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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.

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.

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.

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.