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Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse1,2. Hypomethylating agents (HMAs) are the standard of care in the treatment of older or unfit patients with AML, but responses are modest and not durable3-5. Here we demonstrate that LSCs upregulate the tumor necrosis factor family ligand CD70 in response to HMA treatment resulting in increased CD70/CD27 signaling. Blocking CD70/CD27 signaling and targeting CD70-expressing LSCs with cusatuzumab, a human αCD70 monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity activity, eliminated LSCs in vitro and in xenotransplantation experiments. Based on these preclinical results, we performed a phase 1/2 trial in previously untreated older patients with AML with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine (NCT03030612). We report results from the phase 1 dose escalation part of the clinical trial. Hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission with 4 patients achieving minimal residual disease negativity by flow cytometry at

Introduction Acute myeloid leukemia (AML) is a heterogenous hematological malignancy driven by leukemia stem cells (LSCs) (Lapidot et al, 1994). LSCs resistant against conventional chemotherapy represent the major cause of relapse. Elderly or unfit AML patients not eligible for intensive chemotherapy are treated in a palliative setting with hypomethylating agents (HMA) or low dose Ara-C, but responses are modest and not durable. The reason for the low efficacy of HMA treatment is their insufficient action on the disease initiating- and -maintaining LSC population (Craddock et al, 2013). We recently demonstrated that CD34+ AML cells (progenitors and LSC) consistently express the tumor necrosis factor family ligand CD70 as well as its receptor CD27 and that cell-autonomous CD70/CD27-signaling propagates the disease (Riether et al. 2017). The aim of the current study was to evaluate whether resistance to HMA treatment can be overcome by combining HMA with an anti-CD70 monoclonal antibody treatment. Experimental design The effect of HMA treatment on the expression of CD70 on primary human CD34+CD38- AML LSCs was determined in vitro cultures and in patients treated with HMA in vivo. The therapeutic potential of targeting CD70-expressing LSCs in presence and absence of HMA was assessed using the anti-CD70 ADCC-optimized monoclonal antibody (mAb), cusatuzumab, and an effector-dead anti-CD70 mAb in colony formation and re-plating assays as well as patient-derived xenograft models (Silence et al, 2014). The clinical relevance of the findings was determined in a clinical phase 1 trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine (AZA, NCT03030612). Four different dose levels of cusatuzumab (1, 3, 10 and 20 mg/kg Q2W) were studied; AZA was administered at 75 mg/m² for 7 days every 28 days. Results We found that resistance of AML LSCs to HMA treatment is mediated by the up-regulation of the CD70. The up-regulation of CD70 triggered cell-autonomous CD70/CD27 signaling on AML LSCs. Based on these findings we hypothesized that the upregulation of CD70 by HMA may render LSCs more susceptible to CD70-targeting interventions. Targeting CD70-expressing LSCs by a blocking anti-CD70 mAb and the anti-CD70 mAb cusatuzumab, which blocks CD70/CD27-signaling and additionally mediates ADCC and CDC, eradicated LSCs in colony and re-plating assays in vitro and in xenotransplantation experiments in vivo. HMA in combination with blocking αCD70 mAb synergistically reduced LSC numbers in vivo and this was even more efficient when ADCC-enhanced αCD70 mAb cusatuzumab was added in the presence of NK cells. In order to test the hypothesis that targeting CD70 in combination with HMA eliminates LSCs in AML patients, we initiated a phase 1 dose-escalation trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with azacitidine. No dose-limiting toxicities (DLT) were observed in the dose-escalation phase 1 trial and responses were observed across the dose levels (1-20 mg/kg). A single dose of cusatuzumab reduced bone marrow blasts in just two weeks in all patients on average by 32%. Cusatuzumab monotherapy significantly reduced LSC numbers and frequencies in all patients analyzed in the bone marrow as assessed in limiting dilution colony assays. Single cell sequencing analysis revealed that cusatuzumab induced gene signatures related to myeloid differentiation and apoptosis in LSCs. In combination with azacitidine, cusatuzumab induced CR/CRi in 10 out of 12 patients. Responses were observed at all dose levels of cusatuzumab and median time to response was 3.3 months. Conclusions Blocking CD70/CD27-signaling and targeting CD70-expressing LSCs by the ADCC-optimized mAb, cusatuzumab, eliminated LSCs in vitro and in xenotransplantation experiments. In a phase 1 study promising activity of cusatuzumab in combination with HMA was observed in AML patients, in which translational data indicate that cusatuzumab selectively eliminates CD70-expressing LSCs. Disclosures Van Rompaey: argenx: Employment, Equity Ownership, Patents & Royalties. Moshir:Argenx: Employment, Equity Ownership. Delahaye:argenx: Employment, Equity Ownership. Gandini:Argenx: Employment, Equity Ownership. Erzeel:argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Fung:argenx: Consultancy, Equity Ownership. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties.

Outcomes in elderly patients with acute myeloid leukemia (AML) are still adverse, as the majority does not qualify for intensive therapy or allogenic stem cell transplantation (ASCT). DNA hypomethylating agents (HMAs) induce remissions and prolong survival in a fraction of these patients. However, overall prognosis remains dismal and all patients progress due to therapy-resistant leukemia stem cells (LSCs). We recently demonstrated that HMAs upregulate the expression of CD70 on primary human AML LSCs, potentially contributing to HMA resistance and that blocking the cell-autonomous CD70/CD27 signaling inhibits proliferation and myeloid differentiation of LSCs and contributes to HMA resistance. Consequently, combining HMA treatment with a blocking αCD70 monoclonal antibody potently reduced colony formation of AML LSCs in vitro and effectively eliminated human AML LCSs in xenograft experiments. Based on these results, we initiated an open-label, non-controlled, non-randomized Phase 1/2 trial combining the HMA azacitidine (AZA) with ARGX-110, a human monoclonal antibody targeting CD70, in newly diagnosed AML patients unfit for intensive chemotherapy (ARGX-110-1601, NCT03030612). Trial design ARGX-110 with optimized antibody-dependent cell-mediated cytotoxicity (ADCC), is administered intravenously at 1, 3, 10 or 20 mg/kg once every two weeks (Q2W), in combination with a standard dose of AZA (75 mg/m² subcutaneously for 7 days every 28 days). A 2-week lead-in of ARGX-110 enables studying the effect of αCD70 antibody monotherapy. Primary objectives in the Phase 1 include determining the maximum tolerated dose of ARGX-110 in combination with AZA and the recommended phase 2 dose (RP2D). Secondary objectives comprise safety, pharmacokinetics and anti-leukemic activity of ARGX-110 alone or in combination with AZA (including overall response rate (ORR), frequencies of LSCs and minimal/measurable residual disease (MRD)). Results The trial is ongoing and as of 16th July 2018, 12 newly diagnosed AML patients were treated in the dose-escalation part of the trial, the results of which will be further updated in December 2018. The median age over the dose cohorts was 75 years (range 64-84) and included intermediate (N=6) and adverse (N=6) ELN risk groups. WHO subtypes were AML with recurrent genetic abnormalities (N=2), MDS-related changes (N=6), AML-NOS (N=3) and therapy-related myeloid neoplasms (N=1). No dose-limiting toxicity was observed and the combination of ARGX-110 with a standard dose of AZA was well tolerated. Hematological toxicities, in line with expected AZA toxicities, were the most frequently reported adverse events (AEs). At the cut-off, the ORR (complete remission [CR] + incomplete recovery [CRi] + partial remission [PR] + morphologic leukemia-free status [MLFS]) was 92% (11/12 patients); among the 11 evaluable subjects, the best response was CR/CRi for 9 patients (82%), 1 patient (9%) reached MLFS, and 1 (9%) PR, with 7 patients still on the trial (median duration on the trial at cut-off was 6.9 months [range: 2-14.4 months]). Significantly, 1 patient reached CR and was discontinued after 5 months to undergo ASCT. Five evaluable patients (45%) achieved MRD negativity by flow cytometry. Molecular MRD negativity was achieved in 3/8 patients with available results, two correlating with flow-MRD negativity. The mean time to response for the first 9 patients was 2.8 months and 3.4 months to reach best response. The 3 patients in the 20 mg/kg cohort did not all reach response at the time of cut-off. Based on pharmacokinetics, safety, and pharmacodynamic data, the RP2D of 10 mg/kg was established. Translational studies assessing LSC frequencies in bone marrow aspirates by limiting dilution colony assays and xenograft experiments revealed that ARGX-110 monotherapy and in combination with AZA significantly reduced LSC frequencies in AML patients. Conclusions ARGX-110 in combination with AZA is well tolerated and leads to a higher ORR compared to historical data for AZA alone. Clinical activity of the combination is observed in different AML subtypes and risk categories. Preliminary data indicate that ARGX-110 alone, and in combination with AZA, efficiently eliminates LSCs. These observations warrant further accelerated investigation of ARGX-110 in combination with AZA in AML patients unfit for intensive therapy. Disclosures Ochsenbein: argenx: Research Funding. Riether:argenx: Research Funding. Bacher:argenx: Research Funding. Höpner:argenx: Research Funding. Hinterbrandner:argenx: Research Funding. Van Rompaey:argenx: Employment. Moshir:argenx: Employment. Delahaye:argenx: Employment. Gandini:argenx: Employment. Erzeel:argenx: Employment. Hultberg:argenx: Employment. Fung:argenx: Consultancy. De Haard:argenx: Employment. Leupin:argenx: Employment.

Lysine biosynthesis in plants is tightly regulated by feedback inhibition of the end product on the first enzyme of the lysine-specific branch, dihydrodipicolinate synthase (DHDPS). Three complete DHDPS coding sequences and one partial sequence were obtained in Medicago truncatula via inverse PCR. Analysis of the MtDHDPS sequences indicated the presence of isozymes (MtDHDPS2 and MtDHDPS3) with multiple amino acid substitutions on positions previously shown to be involved in feedback inhibition and of residues important for catalytic activity, possibly affecting the enzymatic properties of these isoforms. Sequences similar to MtDHDPS2 and 3 are present in Lotus japonicus and Glycine max, suggesting the existence of a specific conserved class of DHDPS genes within the Fabaceae family. The MtDHDPS genes were found by quantitative RT-PCR analysis to be expressed in an organ-specific manner in Medicago truncatula. All four MtDHDPS enzymes were expressed separately in Escherichia coli, revealing a strongly reduced sensitivity of the MtDHDPS2 protein to lysine feedback inhibition and a severely reduced activity of the MtDHDPS3 protein. Remarkably, MtDHDPS3 expression in A. thaliana produced transgenic plants with a significantly increased threonine level, suggesting a dominant DHDPS inhibiting role of this isoform. This is supported by co-expression experiments in E. coli which indicate that AtDHDPS and MtDHDPS3 interact and may form hetero-oligomers with strongly reduced enzymatic activity. In conclusion, analysis of DHDPS in Medicago truncatula revealed the presence of unique isozymes displaying novel regulatory properties.