Strategies targeting leukemic stem and progenitor cells (LSPCs) are needed for durable remissions in acute myeloid leukemia (AML) and high-risk myelodysplastic neoplasms (MDS). While CD123 constitutes a promising target on LSPCs and leukemic blasts, previous CD123-targeting approaches showed limited efficacy and challenging safety profiles. Here, we describe the preclinical efficacy and safety of the bispecific CD123/CD16A innate cell engager "AFM28", demonstrating superior activity against AML and MDS patient-derived LSPCs and blasts in vitro compared to an Fc-enhanced CD123-targeting antibody, especially towards CD123low and/or CD64+ leukemic cells. AFM28 induces autologous anti-leukemic activity in fresh AML whole blood cultures, demonstrating its potential to enhance NK cell function from AML patients. Responsiveness can be further enhanced by allogeneic NK cell addition. Anti-leukemic activity of AFM28 is confirmed in xenograft mouse models. In addition, AFM28 is well tolerated and demonstrates pharmacodynamic activity in cynomolgus monkeys. Altogether, our results indicate that AFM28 has the potential to reduce relapse-inducing residual disease and promote long-term remissions for patients with AML and MDS with a favorable safety profile.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are common forms of adult hematologic malignancies with incidences of approximately 1-6/100.000/year. Progress in the management of AML and high-risk MDS has lagged behind other hematological malignancies, and, in particular, treatment for relapsed or refractory (R/R) disease and minimal residual disease (MRD) are needed to induce long-term remission.

Abstract RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4556.

Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.

Natural killer (NK) cells are crucial effector cells of the innate immune system capable of rapidly recognizing and eliminating infected, stressed and malignant cells. NK cells are also the prime mediators of antibody-dependent cell-mediated cytotoxicity (ADCC), a potent mechanism of anti-viral immunity that has been applied to cancer therapy by targeting tumor-expressed surface antigens using monoclonal antibodies (mAbs). Classical ADCC is mediated by low affinity Fc-mediated engagement of NK cells via FcγRIIIA (CD16A) and is modulated by differences in target antigen expression levels. While high potency of therapeutic mAbs is achieved when target antigen is available at high density, potency and efficacy decrease substantially when copy numbers are low. Classical ADCC also needs to overcome the inhibitory effect of competing serum IgG and is negatively affected by a low affinity polymorphism of CD16A (158F) that is prevalent in approximately 8 of 10 individuals. Hence, classical Fc-mediated ADCC does not fully utilize the therapeutic potential of NK cell cytotoxicity.