Results from a phase I/II trial of cusatuzumab combined with azacitidine in patients with newly diagnosed acute myeloid leukemia who are ineligible for intensive chemotherapy

Results from a phase I/II trial of cusatuzumab combined with azacitidine in patients with newly diagnosed acute myeloid leukemia who are ineligible for intensive chemotherapy by Thomas Pabst, Norbert Vey, Lionel Adès, Ulrike Bacher, Mario Bargetzi, Samson Fung, Gianluca Gaidano, Domenica Gandini, Anna Hultberg, Amy Johnson, Xuewen Ma, Rouven Müller, Kerri Nottage, Cristina Papayannidis, Christian Recher, Carsten Riether, Priya Shah, Jeffrey Tryon, Liang Xiu, and Adrian F. Ochsenbein Received: June 10, 2022. Accepted: January 27, 2023. Citation: Thomas Pabst, Norbert Vey, Lionel Adès, Ulrike Bacher, Mario Bargetzi, Samson Fung, Gianluca Gaidano, Domenica Gandini, Anna Hultberg, Amy Johnson, Xuewen Ma, Rouven Müller, Kerri Nottage, Cristina Papayannidis, Christian Recher, Carsten Riether, Priya Shah, Jeffrey Tryon, Liang Xiu, and Adrian F. Ochsenbein. Results from a phase I/II trial of cusatuzumab combined with azacitidine in patients with newly diagnosed acute myeloid leukemia who are ineligible for intensive chemotherapy. Haematologica. 2023 Feb 9. doi: 10.3324/haematol.2022.281563 [Epub ahead of print] Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a r egular peer review and have been accepted for publication. E-publishing of this PDF file has been app roved by the authors. After having E-published Ahead of Print, manuscripts will then un dergo technical and English editing, typesetting, p roof correction and be presented for the aut hors' final approval; the final version of the manuscript will then appear in a regular issue of the journal. All legal disc laimers that apply to t he journal also pertain to this production process. Confidential. Not for further distribution 1 Results from a phase I/II trial of cusatuzumab combined with azacitidine in patients with newly diagnosed acute myeloid leukemia who are ineligible for intensive chemotherapy Thomas Pabst,1 Norbert Vey,2 Lionel Adès,3 Ulrike Bacher,4 Mario Bargetzi,5 Samson Fung,6 Gianluca Gaidano,7 Domenica Gandini,8 Anna Hultberg,8 Amy Johnson,9 Xuewen Ma,9 Rouven Müller,10 Kerri Nottage,11 Cristina Papayannidis,12 Christian Recher,13 Carsten Riether,14 Priya Shah,15 Jeffrey Tryon,11* Liang Xiu11 and Adrian F. Ochsenbein14 (*retired) 1Department of Medical Oncology, University Hospital, Inselspital and University of Bern, Bern, Switzerland 2Hématologie Clinique, Institut Paoli-Calmettes, Marseille, France 3Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris and Université Paris Cité, and Centre d’Investigation Clinique (INSERM CIC 1427), Paris, France 4Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Switzerland 5Division of Hematology and Transfusion Medicine, Kantonsspital Aarau, Aarau, Switzerland 6Fung Consulting Healthcare and Life Sciences, Eching, Germany 7Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont and Maggiore Hospital, Novara, Italy 8argenx, Ghent, Belgium 9Janssen Research & Development, Spring House, PA, USA 10Department of Medical Oncology and Hematology, University Hospital Zurich, Zurich, Switzerland Confidential. Not for further distribution 2 11Janssen R&D, Raritan, NJ, USA 12IRCCS, Azienda Ospedaliero Universitaria di Bologna, Istituto di Ematologia “L e A Seràgnoli”, Bologna, Italy 13Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse Oncopole, Service d'Hématologie, Toulouse, France and Université Toulouse III Paul Sabatier, Toulouse, France 14Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Switzerland; Department of BioMedical Research (DBMR), University of Bern, Bern, Switzerland 15Janssen R&D, High Wycombe, Buckinghamshire, UK Author contribution statement All authors critically reviewed and revised the manuscript and approved the final submitted version; TP and AO contributed to design of study and protocol, study conduct, and interpretation of results; CRiether and UB contributed to design of study and protocol, and interpretation of results; MB, RM, NV, LA, CRecher, CP, GG were involved in study conduct; DG and AH contributed to study protocol, and interpretation of results; SF provided consultancy services to argenx and contributed to interpretation of results; AJ, XM, KN, LX, JT, PS were involved in interpretation of results. Disclosures LX is a Janssen employee. NV has received consulting fees from Janssen; payment or honoraria from argenx. MB has received support for the present manuscript (study materials and patient fee) from Janssen. Confidential. Not for further distribution 3 SF has received support for the present manuscript (consultancy fees, travel expenses) from argenx BV; consulting fees from Bristol-Myers Squibb GmbH & Co. KGaA, Molecular Partners AG (plus travel expenses), InhaTarget Therapeutics, Takeda Pharma AG, Polyphor Ltd, Synteract GmbH, Affimed GmbH (plus travel expenses), Incyte GmbH, IOME BIO SA (no payment), OM Pharma SA, Kiadis Pharma NV (plus travel expenses), ISA Pharma NV (plus travel expenses), Pharmalog GmbH, Novateur Inc., Nagel & Partners Ltd; honoraria for lectures from BioM Biotech Cluster Development GmbH; support for attending meetings from argenx BV (travel expenses); patents: argenx BV (named inventor or several patents); participation on a Data Safety Monitoring Board or Advisory Board from RHEACELL GmbH – DSMB (Honorarium), selectION Therapeutics GmbH (no payment), Immutep Ltd – Ad Board (Honorarium, travel expenses), Simbec-Orion – Ad Board (no payment). GG has received grants or contracts from argenx (payment to my institution), Janssen (payment to my institution and personal fees). DG is an argenx employee; participation on a Data Safety Monitoring Board or Advisory Board: argenx; Stock or stock options: argenx. AH is an argenx employee; patents: argenx; stock or stock options: argenx. AJ has stock or stock options from Johnson & Johnson (employee), Vincerx Pharma (employee). XM is a Janssen employee. RM has received consulting fees from GlaxoSmithKline; payment or honoraria from AbbVie; support for attending meetings and/or travel from Amgen, Janssen; participation on a Data Safety Monitoring Board or Advisory Board from Novartis, Takeda, Celgene/BMS, Janssen, AbbVie, Amgen, Sanofi, Sandoz, Jazz Pharmaceuticals. KN is a Janssen employee and stock holder. CP received payment or honoraria from Amgen, Novartis, Pfizer, AbbVie, Astellas, Janssen. Confidential. Not for further distribution 4 CRecher has received grants or contracts (payments to my institution) from AbbVie, Amgen, Astellas, Celgene BMS, Jazz Pharmaceuticals, Agios, Daiichi-Sankyo, MaaT Pharma, Novartis; consulting fees: AbbVie, Amgen, Astellas, Celgene BMS, Jazz Pharmaceuticals, Agios, Daiichi- Sankyo, Incyte, MacroGenics, Janssen, Novartis, Otsuka, Takeda; payment or honoraria from AbbVie, Astellas, Celgene BMS, Jazz Pharmaceuticals, Daiichi-Sankyo; support for attending meetings and/or travel: Incyte, Celgene BMS, Sanofi, Amgen, Novartis, Daiichi-Sankyo, Gilead; participation on a Data Safety Monitoring Board or Advisory Board from PEVOLAM trial (PETHEMA study group). CRiether Listed as inventor on a patent held by the University of Bern on targeting CD70 for treatment in AML. PS is a Janssen R&D employee. JT has stock or stock options from JNJ Company 401K, JNJ Stock Option Plan (made to JT). AFO has received support for the present manuscript (funding) from SAKK, Rising Tide, Gateway; grants or contracts from argenx (funding); royalties and licenses from argenx/Janssen; patent: Targeting CD70 in myeloid leukemia. TP, LA, and UB have no conflicts of interest to disclose. Running head: Phase I/II trial of cusatuzumab/azacitidine in AML Corresponding author: Thomas Pabst, Department of Medical Oncology, University Hospital, Inselspital, CG-3010 Bern, Switzerland; [email protected] ++41316320378 Abstract word count: 249 Main text word count: 3305 Figures/tables: 1/3 Confidential. Not for further distribution 5 Supplementary files: 1 Trial registration: ClinicalTrials.gov identifier: NCT03030612. Acknowledgments The authors would like to thank all patients and staff who participated in this study. The trial was funded by Janssen Oncology, Pharmaceutical Companies of Johnson & Johnson, in collaboration with argenx. The authors would like to express their sincere gratitude to Christina Guttke of Janssen Research & Development, Spring House, PA, USA for her valuable contribution to the biomarker data in this publication, and to Julie Jacobs of argenx, Ghent, Belgium for her substantial contribution to the discussion and conclusions. Medical writing support for the development of this manuscript was provided by Ranjana Whitlock of Ashfield MedComms, an Ashfield Health company, part of UDG Healthcare plc, and was funded by Janssen Oncology and argenx. Data-sharing statement Janssen has an agreement with the Yale Open Data Access (YODA) Project to serve as the independent review panel for evaluation of requests for clinical study reports and participant- level data from investigators and physicians for scientific research that will advance medical knowledge and public health. Data will be made available following publication and approval by YODA of any formal requests with a defined analysis plan. For more information on this process, or to make a request, please visit The Yoda Project site at http://yoda.yale.edu. The data-sharing policy of Janssen Pharmaceutical Companies of Johnson & Johnson is available at https://www.janssen.com/clinical-trials/transparency. Keywords: Hematologic malignancies; acute myeloid leukemia (AML); cusatuzumab; newly diagnosed AML ineligible for intensive chemotherapy Confidential. Not for further distribution 6 Abstract Cusatuzumab is a high-affinity, anti-CD70 monoclonal antibody under investigation in AML. This two-part, open-label, multicenter, phase I/II trial evaluated cusatuzumab plus azacitidine in patients with newly diagnosed AML ineligible for intensive chemotherapy. Patients received a single dose of cusatuzumab at one of four dose levels (1, 3, 10, or 20 mg/kg), 14 days before starting combination therapy. In phase I dose-escalation, cusatuzumab was then administered on days 3 and 17, in combination with azacitidine (75 mg/m2) on days 1-7, every 28 days. Primary objective in phase I was to determine the recommended phase II dose (RP2D) of cusatuzumab plus azacitidine. Phase II primary objective was efficacy at the RP2D (selected as 10 mg/kg). Thirty-eight patients enrolled: 12 in phase I (three per dose level; four with ELN adverse risk) and 26 in phase II (21 with adverse risk). Objective response (≥ partial remission) was achieved by 19/38 patients (including 8/26 in phase II); 14/38 achieved complete remission. Eleven patients (37.9%) achieved objective response among the 29 patients in phase I and phase II treated at the RP2D. At median follow-up of 10.9 months, median duration of first response was 4.5 months and median overall survival was 11.5 months. Most common treatment-emergent adverse events were infections (84.2%) and hematologic toxicities (78.9%). Seven patients (18.4%) reported infusion-related reactions, including two with grade 3 events. Thus, cusatuzumab/azacitidine appears generally well tolerated and shows preliminary efficacy in this setting. Investigation of cusatuzumab combined with current standard-of-care therapy, comprising venetoclax and azacitidine, is ongoing. Confidential. Not for further distribution 7 Introduction Intensive induction and consolidation chemotherapy with curative intent is recommended for patients with newly diagnosed acute myeloid leukemia (AML), provided they can tolerate it.1 For patients unsuitable for intensive chemotherapy, standard of care is evolving. Hypomethylating agents (HMAs), such as azacitidine and decitabine, have been central to treatment for several years.1-4 However, since the start of this phase I/II trial, other agents have been studied in combination with HMAs, and recent data have established venetoclax plus a HMA as a new standard in this setting.5 Despite this changing landscape, overall survival (OS) is <15 months with venetoclax/azacitidine and even in the subgroup of responding patients, median duration of response is <18 months,5 indicating a need for more effective therapies. AML is driven by leukemic stem cells (LSCs) that have a key role in initiating and sustaining malignancy.6 LSCs also have a capacity for self-renewal and their persistence is believed to be the primary cause of relapse in AML.7-9 Selective elimination of LSCs, without affecting normal hematopoiesis, is challenging owing to the greater resistance of LSCs to conventional chemotherapy compared with more differentiated AML blasts.10,11 CD70, a tumor necrosis factor receptor ligand, is a very promising target due to its consistent expression on LSCs and AML blasts.12,13 In AML, the binding of CD70 to its receptor, CD27, on LSCs and subsequent downstream signaling activates gene-expression profiles that promote LSC proliferation, reduce differentiation, and lead to release of soluble CD27 (sCD27).12,13 Serum sCD27 levels are increased in patients with newly diagnosed AML,12,13 and are a strong, independent negative predictor of cancer prognosis.12 Cusatuzumab (ARGX-110) is a high-affinity, anti-CD70 monoclonal antibody that blocks CD70/CD27 signaling, leading to inhibition of LSC proliferation, a reduction in leukemic blast cells, blockade of regulatory T-cell survival (preventing tumor immune escape), and restoration of normal myeloid differentiation.12-15 It also exerts direct Fc-mediated, effector functions via enhanced antibody-dependent cellular cytotoxicity (modified using POTELLIGENT® Confidential. Not for further distribution 8 Technology), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, leading to apoptosis of leukemic cells and blasts.13,14 Treatment with HMAs upregulates CD70 expression on LSCs isolated from patients with newly diagnosed AML and combined anti-CD70 and HMA treatment can synergistically decrease LSCs to a greater extent than blocking CD70 alone.13 Hence, there is a rationale for studying cusatuzumab in combination with a HMA. First-in-human studies have shown that single-agent cusatuzumab is well tolerated and is biologically active in patients with advanced solid tumors or hematologic malignancies, including AML.16,17 Promising early responses and pharmacodynamic activity at all cusatuzumab doses were demonstrated in interim data from a two-part, phase I/II dose-escalation and expansion study undertaken to investigate the potential of cusatuzumab in combination with azacitidine for the treatment of newly diagnosed patients with AML who were not candidates for intensive chemotherapy.13 Treatment was also well tolerated without reaching a maximum tolerated dose. Here, this publication builds on the interim data from the same study, reporting results for the entire study population, including the phase II expansion. Methods Study design This was an open-label, multicenter, non-randomized, dose-escalation (phase I) and expansion (phase II) study. Phase I employed a 3+3 design with dose increments based on a modified Fibonacci scheme. Phase I enrolled patients in four sequential dose cohorts (1, 3, 10, 20 mg/kg). In each cohort, patients received a single intravenous (IV) dose of cusatuzumab on day -14 followed by combination therapy, comprising cusatuzumab IV on days 3 and 17 plus azacitidine 75 mg/m2 subcutaneously or IV on days 1-7, every 28 days. To mitigate infusion- related reactions (IRRs), all patients were premedicated with acetaminophen, an antihistamine, and an IV glucocorticoid prior to cusatuzumab infusion. The first patient in each cohort was monitored until Cycle 1 day 7; if no dose-limiting toxicities (DLTs) occurred (Online Confidential. Not for further distribution 9 Supplementary Data), further patients were enrolled in the cohort. Subsequent cohorts were opened upon approval from the Data Safety Monitoring Board. Phase II patients received cusatuzumab at the RP2D from phase I plus azacitidine at the same dose/schedule as phase I. Patients were treated for as long as they derived clinical benefit or until disease progression, unacceptable toxicity, death, withdrawal, or loss to follow-up. The study conformed with the Declaration of Helsinki, Good Clinical Practice guidelines, and regulatory and country-specific requirements, and is registered with ClinicalTrials.gov (identifier NCT03030612). The protocol was approved by an Independent Ethics Committee/Review Board. Patients gave written informed consent. Eligibility Adults (≥ 18 years) with newly diagnosed AML (defined by a blast count of ≥ 20%), unsuitable for intensive chemotherapy, were enrolled. Additional eligibility criteria included an expected life expectancy of ≥ 3 months and Eastern Cooperative Oncology Group performance status of 0-2. Patients with any prior chemotherapy/radiotherapy for AML (except hydroxyurea/hydroxycarbamide, which had to be discontinued prior to the first day of azacitidine administration) were excluded. Full eligibility criteria are listed in the Online Supplementary Data. Endpoints and assessments In phase I, the primary endpoint was incidence of DLTs at each dose of cusatuzumab plus standard dose azacitidine (to inform RP2D). In phase II, the primary endpoint was overall response rate (ORR), defined as complete remission (CR) plus CR with incomplete recovery (CRi) plus morphologic leukemia-free state plus partial remission at cusatuzumab dose established in phase I plus standard dose azacitidine. Secondary endpoints in both parts included: treatment-emergent adverse events (TEAEs); pharmacokinetics and immunogenicity of cusatuzumab in peripheral blood; minimal residual disease evaluation by multiparameter flow cytometry performed in one of two laboratories (see Supplementary data); time to, level, and duration of response; OS; 30/60-day mortality; and transfusion independence. Confidential. Not for further distribution 10 Pharmacodynamic markers were also assessed. See Supplementary data. Response evaluation by investigators was based on established criteria (Online Supplementary Table S1). Safety and tolerability were assessed throughout, evaluations included TEAEs (graded using NCI-CTCAE version 4.03), laboratory parameters, electrocardiogram, vital signs, physical examinations, and Eastern Cooperative Oncology Group performance status (Online Supplementary Data). Statistical analysis Using Simon’s two-stage design with a target ORR of 50% versus 25%, 5% type 1 error, and 20% type 2 error, 24 patients were needed in phase II for 80% power. The null hypothesis was to be rejected if the ORR was >37.5% (>9/24 responses). Analyses of the primary endpoint were performed on the full analysis set (patients who received an infusion of any study drug), as well as a combination therapy analysis set (patients from phase I and phase II who received cusatuzumab at the RP2D and azacitidine). Statistical inference according to the Simon’s design was based on the full analysis set as well as the combination therapy analysis set. Time- to-event data were analyzed by Kaplan-Meier methods. Results Patient disposition Between January 2017 and February 2019, 38 patients were enrolled at eight sites across Switzerland, France, and Italy, and treated in the phase I dose escalation (n=12) or phase II expansion (n=26). The dataset used for this analysis includes extended follow-up for 12 phase I patients treated at the 1, 3, 10, and 20 mg/kg dose levels (three per cohort) and 26 phase II patients treated at the RP2D of 10 mg/kg. The data cut-off for this analysis was July 1, 2020. Treatment was discontinued in 34 of the 38 patients (89.5%): two of three patients at 1 mg/kg, three of three at 3 mg/kg, 26/29 at 10 mg/kg, and three of three at 20 mg/kg. Reasons for treatment discontinuation were progressive disease (n=17, 50%), adverse event (AE) (s) (n=6, Confidential. Not for further distribution 11 17.6%), death (n=6, 17.6%), investigator decision (n=2, 5.9%), protocol deviation (n=1, 2.9%), withdrawal of consent (n=1, 2.9%), and other (n=1, 2.9%; patient wished to proceed to allogeneic transplant). Recommended phase II dose The 10 mg/kg dose level of cusatuzumab was selected as the RP2D based on a prespecified interim analysis of phase I data for the 1-10 mg/kg dose cohorts in April 2018. No DLTs were observed in any of these dose cohorts and the maximum tolerated dose was not reached. At the time of the interim analysis, data for the DLT period were incomplete for two of the three patients in the phase I 20 mg/kg dose cohort. None of the three patients treated at 20 mg/kg went on to experience DLTs. Baseline characteristics Baseline demographics and disease characteristics are shown in Table 1. Across all patients, median age was 75 years (range, 59-90), 50% of patients were female, 7.9% had an Eastern Cooperative Oncology Group performance status of 2, 34.2% had secondary AML, and 65.8% had adverse genetic risk per European LeukemiaNet 2017 criteria3 (with risk categories assigned post hoc by an independent reviewer). Median time from diagnosis to treatment was 14.5 days (range, 3-139). Efficacy Response data are presented in Table 2 and Figure 1. An objective response was achieved by 19/38 patients in the full analysis set (both study phases combined), for an ORR of 50% (95% confidence interval [95% CI]: 33.4-66.6). All responding patients achieved CR or CRi (no partial remission or morphologic leukemia-free state): 14 (36.8%) with CR and five (13.2%) with CRi. In phase II, eight of 26 patients responded to treatment at 10 mg/kg (ORR, 30.8%; 95% CI: 14.3-51.8), including five with CR (19.2%) and three with CRi (11.5%). A response of CR/CRi was achieved by four of four patients with favorable ELN risk status, seven of nine with intermediate status, and eight of 25 with adverse status (Online Supplementary Table S2). Of the 19 patients with a best response of CR/CRi, six (31.6%) achieved minimal residual disease Confidential. Not for further distribution 12 negativity. Among patients in phase I and phase II receiving cusatuzumab 10 mg/kg, 11/29 (37.9%) achieved an objective response in the full analysis set. Two of these patients were classified as non-evaluable because they died in the interval after their first dose of cusatuzumab and did not receive azacitidine nor their first post-treatment assessment on Cycle 1 day 1; these deaths were unrelated to cusatuzumab. Excluding these two patients resulted in the combination therapy analysis set and an objective response rate of 11/27 (40.7%). While the total number of patients in these analyses deviates from the original Simon’s design of 24, ad-hoc Simon’s criteria based on the same design parameters calls for rejecting the null hypothesis if ORR is ≥ 12/29 or ≥ 11/27. The null hypothesis is not rejected in the full analysis set but is rejected in the combination therapy analysis set. For the full analysis set, at a median follow-up of 10.6 months (range, 0.3-38.2), median time to first response was 3.2 months (range, 0.5-12.4) and median duration of first response was 4.5 months (range, 0.02-33.7). Median OS was 11.5 months (95% CI: 7.3-17.1), with a 12- month OS rate of 49%. Independence from red blood cell and platelet transfusion (defined as reaching ≥ 8 consecutive weeks without a transfusion from first dose of study drug administered) was observed in 24 patients (63.2%). Twenty-four patients (63.2%) achieved red blood cell transfusion independence and 29 (76.3%) obtained platelet transfusion independence. Median duration of red blood cell /platelet transfusion independence was 13.0 months (range, 2.0-37.9). Safety and tolerability Median duration of study treatment was 5.8 months (range, 0-37.9) and median number of cycles administered was six (range, 1-40). All 38 patients had ≥ 1 TEAE and all experienced a grade ≥ 3 TEAE (Table 3). The most common TEAEs were febrile neutropenia and neutropenia (n=15 each, 39.5%), followed by anemia, thrombocytopenia, pneumonia, and pyrexia (n=14 each, 36.8%). After pneumonia, the next most frequent infectious TEAEs were sepsis (n=11, 28.9%) and urinary tract infection (n=4, Confidential. Not for further distribution 13 10.5%). Thirty-two patients (84.2%) had a serious TEAE; these serious TEAEs led to hospitalization in all but one of these patients (Table 3). The most common serious TEAEs were febrile neutropenia (n=13, 34.2%), sepsis (n=11, 28.9%), and pneumonia (n=10, 26.3%) (Online Supplementary Table S3). TEAEs leading to discontinuation of any study agent were reported in eight patients (21.1%; n=6 at 10 mg/kg, and n=1 each at 3 and 20 mg/kg) and included anal abscess, diverticulitis, pneumonia, general physical health deterioration, multiple organ dysfunction syndrome, cardiac failure, hypopituitarism, enterocolitis, and hypertension (all n=1). There were 10 fatal TEAEs (26.3%; eight at 10 mg/kg and two at 20 mg/kg); none were considered drug-related. TEAEs leading to death were multiple organ dysfunction syndrome (n=3), general physical health deterioration (n=2), and pneumonia, sepsis, acute coronary syndrome, large intestine perforation, and respiratory failure (all n=1). Seven patients (18.4%) reported IRRs, of which chills (n=5, 13.2%) and pyrexia (n=2, 5.3%) were the most common. Two grade 3 IRRs (5.3%; chills n=1 and hypertension n=1) were observed; the hypertension event led to treatment discontinuation. There were no IRRs observed in the 20 mg/kg cohort. Two (5.3%; both due to an AE) deaths occurred within 30 days and four (10.5%; three due to an AE [two multiple organ dysfunction syndrome, one pneumonia], one due to other reasons [assisted-suicide]) deaths occurred within 60 days of first treatment with cusatuzumab (all in the phase II 10 mg/kg cohort). Pharmacokinetics and pharmacodynamics In phase I, after IV administration of the monotherapy dose (on day -14) or second dose (on Cycle 1 day 3, post-azacitidine) of cusatuzumab, mean maximum serum concentration (Cmax) and mean area under the serum concentration–time curve from time 0 to 14 days (AUC14d) increased with increasing doses (Online Supplementary Table S4). Mean serum half-life (t1/2) ranged from 6.1 to 10.4 days across the four dose cohorts in phase I. There was no obvious change in dose-normalized parameters with increasing dose, suggesting exposure increased in an approximately dose-proportional manner over the dose range 1-20 mg/kg. In phase II, after Confidential. Not for further distribution 14 IV administration of the 10 mg/kg monotherapy dose of cusatuzumab, mean Cmax was 195 µg/mL. AUCl4d was 32,932 µg.h/mL, and t1/2 was 11.1 days. After administration of the second dose on Cycle 1 day 3, mean Cmax was 233 µg/mL. AUCl4d was 38,298 µg.h/mL, and t1/2 was 8.3 days. There was a lower median percentage bone marrow blast count from baseline (screening) to Cycle 1 day 1, i.e., following the single monotherapy dose of cusatuzumab, and prior to the first dose of azacitidine and second dose of cusatuzumab (Online Supplementary Figure S1). Analysis of pharmacodynamic markers showed most patients exhibited the biggest decrease in sCD27 levels after the initial cusatuzumab monotherapy dose (Online Supplementary Figure S2). Finally, expression of CD70 on the blasts was confirmed by flow cytometry but could not be associated with clinical response (Online Supplementary Figure S3). Immunogenicity Among 36 cusatuzumab-treated patients with evaluable samples, 11 (30.6%) tested positive for antibodies to cusatuzumab post-dose: 10/11 patients had antibodies first detected in Cycle 1; one of 11 patients had antibodies first detected in Cycle 3. One of four patients with a positive sample at baseline became positive for treatment-boosted antidrug antibodies. The small sample size limits understanding of how cusatuzumab concentration effects immunogenicity. Discussion This study assessed the feasibility of combining the anti-CD70 monoclonal antibody, cusatuzumab, with standard-dose azacitidine in patients with newly diagnosed AML who were ineligible to receive intensive chemotherapy due to advanced age, comorbidities, and/or a poor performance status. Building on the interim results of the phase I dose-escalation period of this study,13 we found that half of the 38 patients (50%) treated with cusatuzumab/azacitidine achieved an objective response (CR or CRi). For the full analysis set of all patients who received the cusatuzumab 10 mg/kg treatment from phase I and phase II (n=29), the null hypothesis is not rejected, but is rejected in the combination therapy analysis set (n=27) after Confidential. Not for further distribution 15 excluding two patients who died before receiving combination therapy. It should be noted that after the data lock for this study, another response of CRi was confirmed for a patient who received the cusatuzumab 10 mg/kg treatment, which would allow for the null hypothesis to be rejected for both the full analysis and combination therapy sets. While the responses herein clearly demonstrate the clinical activity of the combination, response rates in the phase II part were lower than those reported in the initial phase I interim analysis, where high response rates were reported.13 The apparent discrepancy between the phase I interim data and final combined results may be explained, at least in part, by the small number of patients in the two phases of the study and by differences in baseline characteristics, particularly the prevalence of adverse genetic risk by ELN criteria, (33.3% in phase I versus 80.8% in phase II) with higher response rates among favorable- and intermediate-risk patients (11/13 for favorable/intermediate risk compared with 8/25 for adverse risk). Despite the lower than anticipated response rates, durable CRs were observed in a number of patients at all dose levels, including at the 10 mg/kg dose of cusatuzumab selected for expansion, and almost two- thirds of patients (63.2%) achieved transfusion independence, which is a strong prognostic factor in unfit patients with AML.18 Responses were also observed in each ELN 2017 genetic risk group (a good predictor of prognosis in newly diagnosed AML3,19), indicating the feasibility of the combination for all patients, including those with adverse risk profiles. Notably, the median OS time of 11.5 months compares favorably with a recent real-world report for azacitidine alone (7.1 months),20 suggesting that the cusatuzumab/azacitidine combination is worthy of further study. Cusatuzumab combined with azacitidine was generally well tolerated, with most TEAEs consistent with those expected for an AML population undergoing treatment with azacitidine,4,21 and there was no obvious dose dependency for toxicities. The most common TEAEs were infections and hematologic toxicities, which were generally manageable. IRRs, a common side effect of many monoclonal antibodies used to treat hematologic malignancies,22-24 were the only notable addition to the AE profile. These reactions were usually mild or moderate in intensity Confidential. Not for further distribution 16 and generally managed successfully by interrupting the cusatuzumab infusion, providing symptomatic treatment, and restarting the infusion at a reduced rate. Formation of antidrug antibodies have previously been shown to contribute to loss of efficacy,25 however the clinical impact of the antidrug antibodies observed in this cohort remain uncertain since neutralizing assays were not performed. The pharmacodynamic data were consistent with previous assessments for cusatuzumab and support its mechanism of action to reduce AML blasts and decrease serum sCD27 levels.12-14,16 CD70 expression could be detected on baseline peripheral blood blasts but could not be identified as a predictor of response to cusatuzumab/azacitidine in patients with newly diagnosed AML as observed for other immune related molecules.26,27 These data suggest that CD70 expression is not a limiting factor for the efficacy of cusatuzumab treatment. In addition, it has been shown that HMA treatment upregulates CD70.13 The pharmacokinetic evaluations also supported other prior investigations16,17 and showed that cusatuzumab exposure increases in an approximately dose-proportional manner following treatment over the dosing interval 1-20 mg/kg. This approximate dose-proportional increase in systemic exposure, combined with the (limited) response and safety data seen at the 20 mg/kg dose level, provides a rationale for further investigating the higher dose of cusatuzumab. Though the cohort was small, all three patients treated at 20 mg/kg, including two with adverse genetic risk per ELN, achieved CR without evidence of disease progression after >1 year of therapy. There were also no indications of additional toxicity. As these data only became available after the RP2D of 10 mg/kg had been selected, the optimal dose of cusatuzumab for further study remains uncertain. Consequently, the randomized, phase II, CULMINATE trial is evaluating the efficacy and safety of the 10 and 20 mg/kg doses of cusatuzumab combined with azacitidine in a similar AML study population.28 The clinical potential of cusatuzumab is also being investigated in combination with the new standard of care, venetoclax, with or without azacitidine (ClinicalTrials.gov Identifier: NCT04150887). This latter trial is informed by preclinical data showing that cusatuzumab works synergistically with both azacitidine and venetoclax to eliminate primary human AML LSCs.29 Confidential. Not for further distribution 17 In conclusion, our findings suggest that the combination of cusatuzumab and azacitidine is generally well tolerated and may be efficacious in patients with previously untreated AML not eligible for intensive chemotherapy. Studies are ongoing to establish the optimal dose level of cusatuzumab (10 versus 20 mg/kg) plus azacitidine and assessing the feasibility of combining cusatuzumab with venetoclax, with or without azacitidine. Confidential. Not for further distribution 18 References 1. Heuser M, Ofran Y, Boissel N, et al. Acute myeloid leukaemia in adult patients: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2020;31(6):697-712. 2. Sekeres MA, Guyatt G, Abel G, et al. American Society of Hematology 2020 guidelines for treating newly diagnosed acute myeloid leukemia in older adults. Blood Adv. 2020;4(15):3528-3549. 3. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 4. Dombret H, Seymour JF, Butrym A, et al. International phase 3 study of azacitidine vs conventional care regimens in older patients with newly diagnosed AML with >30% blasts. Blood. 2015;126(3):291-299. 5. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617-629. 6. Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367(6464):645-648. 7. Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. Int J Hematol. 2017;105(5):549-557. 8. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730-737. 9. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood. 2017;129(12):1577-1585. 10. Craddock C, Quek L, Goardon N, et al. Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia. Leukemia. 2013;27(5):1028-1036. 11. Zeng Z, Shi YX, Samudio IJ, et al. Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML. Blood. 2009;113(24):6215-6224. Confidential. Not for further distribution 19 12. Riether C, Schürch CM, Bührer ED, et al. CD70/CD27 signaling promotes blast stemness and is a viable therapeutic target in acute myeloid leukemia. J Exp Med. 2017;214(2):359- 380. 13. Riether C, Pabst T, Höpner S, et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat Med. 2020;26(9):1459-1467. 14. Silence K, Dreier T, Moshir M, et al. ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade. MAbs. 2014;6(2):523-532. 15. Claus C, Riether C, Schürch C, Matter MS, Hilmenyuk T, Ochsenbein AF. CD27 signaling increases the frequency of regulatory T cells and promotes tumor growth. Cancer Res. 2012;72(14):3664-3676. 16. Aftimos P, Rolfo C, Rottey S, et al. Phase I dose-escalation study of the anti-CD70 antibody ARGX-110 in advanced malignancies. Clin Cancer Res. 2017;23(21):6411-6420. 17. De Meulenaere A, Vermassen T, Creytens D, et al. An open-label, nonrandomized, phase Ib feasibility study of cusatuzumab in patients with nasopharyngeal carcinoma. Clin Transl Sci. 2021;14(6):2300-2313. 18. Gavillet M, Noetzli J, Blum S, Duchosal MA, Spertini O, Lambert JF. Transfusion independence and survival in patients with acute myeloid leukemia treated with 5- azacytidine. Haematologica. 2012;97(12):1929-1931. 19. Herold T, Rothenberg-Thurley M, Grunwald VV, et al. Validation and refinement of the revised 2017 European LeukemiaNet genetic risk stratification of acute myeloid leukemia. Leukemia. 2020;34(12):3161-3172. 20. Zeidan AM, Wang R, Wang X, et al. Clinical outcomes of older patients with AML receiving hypomethylating agents: a large population-based study in the United States. Blood Adv. 2020;4(10):2192-2201. 21. Fenaux P, Mufti GJ, Hellström-Lindberg E, et al. Azacitidine prolongs overall survival compared with conventional care regimens in elderly patients with low bone marrow blast Confidential. Not for further distribution 20 count acute myeloid leukemia. J Clin Oncol. 2010;28(4):562-569. 22. Lenz HJ. Management and preparedness for infusion and hypersensitivity reactions. Oncologist. 2007;12(5):601-609. 23. Mateos MV, Cavo M, Blade J, et al. Overall survival with daratumumab, bortezomib, melphalan, and prednisone in newly diagnosed multiple myeloma (ALCYONE): a randomised, open-label, phase 3 trial. Lancet. 2020;395(10218):132-141. 24. Marcus R, Davies A, Ando K, et al. Obinutuzumab for the first-line treatment of follicular lymphoma. N Engl J Med. 2017;377(14):1331-1344. 25. Mosch R, Guchelaar HJ. Immunogenicity of monoclonal antibodies and the potential use of HLA haplotypes to predict vulnerable patients. Front Immunol. 2022;13:885672. 26. Subklewe M, Stein A, Walter RB, et al. Preliminary results from a phase 1 first-in-human study of AMG 673, a novel half-life extended (HLE) anti-CD33/CD3 BiTE (bispecific T-cell engager) in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML). Blood. 2019;134(Supplement_1):833. 27. Subklewe M, Stein A, Walter RB, et al. Updated results from a phase 1 first-in-human dose escalation study of AMG 673, a novel anti-CD33/CD3 BiTE® (bispecific T-cell engager) in patients with relapsed/refractory acute myeloid leukemia. EHA. 2020;EP548 https://library.ehaweb.org/eha/2020/eha25th/294466/marion.subklewe.updated.results.from. a.phase.1.first-in- human.dose.escalation.html?f=menu%3D6%2Abrowseby%3D8%2Asortby%3D2%2Amedia %3D3%2Ace_id%3D1766%2Aot_id%3D23221%2Amarker%3D757 Accessed 4 April 2022. 28. Trudel GC, Howes AJ, Jeste N, et al. CULMINATE: A phase II study of cusatuzumab + azacitidine in patients with newly diagnosed AML, ineligible for intensive chemotherapy. J Clin Oncol. 2020;38(15 suppl):abstract TPS7565. 29. Riether C, Chiorazzo T, Johnson AJ, et al. The combination of the BCL-2 antagonist venetoclax with the CD70-targeting antibody cusatuzumab synergistically eliminates primary human leukemia stem cells. Blood. 2019;134(Supplemental_1):3918. Confidential. Not for further distribution 21 Tables Table 1. Patient demographics and baseline characteristics. Characteristic Phase I Phase II Total (N=38) 1 mg/kg (n=3) 3 mg/kg (n=3) 10 mg/kg (n=3) 20 mg/kg (n=3) All doses (n=12) 10 mg/kg (n=26) Age, years, median (range) 77.0 (75-81) 71.0 (71-84) 74.0 (64-75) 76.0 (7 2-77) 75.0 (64-84) 75.5 (59-90) 75.0 (59-90) Sex, n (%) Female 1 (33.3) 2 (66.7) 1 (33.3) 1 (33.3) 5 (41.7) 14 (53.8) 19 (50) Male 2 (66.7) 1 (33.3) 2 (66.7) 2 (66.7) 7 (58.3) 12 (46.2) 19 (50) Race, n (%) White 3 (100) 3 (100) 3 (100) 3 (100) 12 (100) 19 (73.1) 31 (81.6) Not reported 0 0 0 0 0 7 (26.9) 7 (18.4) ECOG performance status, n (%) 0 1 (33.3) 3 (100) 0 0 4 (33.3) 9 (34.6) 13 (34.2) 1 2 (66.7) 0 3 (100) 3 (100) 8 (66.7) 14 (53.8) 22 (57.9) 2 0 0 0 0 0 3 (11.5) 3 (7.9) AML type, n (%) Confidential. Not for further distribution 22 De novo 0 1 (33.3) 3 (100) 2 (66.7) 6 (50) 19 (73.1) 25 (65.8) Secondary 3 (100) 2 (66.7) 0 1 (33.3) 6 (50) 7 (26.9) 13 (34.2) Genetic risk category per ELN 2017 criteria, n (%) Favorable 0 1 (33.3) 0 1 (33.3) 2 (16.7) 2 (7.7) 4 (10.5) Intermediate 2 (66.7) 2 (66.7) 2 (66.7) 0 6 (50) 3 (11.5) 9 (23.7) Adverse 1 (33.3) 0 1 (33.3) 2 (66.7) 4 (33.3) 21 (80.8) 25 (65.8) Time from diagnosis to treatment, days, median (range) 28.0 (3-64) 29.0 (13-69) 8.0 (3-17) 30.0 (6-47) 22.5 (3-69) 13.5 (6-139) 14.5 (3-139) AML: acute myeloid leukemia; ECOG: Eastern Cooperative Oncology Group; ELN: European LeukemiaNet. Confidential. Not for further distribution 23 Table 2. Best response to cusatuzumab plus azacitidine. Phase I Phase II Phase I+II Total (N=38) 1 mg/kg (n=3) 3 mg/kg (n=3) 10 mg/kg (n=3) 20 mg/kg (n=3) All doses (n=12) 10 mg/kg (n=26) 10 mg/kg (n=29) 10 mg/kg (n=27)a ORRb , n (%) [95% CI] 3 (100) [29.2-100] 2 (66.7) [9.4-99.2] 3 (100) [29.2-100] 3 (100) [29.2-100] 11 (91.7) [61.5-99.8] 8 (30.8) [14.3-51.8] 11 (37.9) [20.7-57.7] 11 (40.7) [22.4-61.2] 19 (50) [33.4-66.6] Response category, n (%) CR 2 (66.7) 2 (66.7) 2 (66.7) 3 (100) 9 (75) 5 (19.2) 7 (24.1) 7 (25.9) 14 (36.8) CRi 1 (33.3) 0 1 (33.3) 0 2 (16.7) 3 (11.5) 4 (13.8) 4 (14.8) 5 (13.2) MLFS 0 0 0 0 0 0 0 0 0 PR 0 0 0 0 0 0 0 0 0 SDc 0 1 (33.3) 0 0 1 (8.3) 16 (61.5) 16 (55.2) 16 (59.3) 17 (44.7) NE 0 0 0 0 0 2 (7.7) 2 (6.9) 0 2 (5.3) aExcluding 2 patients who did not receive azacitidine and died before first post-treatment disease assessment. bOverall response includes patients with a response of CR, CRi, MLFS, or PR. cTreatment failure responses were categorized as SD. CI: confidence interval; CR: complete remission; CRi: complete remission with incomplete recovery; MLFS: morphologic leukemia-free state; NE: not evaluable; ORR: overall response rate; PR: partial remission; SD: stable disease. Confidential. Not for further distribution 24 Table 3. Summary of TEAEs following treatment with cusatuzumab plus azacitidine. Patients with ≥ 1 TEAE,a n (%) Dose group Total (N=38) 1 mg/kg (n=3) 3 mg/kg (n=3) 10 mg/kg (n=29) 20 mg/kg (n=3) Any TEAE, n (%) 3 (100) 3 (100) 29 (100) 3 (100) 38 (100) Grade ≥ 3 3 (100) 3 (100) 29 (100) 3 (100) 38 (100) Drug related 3 (100) 3 (100) 27 (93.1) 3 (100) 36 (94.7) Serious TEAE, n (%) 3 (100) 3 (100) 24 (82.8) 2 (66.7) 32 (84.2) Grade ≥ 3 3 (100) 3 (100) 24 (82.8) 2 (66.7) 32 (84.2) Leading to hospitalization 3 (100) 3 (100) 23 (79.3) 2 (66.7) 31 (81.6) TEAE leading to any study drug discontinuation, n (%) 0 1 (33.3) 6 (20.7) 1 (33.3) 8 (21.1) TEAE leading to death, n (%) 0 0 8 (27.6) 2 (66.7) 10 (26.3) Drug related 0 0 0 0 0 Most common TEAEs (≥ 15% of all patients), b n (%) All Gr ≥ 3 All Gr ≥ 3 All Gr ≥ 3 All Gr ≥ 3 All Gr ≥ 3 Febrile neutropenia 2 (66.7) 2 (66.7) 1 (33.3) 1 (33.3) 10 (34.5) 10 (34.5) 2 (66.7) 2 (66.7) 15 (39.5) 15 (39.5) Neutropenia 1 (33.3) 1 (33.3) 3 (100) 3 (100) 9 (31) 9 (31) 2 (66.7) 2 (66.7) 15 (39.5) 15 (39.5) Anemia 1 (33.3) 1 (33.3) 3 (100) 3 (100) 10 (34.5) 10 (34.5) 0 0 14 (36.8) 14 (36.8) Thrombocytopenia 2 (66.7) 2 (66.7) 3 (100) 3 (100) 7 (24.1) 7 (24.1) 2 (66.7) 1 (33.3) 14 (36.8) 13 (34.2) Pneumoniac 2 (66.7) 1 (33.3) 0 0 10 (34.5) 6 (20.7) 2 (66.7) 2 (66.7) 14 (36.8) 9 (23.7) Pyrexia 2 (66.7) 0 2 (66.7) 1 (33.3) 9 (31.0) 0 1 (33.3) 0 14 (36.8) 1 (2.6) Confidential. Not for further distribution 25 Constipation 1 (33.3) 0 1 (33.3) 1 (33.3) 7 (24.1) 0 2 (66.7) 0 11 (28.9) 1 (2.6) Nausea 1 (33.3) 0 0 0 9 (31.0) 0 1 (33.3) 0 11 (28.9) 0 Sepsisd 0 0 0 0 10 (34.5) 10 (34.5) 1 (33.3) 1 (33.3) 11 (28.9) 11 (28.9) Vomiting 0 0 2 (66.7) 0 5 (17.2) 0 2 (66.7) 0 9 (23.7) 0 Leukopenia 2 (66.7) 2 (66.7) 2 (66.7) 1 (33.3) 2 (6.9) 2 (6.9) 1 (33.3) 0 7 (18.4) 5 (13.2) Diarrhea 0 0 0 0 4 (13.8) 0 3 (100) 1 (33.3) 7 (18.4) 1 (2.6) Chills 1 (33.3) 0 1 (33.3) 0 4 (13.8) 1 (3.4) 0 0 6 (15.8) 1 (2.6) Cough 1 (33.3) 0 2 (66.7) 0 3 (10.3) 0 0 0 6 (15.8) 0 Hypokalemia 0 0 1 (33.3) 1 (33.3) 5 (17.2) 0 0 0 6 (15.8) 1 (2.6) aTEAEs are defined as AEs with onset or worsening on or after the date of the first dose of study treatment up to and including 30 days after date of last dose of study medication. bTEAEs are listed in decreasing frequency of any-grade TEAE in the total study population (N=38). cPneumonia includes the following preferred terms: pneumonia, pneumonia bacterial, and pneumonia fungal. dSepsis includes the following preferred terms: Enterobacter sepsis, Escherichia sepsis, Pseudomonal bacteremia, sepsis, septic shock, and Staphylococcal bacteremia. AE: adverse event; Gr: grade; TEAE: treatment-emergent adverse event. Confidential. Not for further distribution 26 Figure legends Figure 1. Swimmer plot illustrating responses and outcomes in patients with newly diagnosed AML treated with cusatuzumab plus azacitidine (N=38, total study population). Death symbols do not represent timing of death. Adv: adverse; AE: adverse event; CR: complete remission; CRi: complete remission with incomplete recovery; EOS: end of study; EOT: end of treatment; Fav: favorable; ID: investigator decision; Int: intermediate; MLFS: morphologic leukemia-free state; MRD: minimal residual disease; NE: not evaluable; PD: progressive disease; PR: partial remission; SD: stable disease. Supplementary data Supplementary methods Dose-limiting toxicities Dose-limiting toxicities (DLTs) were assessed from day -14 (following administration of the loading dose of cusatuzumab) to the end of Cycle 1. They were defined as any of the following drug-related events: • Any grade ≥3 drug-related non-hematologic toxicity with a duration >14 days, except grade 3 events for which the patients had responded optimally to treatment with standard medication, with a duration >14 days • Grade ≥3 infusion-related reactions o Reversible grade 3 infusion-related reactions (defined as an allergic reaction/hypersensitivity, fever, pain, bronchospasm, wheezing, or hypoxia) occurring during or within 24 hours after completion of the infusion and not recurring following a reduction in the infusion rate, provision of supportive care, and/or administration of corticosteroids were not considered to be DLTs • Inability to administer the next dose due to a drug-related adverse event or a delay of the administration of the next dose due to toxicities for >14 days despite adequate medication • Drug-related grade 4 febrile neutropenia • Drug-related grade 4 anemia that could not be treated adequately by blood transfusions. Isolated grade ≥3 laboratory abnormalities (including isolated lymphopenia) that resolved to baseline or to grade 2 within 21 days, without clinical sequelae or the need for therapeutic intervention, and grade 3 fatigue lasting for ≤14 days were not considered to be DLTs. Eligibility criteria Inclusion criteria: 1. Signed informed consent form indicating an understanding of the purposes, risks, and procedures required for the study, and a willingness and ability to participate in the study 2. Acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS), according to the 2016 World Health Organization classification definition of ≥20% bone marrow blasts,1 unsuitable for intensive treatment (including stem cell transplantation) with curative intent, but eligible to receive azacitidine treatment. Note: MDS patients could be rescreened if previously excluded on blast count 3. Aged ≥18 years 4. Expected life expectancy ≥3 months, at the discretion of the investigator 5. Eastern Cooperative Oncology Group performance status of 0, 1, or 2. 6. Women of childbearing potential had to have a negative serum pregnancy test at screening and within 48 hours before infusion of cusatuzumab on day -14, and be willing to use an effective contraceptive method (intrauterine devices, hormonal contraceptives, contraceptive pill, implants, transdermal patches, hormonal vaginal devices, or infusions with prolonged release) during the study and for at least 3 months after the last study drug administration 7. Men had to be willing to use an effective contraceptive method (e.g., condom, vasectomy) during the study and for at least 3 months after the last study drug administration. Exclusion criteria: 1. Prior or concurrent malignancy, except for the following: ‒ Adequately treated basal cell or squamous cell skin cancer ‒ Carcinoma in situ of the cervix ‒ Carcinoma in situ of the breast, or ‒ Incidental histological finding of prostate cancer (TNM stage T1a or T1b), or ‒ Any other cancer from which the patient had been disease-free for >2 years 2. Any previous chemotherapy or radiotherapy for AML or MDS, except hydroxyurea/hydroxycarbamide for leukocyte control (which had to be discontinued by the first day of azacitidine administration), local radiation therapy, and therapy for basal or squamous cell carcinoma of the skin 3. Treatment with any investigational product within 4 weeks before the first administration of cusatuzumab 4. Abnormal organ function, defined as follows (any single parameter to fulfill condition): ‒ Aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) >3 × the upper limit of normal (ULN); or in case of liver infiltration by AML, AST and/or ALT >5 × ULN ‒ Alkaline phosphatase (AP) >2.5 × ULN; or in case of liver infiltration by AML, AP >5 × ULN ‒ Serum (total) bilirubin >1.5 × ULN; or in case of liver infiltration by AML, serum (total) bilirubin >5 × ULN ‒ Serum creatinine >2.5 × ULN or glomerular filtration rate (Modification of Diet in Renal Disease) <40 mL/min for patients with creatinine levels above the normal limit 5. Use of immunosuppressive agents in the past 4 weeks before the first administration of cusatuzumab on day -14. For regular use of systemic corticosteroids, patients could only be included after stepwise discontinuation and had to be free of steroids for a minimum of 5 days before the first administration of cusatuzumab 6. Any known active or chronic infection, including human immunodeficiency virus, and hepatitis B or C virus infection. 7. Any other concurrent disease or medical condition that was likely to interfere with study procedures or results, or that in the opinion of the investigator would constitute a hazard for participating in this study 8. Known hypersensitivity to cusatuzumab or azacitidine and its analogs in general, or to any other component of the study drug formulation 9. Congestive heart failure New York Heart Association (NYHA) Class III and IV, cardiac arrhythmias (except atrioventricular block type I and II, atrial fibrillation/flutter, or bundle brunch block) or other signs and symptoms of relevant cardiovascular disease 10. Pregnant women, nursing mothers, lactating women, and women of childbearing potential who were unwilling to use effective contraceptive methods (intrauterine devices, hormonal contraceptives, contraceptive pill, implants, transdermal patches, hormonal vaginal devices, or infusions with prolonged release) during the study from the time of consent and for at least 3 months after the last study drug administration 11. Men who were unwilling to use effective contraception for the duration of the study and for at least 3 months after the last study drug administration 12. Patients who were unwilling or unable to follow the protocol requirements. Assessments Disease assessments were based on peripheral blood counts and bone marrow (aspirate or biopsy) evaluations, as per clinical practice guidelines,2-4 and were classified according to established criteria for AML (Table S1). Bone marrow samples for disease assessment were taken on Cycle 1 day 1 (pre-azacitidine), and on day 1 (pre-azacitidine) of every odd cycle from Cycle 3 onwards until complete remission (CR) or CR with incomplete recovery (CRi), and at the end-of-treatment visit. Additional samples could be taken as instructed by the treating physician. Bone marrow aspirate was assessed by experienced hematopathologists at local pathology laboratories and there was no central assessment performed. Minimal residual disease assessments on bone marrow aspirates were performed at either Inselspital Bern or Covance Laboratory using multiparameter flow cytometry (based on quantification of leukemia-associated immunophenotype [LAIP]-positive cells) according to European LeukemiaNet guidelines.5 Samples were collected as per the response evaluations. Minimal residual disease-negative status was defined as <10-3 LAIP-positive cells. Blood samples for pharmacokinetic assessments of cusatuzumab were taken on day -14 (pre-, and 0, 2, 24, and 96 hours post-cusatuzumab), Cycle 1 day 1 (pre-azacitidine), Cycle 1 day 3 (pre-azacitidine, and 0, 2, and 24 hours post-cusatuzumab), Cycle 1 day 7 (pre-azacitidine), Cycle 1 day 17 (pre- and 0 hours post-cusatuzumab), Cycle 2 day 3 (pre-azacitidine and 0 hours post-cusatuzumab), Cycle 2 day 17 (pre- and 0 hours post-cusatuzumab), Cycle ≥3 day 3 (pre-azacitidine and 0 hours post-cusatuzumab), Cycle ≥3 day 17 (pre- and 0 hours post- cusatuzumab), and at the end-of-treatment and follow-up visits. Serum concentrations of cusatuzumab were analyzed using a validated enzyme-linked immunosorbent assay (ELISA) method. The immunogenicity of cusatuzumab was evaluated in venous blood samples, taken up to 4 hours prior to cusatuzumab infusion on day -14, on days 3 (pre-azacitidine) and 17 (pre- cusatuzumab) in Cycles 1-4 and in Cycle 8 (if applicable), and at the end-of-treatment and follow-up visits. Antidrug antibodies were detected in serum using an enzyme-linked immunoassay (ELISA) method. The primary antibody was derived from llama immunizations and germlined to human antibody framework. Bone marrow aspirate and/or whole blood samples were used for the pharmacodynamic evaluations. Soluble CD27 (sCD27) levels in serum were measured on days 1 and 17 of each treatment cycle and at the end-of-treatment and follow-up visits using electrochemiluminescence methodology with the R-PLEX Human CD27 Antibody Set [Meso Scale Discovery]; the lower and upper assay detection limits were 12.2 and 50,000 pg/mL, respectively. To determine the cell-surface expression of pharmacodynamic markers, flow cytometric analysis was performed on bone marrow samples following red blood cell lysis, as described previously.6 Supplementary tables and figures Table S1. Response criteria (investigator assessed). Response Definitiona Complete remission (CR)b Bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count >1.0 × 109/L (1,000/µL); platelet count >100 × 109/L (100.000/µL); independence of red cell transfusions CR with incomplete recovery (CRi) All CR criteria except for residual neutropenia (<1.0 × 109/L [1,000/µL]) or thrombocytopenia (<100 × 109/L [100.000/µL]) Morphologic leukemia- free state (MLFS) Bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; no hematologic recovery required Partial remission (PR) Relevant in the setting of phase I and II clinical trials only; all hematologic criteria of CR; decrease in bone marrow blast percentage to 5-25%; and decrease in pretreatment bone marrow blast percentage by at least 50% Treatment failure Resistant disease Death in aplasia Death from indeterminate cause Failure to achieve CR or CRi (general practice; phase II/III trials), or failure to achieve CR, CRi, or PR (phase I trials); only includes patients surviving >7 days following completion of initial treatment, with evidence of persistent leukemia by blood and/or bone marrow examination Deaths occurring >7 days following completion of initial treatment while cytopenic; with an aplastic or hypoplastic bone marrow obtained within 7 days of death, without evidence of persistent leukemia Deaths occurring before completion of therapy, or <7 days following its completion; or deaths occurring >7 days following completion Relapse Bone marrow blasts >5%; or reappearance of blasts in the blood; or development of extramedullary disease aDefinitions were based on those given by Cheson et al2, Döhner et al3, and NCCN4. bAll criteria must be fulfilled; marrow evaluation should be based on a count of 200 nucleated cells in an aspirate with spicules; if ambiguous, consider repeat exam after 5-7 days; flow cytometric evaluation may help to distinguish between persistent leukemia and regenerating normal marrow; a marrow biopsy should be performed in cases of dry tap, or if no spicules are obtained; no minimum duration of response required. Table S2. Responses to cusatuzumab plus azacitidine by genetic risk classification per ELN 2017 criteria. ELN 2017 risk classification Response, n (%) Phase I Phase II Total (N=38) 1 mg/kg (n=3) 3 mg/kg (n=3) 10 mg/kg (n=3) 20 mg/kg (n=3) All doses (n=12) 10 mg/kg (n=26) Favorable n=0 n=1 n=0 n=1 n=2 n=2 n=4 CR 0 1 (100) 0 1 (100) 2 (100) 1 (50) 3 (75) CRi 0 0 0 0 0 1 (50) 1 (25) Intermediate n=2 n=2 n=2 n=0 n=6 n=3 n=9 CR 2 (100) 1 (50) 2 (100) 0 5 (83.3) 2 (66.7) 7 (77.8) Adverse n=1 n=0 n=1 n=2 n=4 n=21 n=25 CR 0 0 0 2 (100) 2 (50) 2 (9.5) 4 (16) CRi 1 (100) 0 1 (100) 0 2 (50) 2 (9.5) 4 (16) NE 0 0 0 0 0 2 (9.5) 2 (8) CR: complete remission; CRi: complete remission with incomplete recovery; ELN: European LeukemiaNet; NE: not evaluable. Table S3. Summary of the most common serious TEAEs (occurring in ≥5% of all patients) following treatment with cusatuzumab plus azacitidine. Patients with ≥1 serious TEAE,a n (%) Dose group Total (N=38) 1 mg/kg (n=3) 3 mg/kg (n=3) 10 mg/kg (n=29) 20 mg/kg (n=3) Febrile neutropenia 2 (66.7) 0 9 (31) 2 (66.7) 13 (34.2) Sepsisb 0 0 10 (34.5) 1 (33.3) 11 (28.9) Pneumoniac 1 (33.3) 0 7 (24.1) 2 (66.7) 10 (26.3) General physical health deterioration 0 0 1 (3.4) 2 (66.7) 3 (7.9) Acute coronary syndrome 0 0 1 (3.4) 1 (33.3) 2 (5.3) Cardiac failure 0 0 1 (3.4) 1 (33.3) 2 (5.3) Cellulitis 0 1 (33.3) 1 (3.4) 0 2 (5.3) Device-related infection 0 0 2 (6.9) 0 2 (5.3) Pyrexia 0 1 (33.3) 1 (3.4) 0 2 (5.3) aTEAEs are defined as AEs with onset or worsening on or after the date of the first dose of study treatment up to and including 30 d ays after date of last dose of study medication. TEAEs are listed in decreasing frequency of any-grade TEAE in the total study population (N=38). bSepsis includes the following preferred terms: Enterobacter sepsis, Escherichia sepsis, Pseudomonal bacteremia, sepsis, septi c shock, and Staphylococcal bacteremia. cPneumonia includes the following preferred terms: pneumonia. AE: adverse event; TEAE: treatment-emergent adverse event. Table S4. Summary of pharmacokinetic parameters following cusatuzumab administration on day -14 (loading dose) and on Cycle 1 day 3. Phase I Phase II 1 mg/kg 3 mg/kg 10 mg/kg 20 mg/kg 10 mg/kg Post loading dose (day -14) (n=3) (n=3) (n=3) (n=3a) (n=25a) Cmax, µg/mL 28.3 (13.5) 61.4 (13) 188 (38.4) 405 (32.4) 195 (81.3) tmax, day 0.013 (0.135-0.170) 0.233 (0.205-0.245) 0.215 (0.143-0.226) 0.229 (0.153-0.293) 0.219 (0.132-2.86) AUC14d, µg.h/mL 2,682 (237) 9,911 (1,689) 28,170 (3,135) 52,149 (13,479) 32,932 (25,722) t1/2, days 6.1 (1.6) 8.2 (1.3) 9.7 (2) 10.4 (1.4) 11.1 (4.1) Cmax, dn, µg/mL/mg 28.3 (13.5) 20.6 (4.37) 18.8 (3.78) 20.2 (1.53) 19.5 (8.05) AUC14d,dn, µg.h/mL/mg 2,682 (237) 3,316 (574) 2,825 (301) 2,603 (682) 3,308 (2,564) Cycle 1 day 3 (n=3) (n=3) (n=3) (n=3) (n=23b) Ctrough, µg/mL 3.09 (1.22) 12.2 (1.35) 40.3 (8.46) 64.1 (6.83) 40.6 (17.6) Cmax, µg/mL 22.2 (2.99) 79.2 (10.1) - 452 (49.5) 233 (59.2) tmax, day 0.0658 (0.0625-0.0692) 0.0763 (0.0658-0.193) - 0.260 (0.212-0.979) 0.139 (0.0692-0.910) AUC14d, µg.h/mL 2,351 (554) 10,851 (4,636) - 78,762 (10,488) 38,298 (11,991) t1/2, days - 7.4 (4.2) - - 8.3 (2.4) Cmax, dn, µg/mL/mg 22.2 (2.99) 26.4 (3.45) - 22.6 (2.48) 23.2 (5.93) AUC14d,dn, µg.h/mL/mg 2,351 (554) 3,625 (1,555) - 3,938 (524) 3,811 (1,192) an=23 for AUC14d and AUC14d,dn, and n=18 for t1/2. bn=21 for AUC14d and AUC14d,dn, and n=11 for t1/2. AUC14d: area under the serum concentration–time curve from time 0 to 14 days; Cmax: maximum serum concentration; Ctrough: minimum serum concentration; Dn: dose normalized to 1 mg/kg; t1/2: terminal elimination half-life; tmax:, time to maximum serum concentration. Figure S1. Box-and-whisker plot of bone marrow blasts at baseline (day -14, pre- cusatuzumab), on Cycle 1 day 1 (pre-combination treatment), and at best response in all patients treated with cusatuzumab plus azacitidine (both study phases combined), as determined by cytomorphology. The BR timepoint only includes patients achieving an objective response (complete remission or complete remission with incomplete recovery). Bone marrow data are based on aspirate blast percentage; if aspirate was not available due to dry tap, biopsy blast percentage was used. BL: baseline; BR: best response; C: Cycle; D: day. Figure S2. Change in serum sCD27 levels in phase I (A) and phase II (B) patients treated with cusatuzumab monotherapy (day -14), all dose levels and responses combined per phase. Phase I and phase II sCD27 assays were run in different laboratories. BL: baseline; C: Cycle; D: day; N: number; sCD27: soluble CD27. Figure S3. Box-and-whisker plot of baseline CD70 expression in peripheral blood blasts in responders and non-responders to cusatuzumab plus azacitidine (all dose levels combined). Responders include patients achieving complete remission or complete remission with incomplete recovery. Abbreviations: MFI, mean fluorescence intensity. References 1. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391-2405. 2. Cheson B, Bennett J, Kopecky K, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol. 2003;21(24):4642-4649. 3. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373(12):1136-1152. 4. National Comprehensive Cancer Network® (NCCN). NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). Acute Myeloid Leukemia. Version 1.2015. 5. Schuurhuis GJ, Heuser M, Freeman S, et al. Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party. Blood. 2018;131(12):1275-1291. 6. Riether C, Pabst T, Höpner S, et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat Med. 2020;26(9):1459-1467.

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