical trial identification NCT02689284. Legal entity responsible for the study MacroGenics, Inc. Funding MacroGenics, Inc. Disclosure H. Park: Research grant / Funding (institution): Ambrx; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): BeiGene; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): EMD Serono; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Medivation; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Millennium; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Puma Biotechnology; Research grant / Funding (institution): Regeneron; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Vertex. H. Uronis: Shareholder / Stockholder / Stock options, Full / Part-time employment: GeneCentric; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Research grant / Funding (institution): Advaxis; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Merck. Y. Kang: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): DAE HWA Pharmaceutical; Advisory / Consultancy: Lilly/Imclone; Advisory / Consultancy: Merck/Serano; Advisory / Consultancy: Ono Pharmaceutical; Advisory / Consultancy: Roche/Genentech; Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): LSK Biopharma. M.C.H. Ng: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD Oncology; Honoraria (self), Travel / Accommodation / Expenses: Taiho Pharmaceutical; Advisory / Consultancy: Bristol-Myers Squibb; Research grant / Funding (institution): ASLAN Pharmaceuticals. P. Enzinger: Advisory / Consultancy: Astellas Pharma; Advisory / Consultancy: Five Prime Therapeutics; Advisory / Consultancy: Merck; Advisory / Consultancy: Taiho Pharmaceutical. K.W. Lee: Research grant / Funding (institution): Array BioPharma; Research grant / Funding (institution): ASLAN Pharmaceuticals; Research grant / Funding (institution): AstraZeneca/MedImmune; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Green Cross Corp; Research grant / Funding (institution): LSK BioPharma; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Merck KGaA; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Pharmacyclics; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Lilly; Research grant / Funding (institution): ALX Oncology. S. Rutella: Leadership role: Clinical & Biomarkers Data Sharing Subcommittee, Society for Immunotherapy of Cancer; Research grant / Funding (institution): John and Lucille van Geest Foundation. S.E. Church: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. J. Nordstrom: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. T. Wu: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. J. Yen: Shareholder / Stockholder / Stock options, Full / Part-time employment: Guardant Health. A. Franovic: Shareholder / Stockholder / Stock options, Full / Part-time employment: Guardant Health. J. Muth: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. M. Rosales: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. J. Davidson-Moncada: Shareholder / Stockholder / Stock options, Full / Part-time employment: MacroGenics, Inc. Y. Bang: Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca/MedImmune; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Beigene; Advisory / Consultancy, Research grant / Funding (institution): Green Cross; Advisory / Consultancy: Hanmi; Advisory / Consultancy, Research grant / Funding (institution): Merck Serono; Advisory / Consultancy, Research grant / Funding (institution): MSD Oncology; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Samyang; Advisory / Consultancy, Research grant / Funding (institution): Taiho Pharmaceutical; Research grant / Funding (institution): Astellas Pharma; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): CKD; Research grant / Funding (institution): Curis; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Takeda. D.V. Catenacci: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Bristol-Myers Squibb; Honoraria (self): Five Prime Therapeutics; Honoraria (self), Speaker Bureau / Expert testimony: Foundation Medicine; Honoraria (self), Advisory / Consultancy: Genentech/Roche; Honoraria (self): GenMab; Honoraria (self): Gritstone Oncology; Honoraria (self), Speaker Bureau / Expert testimony: Guardant Health; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self): Merck; Honoraria (self): NantOmics; Honoraria (self): OncoPlex Diagnostics; Honoraria (self), Advisory / Consultancy: Taiho Pharmaceutical; Advisory / Consultancy: Astellas Pharma. All other authors have declared no conflicts of interest.

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FceRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3-CD56bright, canonical, or adaptive CD3-CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

65 Background: Trastuzumab (T) + chemo is standard first-line therapy (tx) for HER2+ gastroesophageal adenocarcinoma (GEA) pts, though progression ensues in 6-8 months. The approved second-line tx is ramucirumab +/- paclitaxel (R+PAC). Pts with GC are less responsive to R+PAC than gastroesophageal junction (GEJ) pts, in particular HER2+ GC, and no anti-HER2 agents are approved in post-T setting. We report results of combination M+P in HER2+ GC pts and describe a biological rationale for this population. M is an anti-HER2 mAb Fc optimized for enhanced binding to activating FcgRIIIa (CD16A) and decreased binding to inhibitory FcgRIIb (CD32B). M demonstrated an enhanced Fc-dependent MoA, including enhanced ADCC. Methods: HER2+, PD-L1-unselected, second-line GEA pts post T progression received M (15 mg/kg) + P (200 mg) Q3wk. Safety, objective response rate (ORR), median overall & progression-free survival (mOS, mPFS), disease control rate (DCR), circulating tumor DNA, & tumor PD-L1 expression were assessed. Results: To date, 66 GEA pts were dosed; 35 (53%) GC and 31 (47%) GEJ. Overall, 12/66 (18.2%) had tx-related adverse events ≥ grade 3; 5 had drug-related SAEs: dehydration, diabetic ketoacidosis, hypotension and pneumonitis, each a single event, and 2 events of autoimmune hepatitis. Eligibility was based on archival HER2 expression; an exploratory endpoint measured retention of HER2 expression post-T by ERBB2 ctDNA. HER2 expression was lost in 23/56 (41.1%) of pts tested post T. HER2 retention was higher in pts with GC versus GEJ (65.8% vs. 44.8%) and in GEA pts with IHC 3+ vs 2+ archival tumors (61.7% vs 47.4%, respectively). Furthermore, GC had higher PD-L1 expression than GEJ, 53.3 vs. 33.3%, respectively. This coincided with more responses in IHC3+ GC pts, ORR 12/29 (41.4%; 95% CI 23.5-61.1), DCR 21/29 (72.4%; 95% CI 52.8-87.3), mPFS 5.5 months (95% CI 2.3-7.6), mOS not reached, with lower bound of 9.1 months for 95% CI. Enrollment of an additional 25 pts enriched for IHC3+ GC is ongoing. Conclusions: Results suggest that M+P, a chemo-free regimen, demonstrates acceptable tolerability and has encouraging preliminary activity in second-line HER2+ GEA, specifically in GC pts who retain ERBB2 amp prior to second-line tx. Clinical trial information: NCT02689284.

Background: Flotetuzumab (FLZ; MGD006/S80880) is a novel CD123 x CD3 bispecific DART® protein being tested in a Phase 1/2 study (NCT02152956) in patients with relapsed/refractory acute myeloid leukemia (AML). As with all T-cell redirecting therapies, cytokine secretion, inherent in T-cell activation, with ensuing potential for cytokine release syndrome (CRS), remains an important side effect. We have previously reported that multi-step dosing mitigates CRS severity (1). CRS diagnosis and treatment is guided by the occurrence of non-specific clinical signs, such as fever, chills, hypotension and tachycardia. Therefore, identification of predictors of CRS will be useful for optimal pt. management. Here we report on potential biomarkers of CRS severity that may help guide CRS management. Methods: Data from pts treated with FLZ at RP2D (lead-in dose of 30ng/kg/d for 3d, 100ng/kg/d for 4d for week 1 followed by 500ng/kg/d CIV week 2-4 of cycle 1, and a 4d-on/3d-off schedule for Cycle 2 and beyond in 28-day cycles) was collected and analyzed. Incidence and severity of CRS were analyzed for correlation with cytokine levels and changes in BM blasts. Relation between immune cells (T-cell subsets, monocytes) with tumor burden, percent CD123+ AML blasts, and CD123 expression, were interrogated as potential determinants of CRS. Administration, dose and frequency of tocilizumab (TCZ), an IL-6 receptor antagonist, were evaluated for their relationship with CRS severity, frequency, CRP and cytokine levels. Results: 30 pts were dosed at RP2D. Most pts experienced mild to moderate CRS (G1 26.7%, G2 60%) of short duration (median 1 day(d), range 1-26 d) and were conservatively managed to full resolution. Grade ≥ 3 events occurred in 4/30 pts (13%), with vasopressors use in 2 pts, and a median duration of 2.5 d (range 2-13 d). CRS frequency decreased with time on treatment: 42% occurred within the first week (LID), 39.6% occurring in week 2 (step up to 500ng/kg/day) and 18% occurring during week 3 and 4. IL-6 levels showed the best relationship with CRS severity, as previously shown (1). IL-6 levels, however, did not correlate with response. Twenty pts (67%) received at least one dose of TCZ (median 2 doses/pt; range 1-12 doses). As anticipated, mean IL-6 levels increased after administration of TCZ. CRS severity showed a relationship with the baseline frequency of circulating CD4+ cells (median 47% in G1 vs 73% in G ≥2, p = 0.0082), while CD8+ cell frequency did not correlate with CRS. Disease burden (absolute AML blasts, % CD123 AML blasts), CD123 expression on AML blasts, monocytes levels or effector-to-target ratio in the peripheral blood did not show a relationship with CRS severity. Importantly, CRS severity was not correlated with FLZ anti-leukemic activity. Conclusion: The frequency of CD4+ cells at baseline may be a potential biomarker for identifying pts at risk of more severe CRS. Early use of TCZ can effectively modify the activity of IL-6, a significant contributor to CRS, and blunt CRS severity. Since severity of CRS and IL-6 levels did not correlate with FLZ anti-leukemic activity, blunting its severity should be aggressively pursued. Early identification of pts at greater CRS risk together with multistep dosing (1) and early use of TCZ can effectively manage CRS with no impact on FLZ anti-leukemic activity.Jacobs et al. ASH 2017, abstract #3856 Disclosures Jacobs: MacroGenics: Employment. Viero:Servier: Employment. Baughman:MacroGenics: Employment. Sun:MacroGenics: Employment. Ying:Macrogenics: Employment. Muth:MacroGenics: Employment. Hong:MacroGenics: Employment. Sweet:BMS: Honoraria; Agios: Consultancy; Phizer: Consultancy; Astellas: Consultancy; Jazz: Speakers Bureau; Jazz: Speakers Bureau; BMS: Honoraria; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Agios: Consultancy; Phizer: Consultancy; Celgene: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Uy:Curis: Consultancy; GlycoMimetics: Consultancy. Ravandi:Xencor: Research Funding; Macrogenix: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; Jazz: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Abbvie: Research Funding; Orsenix: Honoraria; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Sunesis: Honoraria; Sunesis: Honoraria; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding; Jazz: Honoraria. Foster:Celgene: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding; Shire: Honoraria. Rizzieri:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Arog: Consultancy, Membership on an entity's Board of Directors or advisory committees; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees. Arellano:Cephalon: Research Funding. Rettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding. Topp:F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron Pharmaceuticals, Inc.: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding. Lelièvre:Servier: Employment. Lowenberg:Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees. Wigginton:MacroGenics: Employment. Davidson-Moncada:MacroGenics: Employment.

Therapy-related acute myelogenous leukemia (t-AML) is associated with adverse genetic lesions, complex karyotype, and TP53 mutation; it is challenging to treat and confers a poor prognosis. We describe a 74-year-old female patient with AML that developed 9 years after receiving 6 cycles of cytotoxic (FOLFOX) chemotherapy as adjuvant treatment of colorectal carcinoma. At diagnosis, the bone marrow (BM) biopsy revealed blast count of 35%, with normal karyotype, 5q loss and AML1 (RUNX1) locus (21q22) amplification by fluorescence in situ hybridization (FISH). Initial treatment with 5 cycles of azacitidine (AZA) failed to induce a response. The patient was subsequently treated on a Phase 1 trial of flotetuzumab (MGD006/S80880) (FLZ), a novel T-cell redirecting (CD123 x CD3) DART protein [NCT02152956]. Prior to FLZ, BM blasts demonstrated clonal evolution with a complex karyotype (92,XXXX, t(14;21)(q22;q22)) and new IDH1, and TET2 mutations. Patient received a total of 3 cycles (28 days/cycle) of FLZ. After one FLZ cycle, the patient achieved a (complete response) CR, with normal cytogenetics, and residual IDH1 and TET2 mutations. After 2 additional cycles of FLZ consolidation, the CR was maintained, with loss of the IDH1 mutation, but persistence of the TET2 mutation. CR was maintained with no additional therapy for approximately 7 months. Serial BM samples were evaluated for T cells (CD3, CD4, CD8, FOXP3 and PD-L1) using multiplex IHC (Fig 1). BM T cells were unchanged during AZA treatment. At the initial bone marrow on FLZ (CR), significant increases in CD3+, CD3+CD8-, CD3+ CD8+, and FOXP3-positive T cells and PD-L1 expression compared to baseline were noted. CD3+CD8+ T cells persisted in the BM 1 month beyond completion of the additional 2 cycles of consolidation with FLZ, while other T cells subsets and PD-L1 expression returned to baseline (Fig. 1) Three months after the last FLZ treatment, all T cell subsets had returned to baseline, with an early increase in leukemic blasts (8%), but normal cytogenetics. Five months after FLZ treatment blasts were unchanged, but a new abnormal cytogenetic clone, with additional mutations, accompanied by a rise in PD-L1 expression cells was observed. Seven months after FLZ treatment, frank leukemia with 50% blasts developed, and all T cell subsets and PD-L1 expression had returned to pre-treatment levels. Consistent with its proposed mechanism of action, these data highlight for the first time the induction of an increase in T-cell infiltration, which persist beyond FLZ, in the bone marrow microenvironment of an AML patient that also achieved a complete and durable anti-leukemic response after treatment with FLZ. Figure 1. Figure 1. Disclosures Lelièvre: Servier: Employment. Wigginton:MacroGenics: Employment. Davidson-Moncada:MacroGenics: Employment. Fox:MacroGenics: Research Funding.

Acute myeloid leukemia (AML) blasts and leukemia stem cells highly express the α chain of the IL-3 receptor (CD123), compared to normal hematopoietic stem cells. CD123 expression is associated with high-risk features, increased risk of induction failure and poor prognosis (Vergez F, et al. Haematologica 2011; 96: 1792-8). Flotetuzumab (FLZ), a CD123 × CD3 bispecific DART molecule, is being tested in a phase 1/2 study in patients with relapsed/refractory (R/R) AML. The recommended Phase 2 dose (RP2D) of FLZ is 500 ng/kg/day (d) administered as a 7-day/week continuous infusion. Patients receive a lead-in dose (30 ng/kg/d x 3d then 100 ng/kg/d x 4d) during week (W) 1, followed by 500 ng/kg/d during W2-4 of cycle 1, and a 4d on/3 d off schedule for cycle 2 and beyond. Disease status was assessed by modified IWG criteria; samples were collected to investigate candidate biomarkers, including CD123 receptor density/cell (RD), and gene expression profiling using the NanoString® PanCancer IO 360™ assay. This platform was used to assess the expression of 770 genes, including 14 immune cell types and 32 immuno-oncology biological signatures in bone marrow (BM) samples from patients treated with FLZ. Thirty patients with R/R AML, median age 64.5 years, received FLZ at the RP2D. Most patients enrolled had primary refractory disease (60% [18/30; 14 to cytotoxic chemotherapy (refractory to ≥ 2 induction attempts, or first CR with initial CR In conclusion, among this initial cohort of patients treated at the RP2D (500 ng/kg/day), FLZ demonstrates antileukemic activity with an acceptable safety profile, in particular, among refractory patients, a difficult-to-treat patient population that represents a significant area of unmet medical need. Enrollment to the current study has now been expanded to further define the antileukemic activity of FLZ in patients with refractory AML, and investigate candidate biomarkers to enable identification of patients more likely to respond to FLZ. In addition, studies are now being initiated to investigate opportunities to expand the antileukemic activity of FLZ via combined administration with either concurrent or sequenced anti-PD-1 checkpoint blockade. Disclosures Uy: GlycoMimetics: Consultancy; Curis: Consultancy. Rettig:Novimmune: Research Funding; Amphivena Therapeutics: Research Funding. Foster:Celgene: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding; Shire: Honoraria. Rizzieri:Arog: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees. Arellano:Cephalon: Research Funding. Topp:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Boehringer Ingelheim: Research Funding; Regeneron Pharmaceuticals, Inc.: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sweet:Phizer: Consultancy; BMS: Honoraria; BMS: Honoraria; Agios: Consultancy; Jazz: Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy; Astellas: Consultancy; Jazz: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Agios: Consultancy; Celgene: Honoraria, Speakers Bureau; Phizer: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Ravandi:Abbvie: Research Funding; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Jazz: Honoraria; Jazz: Honoraria; Macrogenix: Honoraria, Research Funding; Sunesis: Honoraria; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Macrogenix: Honoraria, Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Xencor: Research Funding; Orsenix: Honoraria; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Abbvie: Research Funding. Church:NanoString Technologies: Employment. Rutella:NanoString Technologies: Research Funding. Sun:MacroGenics: Employment. Yang:MacroGenics: Employment. Baughman:MacroGenics: Employment. Curtis:MacroGenics: Employment. Timmeny:MacroGenics: Employment. Cali:MacroGenics: Employment. Tran:MacroGenics: Employment. Muth:MacroGenics: Employment. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Poirot:Servier: Employment. Pallis:Servier: Employment. Cesano:NanoString Technologies: Employment. Bonvini:MacroGenics: Employment, Equity Ownership. Wigginton:MacroGenics: Employment. Lowenberg:Astex: Consultancy; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:MacroGenics: Employment.

Background. The therapeutic approach in patients (pts) with acute myeloid leukemia (AML) has not changed substantially in >30 years. The introduction of new treatment strategies, including immunotherapy, remains a priority. Flotetuzumab, a CD123 × CD3 bispecific DART immunotherapy, is being tested in a phase 1/2 study of relapsed/refractory (R/R) AML. We previously showed that AML pts with an immune-enriched and IFN-γ-dominant tumor microenvironment (TME) experience significantly shorter relapse-free survival, suggesting refractoriness to standard induction chemotherapy (Vadakekolathu J, et al. Blood 2017; 130: 3942A). Herein, we report that an IFN-γ-dominant TME, while predicting resistance to standard therapy, is favoring response of AML to flotetuzumab. Methods. Gene expression was analyzed in 78 bone marrow (BM) samples (36 at baseline, 27 post-cycle 1 and 15 post-cycle 2) from 40 pts with relapsed or refractory AML enrolled in a phase 1/2 clinical trial of flotetuzumab (NCT#02152956). Thirty-six baseline BM samples were included in the analysis, of which 34 from pts who were treated at the target dose of ≥500 ng/kg/day. The NanoString PanCancer IO360™ assay was used to assess the expression of 770 genes, including the levels of 14 immune cell types and of 32 immuno-oncology signatures, and their correlation with response to flotetuzumab. Data are presented as score means per group±SEM and analyzed by unpaired t-test. Results. Gene expression analysis of BM samples at baseline stratifies flotetuzumab-treated pts into 3 clusters within an immunological continuum: immune-depleted, immune-exhausted and immune-enriched (Fig. 1A). Pts with primary-refractory disease (refractory to ≥2 induction attempts, first CR of Conclusions. We provide evidence for a range of immune gene expression profiles in AML, with primary refractory pts displaying an enhanced immune infiltration signature compared with relapse pts. Furthermore, an IFN-γ-related gene signature at baseline, a feature of primary refractory pts, was associated with clinical response to flotetuzumab. HMA-refractory pts showed an immune-rich but exhausted phenotype with PD-L1 expression, suggesting these pts may further benefit from flotetuzumab combination therapy with checkpoint inhibition (Rettig M, et al. Blood 2017; 130: 1365). Lastly, treatment with flotetuzumab further shifted the immune signature toward a more immune rich phenotype. The AML TME immune gene expression can influence susceptibility to therapy, with primary refractory AML showing an IFN-γ-dominant signature associated with response to flotetuzumab. Figure Figure. Disclosures Rutella: NanoString Technologies: Research Funding. Church:NanoString Technologies: Employment. Viboch:NanoString Technologies: Employment. Sullivan:NanoString Technologies: Employment. Hood:NanoString Technologies: Employment. Warren:NanoString Technologies: Employment. Cesano:NanoString Technologies: Employment. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Muth:MacroGenics: Employment. Lelièvre:Servier: Employment. Lowenberg:Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:MacroGenics: Employment.

Acute myeloid leukemia (AML) is a molecularly heterogeneous hematological malignancy with variable response to treatment. Recurring cytogenetic abnormalities and molecular lesions identify AML patient subgroups with different survival probabilities; however, 50–70% of AML cases harbor either normal or risk-indeterminate karyotypes. The discovery of better biomarkers of clinical success and failure is therefore necessary to inform tailored therapeutic decisions. Harnessing the immune system against cancer with programmed death-1 (PD-1)-directed immune checkpoint blockade (ICB) and other immunotherapy agents is an effective therapeutic option for several advanced malignancies. However, durable responses have been observed in only a minority of patients, highlighting the need to gain insights into the molecular features that predict response and to also develop more effective and rational combination therapies that address mechanisms of immune evasion and resistance. We will review the state of knowledge of the immune landscape of AML and identify the broad opportunity to further explore this incompletely characterized space. Multiplexed, spatially-resolved immunohistochemistry, flow cytometry/mass cytometry, proteomic and transcriptomic approaches are advancing our understanding of the complexity of AML-immune interactions and are expected to support the design and expedite the delivery of personalized immunotherapy clinical trials.

4030Background: T + chemo is standard 1st line therapy (tx) for HER2+ GEA patients (pts). However, pts typically progress within 6-8 months, with up to 30% demonstrating loss of HER2 positivity. No...

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by binding to complementary target mRNAs and either promoting their decay or inhibiting their translation. Most eukaryotic genomes studied encode miRNAs, which are processed from longer, non-coding transcripts through pathways conserved from fungi to plants to animals. MiRNAs are now understood to be key mediators of developmental transitions in a number of model organisms. With respect to the immune system, miRNAs affect all facets of immune system development, from hematopoiesis to activation in response to infection, both during the innate and the adaptive immune response. At the same time, miRNA dysregulation is a central event in the development and pathophysiology of a number of cancers of the immune system. Here we will discuss our current understanding of this general regulatory mechanism focusing on its involvement in inflammation and in oncogenesis.

Background aims Umbilical cord blood transplantation (UCBT) is increasingly used to treat acute leukemias. UCB units are thawed and infused in their entirety at transplant, precluding later use as immunotherapy to prevent or treat leukemia relapse. Methods We developed a device that selectively thaws only 1 mL of the UCB unit, leaving the remaining UCB unit cryopreserved for subsequent transplantation. We also show that large numbers of CD56 + natural killer (NK) cells can be expanded from these 1-mL fractions of selectively accessed UCB. Immunomagnetic depletion of CD3 + cells of the 1-mL fraction was performed, and the cells were subsequently stimulated with irradiated Epstein-Barr virus–transformed lymphoblastoid cell lines (EBV-LCLs) and set to culture in media containing interleukin (IL)-2. Results When a 1:20 ratio of total nucleated cells to EBV-LCL feeder cells was used, day-21 and day-35 NK cell cultures initiated from 1 mL of UCB contained a median of 430 × 10 6 (range: 44–4321 × 10 6 ) and 6092 × 10 6 (range: 165–20947 × 10 6 ) CD3 – CD56 + NK cells. These cells expressed high levels of CD161, LFA-1, CD69, NKG2D, NKp30, NKp44, NKp80 and NKp46. UCB-derived NK cells were highly cytotoxic against K562 leukemia cells, although cytotoxicity was slightly lower than in expanded PBMC-derived NK cells. Conclusions We have developed and optimized a strategy to selectively access a small fraction from cryopreserved UCB and show that large numbers of CD56 + cells can be expanded from this selectively accessed fraction. This strategy presents a method to explore whether early adoptive transfer of NK cells expanded from the same UCB unit used for transplantation can prevent leukemic relapse and decrease graft-versus-host disease after UCBT.

140 Background: Trastuzumab (T) + chemotherapy (ctx) is standard for 1st line advanced HER2+ GEA, yet subsequent targeted options are lacking. M is an anti-Her 2 monoclonal antibody with an optimized Fc domain to increase affinity for activating CD16A Fc-receptors (FcR) on NK cells. Outcomes for T-treated patients (pts) carrying the low-affinity CD16A-F allele are generally worse than pts homozygous for the high-affinity V allele. M is designed to be FcR genotype independent. Evidence of clinical activity of M alone has been seen in HER2+ GEA pts post T, while P has demonstrated durable activity. Loss of HER2 amplification may occur after T failure in a subset of initially HER2+ GEA pts. Preclinical studies suggest that engagement of innate and adaptive immunity with the combination of anti-HER-2 antibodies and T-cell checkpoint inhibition could achieve greater antitumor activity than either agent alone. Methods: Advanced HER2+, PD-L1-unselected GEA pts post T failure were eligible. Dose escalation evaluated 10 and 15 mg/kg M and 200 mg P for 2nd line or higher pts. Cohort expansion evaluates safety and objective response rate (ORR) by RECIST v1.1 in 2nd line pts. M + P is given every 21 days; response assessed every 6 weeks. HER2 amplification status was assessed in a subset of pts by plasma circulating tumor (ct) DNA analysis prior to Cycle 1 of M+P. Results: Dose escalation enrolled 9 pts; cohort expansion 48 pts at 15 mg/kg M: 30 in North America (NA) and 18 in Asia (A). Treatment was well tolerated, with 1 drug-related serious adverse event. Of 38 evaluable pts to date in expansion (24 NA and 14 A), the best overall responses include 7 pts (18.4%) with PR (4 confirmed and 3 unconfirmed) and 11 (28.9%) with SD. Higher ORR trends were observed in A vs NA (35.7% vs 8.3%) and G vs GEJ (31.6% vs 5.3%). Of 25 pts with ctDNA results, HER2 amplification detection was higher in GC than GEJ (80% vs 53%). Responses were independent of FcR genotype; CD16A genotype for evaluable pts with PR: 1 V/V, 2 V/F, 2 F/F with similar allelic distribution among non-responders. Conclusions: M+P is a well-tolerated ctx-free regimen that has shown preliminary antitumor activity in 2nd line pts with advanced/metastatic GEA. Clinical trial information: NCT02689284.

Protein kinases involved in the activation of the NADPH oxidase by Fcγ receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70–80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fcγ receptors. Down-regulation of PKC isotypes resulted in inhibition of Fcγ-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-μ (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fcγ-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40phox and p47phox. Taken together, these data indicate that Fcγ receptors engage PKD in the regulation of the NADPH oxidase.

Background: Acute myeloid leukemia (AML) blast and leukemic stem cells highly express CD123, which is associated with high-risk disease and disease progression. CD123 expression on normal hematopoietic stem cells is minimal, enabling a strategy of preferential ablation of AML with a CD123-targeted approach. Flotetuzumab (MGD006/S80880) is a novel T-cell redirecting (CD123 x CD3) bispecific DART® protein being tested in a phase 1 study in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: This phase 1 dose-escalation study is designed to define the safety profile, maximum tolerated dose and schedule (MTDS), and preliminary anti-leukemic activity of flotetuzumab. Relapsed/refractory (R/R) AML or intermediate-2/high-risk MDS patients (pts) are treated on 28-day cycles at doses from 3-1000ng/kg/day on one of 2 dosing schedules (4-day on/3-day off or a continuous 7-day on schedule). To mitigate cytokine-release syndrome (CRS), a one-step lead-in dose (LID) (100ng/kg/day for 4 days) or two-step LID (30 ng/kg/day for 3 days followed by 100ng/kg/day for 4 days) was instituted during Cycle 1/Week 1 (C1W1), followed by the cohort target dose (300-1000ng/kg/day) on either of the dosing schedules on W2-4. Cycle 2 and beyond, all pts were treated on a 4-day on/3-day off schedule at the cohort target dose for a maximum of 12 cycles, with 2 cycles after a complete response or complete remission with incomplete blood count recovery. Steroid-sparing, anti-cytokine therapy was used, if clinically indicated, to manage CRS symptoms. Disease status was assessed by International Working Group (IWG) criteria. Samples were collected for pharmacokinetics, anti-drug antibodies (ADA) and cytokine analysis, including IL-2, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma and GM-CSF. Results: 45 pts with R/R AML/MDS (89% AML and 11% MDS) have been treated with flotetuzumab, median age of 64 (29-84), and 44% female. The MTDS has been reached at 500ng/kg/day. Overall flotetuzumab has demonstrated manageable toxicity; drug-related adverse events ≥G3 were observed in 20/45 (44%) pts; infusion-related reaction/CRS (IRR/CRS), the most common toxicity, was observed in 34/45 (76%) pts (G3 in 6/45, 13%). The most frequent CRS symptoms were pyrexia (15), chills (10), tachycardia (10), and hypotension (4). Cytokine levels were higher in pts with CRS than in pts without (median IL-6, 116.2 vs. 67.9 pg/mL; IL-8, 191.1 vs. 144.6 pg/mL; IL-10, 867.6 vs. 348.7 pg/mL) and were generally higher with increasing CRS grade. A two-step LID during week 1 appeared to decrease the severity of CRS grade (mean grade reduction 0.54) compared to pts that received a one-step LID during cycle 1. In addition, lower median peak cytokine levels are observed with two-step LID during W1 and after achieving W2 target dose. Fourteen pts treated at the threshold 500 ng/kg/day dose cohort and beyond (700ng/kg/day dose cohort) have completed at least one cycle of treatment and had a post-treatment bone marrow (BM) biopsy. Anti-leukemic activity was documented in 57% (8/14) pts, 6/14 reached IWG criteria (3 CR, 1 CRi, 1 MLF, 1 PR) for an overall response rate (ORR) of 43%, and 2 pts had stable disease and bone marrow (BM) blast reduction of 20 and 25% from baseline, respectively. Blast reduction occurred rapidly, often within one cycle of therapy, and extended beyond flotetuzumab discontinuation. Multispectral immunohistochemistry analysis of BM samples showed flotetuzumab in situ with a significant increase (in CD-8 T cells (1.58-fold increase, p=0.0013). Consistent with T-cell activation, CD-25, CD-69 and PD-1 upregulation on both CD-4 and CD-8 T-cells was also observed in peripheral blood samples. Conclusions: Flotetuzumab in R/R AML and MDS demonstrated evidence of anti-leukemic activity (ORR 43%) with a manageable safety profile. This program advances an immune-activating agent in treating AML and continues to enroll patients in cohort expansion (24 AML and 24 MDS patients at the MTDS) in the US and Europe. clinicaltrials.gov NCT02152956. Disclosures Uy: Boehringer Ingelheim: Consultancy; GlycoMimetics: Consultancy; Novartis: Consultancy, Other: Travel Suppport. Foster: Macrogenics: Research Funding; Shire: Honoraria; Pfizer: Research Funding; Amgen: Honoraria; Incyte: Honoraria; Celgene: Research Funding; Celator: Research Funding. Arellano: Cephalon Oncology: Research Funding. Rizzieri: Shire: Research Funding; Erytech: Research Funding. Topp: Roche: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Research Funding; Macrogenics: Consultancy, Research Funding; Celgene: Other: Travel; Regeneron: Consultancy, Honoraria, Research Funding. Martinelli: Incyte, Pfizer, MSD, Abbvie, J&J, Seattle Genetics, Jazz Pharmaceuticals, Astellas: Consultancy, Other: Advisory Board, Speakers Bureau. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Lelièvre: Institut de recherches international Servier: Employment. La Motte-Mohs: Sunnybrook Health Sciences Centre: Patents & Royalties; MacroGenics: Employment, Equity Ownership, Patents & Royalties. Sun: Macrogenics Inc: Employment, Equity Ownership. Baughman: MacroGenics, Inc.: Employment. Shannon: MacroGenics, Inc.: Employment. Fox: Bristol Myers-Squibb: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy, Research Funding; PerkinElmer: Consultancy, Research Funding; Janssen/Johnson and Johnson: Consultancy, Research Funding; Argos: Consultancy; Bayer: Consultancy; Definiens: Consultancy; OncoSec: Consultancy, Research Funding; PrimeVax: Consultancy, Equity Ownership; Peregrine: Consultancy; UbiVac: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and managing Member of LLC; Ventana/Roche: Consultancy; Viralytics: Consultancy, Research Funding. Bonvini: MacroGenics, Inc.: Employment, Equity Ownership, Research Funding. Wigginton: MacroGenics: Employment, Equity Ownership. Davidson-Moncada: MacroGenics: Employment, Equity Ownership.

TPS7070 Background: MGD006/S80880 is a novel CD123 x CD3 DART molecule designed to target CD123-positive cells for recognition and elimination by CD3-expressing T lymphocytes as effector cells. CD123, the alpha chain of the interleukin 3 receptor (IL-3Ra) is known to be highly expressed in > 90% of AML patients and at least 50% of MDS patients. Based on these observations, targeting CD123 could be a promising strategy in the preferential ablation of AML and MDS cells. Methods: MGD006 is currently being evaluated in a Phase 1 dose-escalation and cohort expansion study in relapsed/refractory (R/R) AML or intermediate-2/high risk MDS. The objectives of the study are to determine the MTDS and safety profile, and describe the pharmacokinetics and preliminary activity of MGD006. Patients are dosed in 28-day cycles. All patients start with a lead-in continuous IV infusion of 30ng/kg/day for 3 days followed by 100ng/kg/day for 4 days. Subsequent weeks (2-4) are dosed in two different schedules. One arm receives MGD006 for 4 days on/3 days off and the second arm receives MGD006 for 7 days continuously at the maximal dose assigned to each cohort (up to 1000ng/kg/day). Beginning with the second cycle, all patients are administered MGD006 for 4 days on/3 days off at the maximal dose/cohort. Patients can continue on treatment until 2 cycles (8 weeks) after the attainment of a CR, maximum of 12 cycles of MGD006, DLT or treatment failure. Once the MTDS is identified, two cohorts of 24 patients each, one in AML and one in MDS, will be enrolled. Response is assessed by IWG or IPSS criteria for AML and MDS, respectively. Signs and symptoms of cytokine release syndrome (CRS), a common AE, are graded according to Lee criteria. The study continues to enroll patients with open sites in the US, France, Germany, Italy, and The Netherlands. ClinicalTrials.gov #NCT02152956. EudraCT #2015-003813-11.

TPS219 Background: Prognosis for advanced HER2+ GEJ and G cancers remains poor, with median survival just beyond one year. Trastuzumab (T) in combination with chemotherapy is the initial treatment of choice, but therapeutic options targeting HER2 beyond T are poorly defined. M is an Fc-enhanced monoclonal antibody (Mab) to HER2 that recognizes with similar affinity the same epitope as T and whose Fc domain, compared to T, binds with increased affinity to the activating CD16A Fc-receptor (FcR) and decreased affinity to the inhibitory CD32B FcR. Preliminary data shows that M monotherapy has clinical activity against HER2+ tumors in GEJ and G cancer patients previously treated with T or other anti-HER2 agents. P is a Mab that blocks the interaction of the immune checkpoint molecule, PD-1, with its ligands, facilitating tumor cell elimination by releasing tumor-associated T cells from exhaustion. Monotherapy P has demonstrated remarkable and durable clinical activity in a Phase I study. Safety profiles of M and P are acceptable and non-overlapping. Methods: This study advances a chemotherapy free combination of M + P treatment for advanced HER2+ GEJ and G cancer patients. Enrolled patients will have relapsed/refractory HER2+ GEJ or G adenocarcinoma with measurable disease that has progressed on T plus first line chemotherapy. HER2+ (IHC 3+ or ISH+) will be confirmed by central review. Two dose levels of M (10mg/kg and 15mg/kg) and a fixed dose of P (200mg) will be evaluated for safety and tolerability. Patients will receive combination treatment once every 21 days for up to 24 months, until confirmed disease progression or intolerable toxicity. Dose expansion will enroll up to 60 patients, with 20 undergoing pre- and on-treatment biopsy. Response will be assessed every 6 weeks for the first 6 months and every 12 weeks thereafter per RECIST v1.1 and immune RECIST to account for response patterns observed with immunotherapies. Primary endpoint is ORR and duration of response, and secondary endpoints include PFS and OS. The study was initiated on January 2016 and is ongoing in North America and Asia. Clinical trial information: NCT02689284.

Natural killer (NK) cells are defined as lymphocytes capable of target cell killing and cytokine production independent of somatic antigen receptor recombination. Challenging the paradigm of continuous production of non-specific cytotoxic NK cells from undifferentiated precursors, a subset of murine NK cells sharing many properties with cytotoxic effector T cells was shown to specifically respond to a viral infection, resulting in expansion and protective immunity upon adoptive transfer, uncovering unexpected "adaptive" capabilities for NK cells. In humans, CD56dimCD16+ NK cells expressing NKG2C and variegated patterns of KIR are associated with Cytomegalovirus (CMV) infection, and may represent human analogs of murine adaptive NK cells. In contrast to CD56bright and canonical CD56dim NK cells, we recently showed that human adaptive NK cells lack expression of specific signaling proteins and transcription factors, and epigenetically resemble CD8+ effector T cells, suggesting shared pathogen-driven developmental pathways (Schulms et al, 2015). NK cell heterogeneity, ontogeny, and mechanisms sustaining NK cell memory are poorly understood, particularly in humans, because in contrast to adaptive T and B cells, clonal tracking of NK cells based on receptor gene structure is not possible. We applied autologous transplantation of genetically barcoded CD34+ rhesus macaque hematopoietic stem and progenitor cells (HSPC) to interrogate the landscape of NK cell production and expansion at a clonal level. We followed 3 monkeys for up to 4 years post-transplantation, and observed a distinct clonal ontogeny for CD56-CD16+ (corresponding to human CD56dimCD16+ NK) versus CD56+CD16- rhesus NK cells. CD56-CD16+ NK were oligoclonal, and dominated by small numbers of highly biased clones, a pattern showing little overlap with highly polyclonal multipotent contributions to T, B, myeloid and CD56+CD16- NK cell lineages. There were clear expansions and contractions of groups of these highly biased CD56-CD16+ NK cell clones over time, with individual clones contributing up to 20% to this lineage, similar to patterns of waxing and waning T cell clones, suggesting responses of both cell types to extrinsic cues such as viruses. In contrast, all other lineages, including CD56+CD16- NK cells, were characterized by very stable contributions from long-term engrafting multipotent HSPC clones over time. The highly biased, greatly expanded, and waxing and waning CD56-CD16+ NK cell clones were specifically enriched in a subpopulation of CD56+CD16- NK with high KIR and low SYK expression,and decreased methylation at IFNG the CNS1 locus, analogous to human CMV-associated adaptive NK cells. This population of cells was increased in CMV+ compared to CMV- macaques; our transplanted macaques were CMV+. Canonical CD56+CD16- NK cells, lacking an adaptive phenotype, had clonal patterns similar to CD56+CD16- NK cells. We profoundly depleted CD56-CD16+ NK cells with an anti-CD16 antibody, and observed rapid regeneration of the same CD56-CD16+ NK clonal pattern, without recruitment of contributions from multipotent HSPC via CD56+CD16- putative precursor NK cells. There was a marked increase in cycling of CD56-CD16dim NK cells just prior to reappearance of CD56-CD16bright NK cells, and no change in cycling of CD56+CD16- cells. Thus, adaptive NK cell clones, both at steady state and under regenerative stress do not appear to require ongoing production from HSPC via CD56+CD16- precursors. These results suggest that adaptive NK cells or their immediate precursors have acquired self-renewal properties and high proliferative potential, with clonal restriction potentially based selection and expansion of cells with specific, epigenetically-controlled receptor expression able to interact with external stimuli such as viruses. In conclusion, via tracking of individual NK cell clonal histories in rhesus macaques, we provide the first direct evidence for persistence of adaptive NK cell clones in any species, offering potential insights into the acquisition of NK cell memory. Disclosures Dunbar: GSK/Novartis: Research Funding.

Members of the protein kinase C family are major effectors of lipid second messengers. We describe three protocols to assess protein kinase C activity in polymorphonuclear leukocytes (neutrophils). These methods are useful to study the activation and function of protein kinase C in these immune cells. Since neutrophils provide a ready source of human primary tissue, these methods are also useful for pharmacological studies on the protein kinase C system and for evaluation of protein kinase C activators and inhibitors in the context of human primary cells. Furthermore, since protein kinase C activity is determined by a number of lipid-generating signaling systems, the methods described here can also be employed to study the pharmacology of these "upstream" signaling systems.