Despite promising anti-leukemic activity of MCL-1 inhibitors in preclinical studies of acute myeloid leukemia (AML), clinical progress has been hindered by limited knowledge of target patient subgroups. To stratify patients for MCL-1 inhibitor treatment, we evaluated the sensitivity of 42 primary AML samples to MCL-1 inhibitor MIK665 (S64315) and analyzed their molecular profiles. We observed that MIK665-sensitive samples had a more differentiated phenotype, whereas resistant samples displayed higher levels of ABCB1 (MDR1) and the anti-apoptotic protein BCL-XL. Moreover, ABCB1 expression had good predictive performance in identifying resistant samples. To induce sensitivity, we treated MIK665-resistant samples with ABCB1 inhibitors elacridar or tariquidar, BCL-XL inhibitor A1331852, or BCL-2 inhibitor venetoclax in combination with MIK665. The combination of MIK665 with each of elacridar, tariquidar, or venetoclax effectively eliminated AML blasts compared to the agents alone, while the combination with A1331852 showed limited efficacy for this patient subgroup. Additionally, the combination of MIK665 with venetoclax restored sensitivity in samples with primary venetoclax resistance. Overall, this study indicates that elevated ABCB1 expression is a potentially targetable resistance mechanism in the context of MIK665 resistance, and that a combination of MIK665 with venetoclax may be effective for overcoming resistance to either MCL-1 or BCL-2 inhibition.

Introduction Treatment of myelodysplastic syndrome (MDS) with primary refractory disease or relapse after frontline treatment (r/r MDS) represents a high unmet medical need. Hypomethylating (HMA) agents, including azacitidine, are used in the frontline setting for higher-risk (HR) MDS patients with response rate reported as 16%1. After HMA-failure, the median overall survival (mOS) is poor, with 5.6 months.2 Bexmarilimab, a macrophage checkpoint inhibitor, blocks Common lymphatic and vascular endothelial receptor-1 (Clever-1) to enhance macrophage antigen presentation and T cell activation. In myeloid malignancies, Clever-1 is also abundant on myeloid blasts. Pre-clinical data suggests that by blocking Clever-1 on blasts, bexmarilimab hampers the energy production of the malignant cells, allowing enhanced killing of generally resistant blasts by cytotoxic agents, such as HMAs. Thus, bexmarilimab may alter the bone marrow (BM) microenvironment making the blasts susceptible to other available therapies, thereby enhancing their effectiveness in patients with MDS, including r/r MDS. Methods As of 15 May 2024, 14 patients with r/r MDS were enrolled in the ongoing Phase 1 dose escalation and Phase 2 dose optimization of the BEXMAB study (NCT05428969). All patients had IPSS-R >3.5 and 7/14 were TP53 mutated. Bexmarilimab was administrated weekly in 28-day cycles, at 1,3, and 6 mg/kg, in combination with a standard regimen of azacitidine (75 mg/m2 D1-7 each cycle). Bayesian optimal interval (BOIN) design was used for dose escalation and r/r MDS was selected as the population for dose optimization and expansion following a Simon's 2-stage design. The primary objectives of the study are to evaluate safety, tolerability, and preliminary efficacy of bexmarilimab plus azacitidine. BM was evaluated at the end of Cycles 1, 3 and then every third cycle for response and exploratory biomarkers. Results Altogether, 113 treatment-emergent adverse events (TEAE) were reported, of which 74 (65%) were grade 1-2. Seventeen serious adverse events (SAE) were reported from 7 r/r MDS patients, with febrile neutropenia as most frequent event (n=9). Four bexmarilimab-related events were reported from 3 r/r MDS patients (fever, infusion related reaction, intermittent nausea and peripheral edema), all grade 1. The objective response rate (ORR) was 79% in r/r MDS patients (11/14). The responses consisted of 1 complete remission (CR), 7 marrow CRs (mCR), 1 partial response (PR) and 2 hematological improvements (HI). In addition, 2 responses of stable disease (SD) and 1 progressive disease (PD) were reported. Clinical activity was observed across all dose levels. Two patients proceeded to allogenic stem cell transplantation. The current median overall survival estimate (mOS) for the 14 Phase 1 r/r MDS patients is 13.4 months. Supporting our hypothesis of modulation of the BM microenvironment, the expression of human leukocyte antigen DR isotype (HLA-DR), was increased up to 6-fold on BM monocytes upon treatment. In addition, the number of CD4 and CD8 T cells was increased in the BM compared to baseline in 50% and 75% of patients, respectively. Both HLA-DR and T cell increases were observed in most patients after one treatment cycle. For further insight on the MoA of bexmarilimab plus azacitidine in r/r MDS, single-cell RNA sequencing analysis of pre- and post-treatment (Cycle 1 and 3) BM samples is ongoing. Preliminary data suggest significant changes in the expression of genes related to blast cell energy production and macrophage activity induced by bexmarilimab and azacitidine treatment. Conclusions Bexmarilimab plus azacitidine is well tolerated and results in promising clinical efficacy in r/r MDS patients after HMA failure. 1 Hasegawa et al. Azacitidine Monotherapy in Patients With Treatment-Naïve Higher-risk Myelodysplastic Syndrome: A Systematic Literature Review and Meta-analysis.Clin Lymphoma Myeloma Leuk. 2023. 2 Prébet et al. Outcome of High-Risk Myelodysplastic Syndrome After Azacitidine Treatment Failure. J Clin Onc. 2011.

The potential of immunotherapies in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains still under investigation. Clever-1 (also known as Stabilin-1) is a multifunctional scavenger and adhesion receptor expressed by monocytes and immunosuppressive macrophages. Bexmarilimab, a Clever-1 targeting antibody, demonstrates many immunomodulatory effects1 along with promising anti-tumor activity against solid tumors in patients with multiple lines of previous treatment2. In AML, high STAB1 (mRNA) levels associate with poor survival and resistance to therapy3. The aim of this study was to profile Clever-1 expression in AML and MDS and test in preclinical models the growth inhibitory and immunomodulatory potential of bexmarilimab, as a single agent and in combination with azaciditine and venetoclax. AML cell lines (n=11) and frozen mononuclear cells, extracted from AML (n=42) and very high risk MDS (n=4) patient bone marrow (BM) aspirates provided by the Finnish Hematology Registry and Biobank, were used. Samples were treated for 48h with bexmarilimab alone, or in combination with azacytidine and/or venetoclax. Flow cytometry was used to detect different myeloid and TBNK cell populations along with Clever-1, HLA-DR, PD-(L)1 and additional T cell activation markers. Our results confirmed Clever-1 protein expression in AML cell lines and bexmarilimab treatment induced metabolic and growth inhibition of KG1 blasts (Clever-1high). In patients with AML, FAB M4/M5 subtypes exhibited highest Clever-1 levels, along with FAB M2 patients with consequent rapid relapse. Clever-1 expression correlated negatively with monocyte MHC class II molecule, HLA-DR, expression, and BM T-cell frequency, in line with the immunosuppressed state associated with high Clever-1. Ex vivo treatment of the primary AML BM cells with bexmarilimab resulted in a notable, 5-10x fold increase in monocyte HLA-DR in samples with low basal HLA-DR and high Clever-1. The combination of azacitidine with bexmarilimab augmented HLA-DR induction by 20-110% (mean 44%). FAB M1/M2 AML showed increase in activation markers, such as Ki67, CXCR3 and Granzyme B after ex vivo bexmarilimab treatment in CD8+ T-cells. Furthermore, bexmarilimab reduced PD-1 expression in NK- and CD8+CXCR3+ T-cell populations of FAB M0-M2 AML and MDS-EB2. This project is the first comprehensive investigation of Clever-1 expression in AML and MDS patient BM blasts and monocytes. Ex vivo treatment with bexmarilimab, alone or in combination with azacytidine or venetoclax, indicate enhanced antigen presentation capability and immunological activation. These results validate the therapeutic potential of bexmarilimab in myeloid malignancies. The safety, tolerability and preliminary efficacy of bexmarilimab is now further investigated in combination with venetoclax and/or azacytidine in a phase I/II clinical trial BEXMAB (NCT05428969).1Viitala et al., Clin Cancer Res 2019;25:3289-303; 2 Bono et al., Annals of Oncology; 2021. p 32, 5: S1283-S346; 3 Lin et al., Mol Ther Nucleic Acids 2019;18:476-84 Citation Format: Arno Ylitalo, Sofia Aakko, Heikki Kuusanmäki, Mari Björkman, Juho Jalkanen, Marie-Louise Fjällskog, Caroline Heckman, Maija Hollmén, Mika Kontro. Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A14.

Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Background: Clever-1 constitutes a novel macrophage checkpoint. Its expression in AML BM cells is a prognostic factor for poor outcome and its suppression inhibits leukemia cell growth (1). Analyses suggests lower expression is associated with venetoclax sensitivity (2). Bexmarilimab (BEX), a humanized IgG4 monoclonal antibody, binds Clever-1 and alters the function of macrophages. BEX has been shown to increase antigen presentation, induce secretion of proinflammatory cytokines and increase activation of T cells (3). The therapeutic potential of BEX is supported by data from the MATINS study (NCT03733990) evaluating single agent activity in advanced solid tumors (n>200) that showed good tolerability as well a clinical activity. Recent data show that Clever-1 is expressed at high levels on malignant blasts and monocytes of AML and MDS patients. Treatment of AML bone marrow cells with BEX alone or in combination with azacitidine/venetoclax results in enhanced antigen presentation capacity and increased activation markers on effector T cells with synergistic effects (4). Aims: The PhI/II study evaluates safety, tolerability and preliminary efficacy of BEX plus standard of care in frontline or hypomethylating agent (HMA) failed MDS/CMML patients, newly diagnosed AML or in relapsed/refractory AML patients. Analyses of parameters for immunological and cellular read-outs and the association of predictive biomarker for BEX clinical activity in patient sub-groups is part of the exploratory endpoints. Methods: BEXMAB (NCT05428969) consists of a dose escalation PhI using a Bayesian Optimal Interval (BOIN) design to explore 4 dose levels of BEX in cohort sizes of n=5 with 1mg/kg as starting dose. BEX plus azacitidine (doublet) is assessed in a mixed cohort of MDS, CMML frontline and HMA-failure patients or r/r AML patients. Dose determination may then be performed in separate indications. BEX plus azacitidine/venetoclax (triplet) will follow a similar BOIN design for dose determination in patients with newly diagnosed AML unfit for induction chemotherapy. BEX is dosed in 28-day cycles Q1W during cycles 1-3 followed by Q2W dosing, until disease progression or intolerable toxicity. Standard of care therapy is administered as per label. During PhII, selected indications are explored for efficacy when BEX is administered at recommended phase 2 dose (RP2D) following a 2-Stage design with indication-specific target efficacy rates. Key eligibility includes patient ≥ 18 years of age presenting with morphologically confirmed diagnosis of either MDS (intermediate-very high rIPSS risk), CMML-2 with indication for azacitidine treatment; CMML and MDS patients with failure to HMA therapy, confirmed diagnosis of r/r AML following at least 1 prior treatment line or diagnosis of AML in patients unfit for induction therapy with indication for azacitidine/venetoclax. Approximately up to 181 patients at 10 US and EU sites will be enrolled. Results: As of 13Feb2023, 14 patients have been treated with BEX in the 1 and 3mg/kg doublet cohorts (n=5 each) and the 1mg/kg triplet cohort (n=4). The majority of AEs are Grade 1-2 and no bexmarilimab-related Grade ≥3 AEs or DLTs have been reported. Preliminary efficacy of the 1mg/kg doublet patients showed CR in frontline MDS, PR in HMA failure MDS, CRi in r/rAML and SD in r/r AML (n=2). Biomarker data (n=4) shows up to 70% reduction of sClever-1 in blood and decreased Clever-1 expression on BM blasts after BEX treatment in patients of the 1mg/kg doublet cohort. Summary/Conclusion: The initial data show that BEX treatment is well-tolerated without additional toxicity to standard treatment. Preliminary efficacy shows 3 responses out of 5 patients in the first dose cohort. The study is ongoing.Keywords: Acute myeloid leukemia, MDS, Macrophage, Immunotherapy

Background: This study aims to explore patients' with acute myeloid leukemia perceptions about precision medicine and their preferences for involvement in this new area of shared decision-making.Methods: Individual semi-structured interviews were conducted in Finland, Italy and Germany (n = 16). The study population included patients aged 24-79 years. Interviews were analyzed with thematic content analysis.Results: Patient's perceived lack of knowledge as a barrier for their involvement in decision-making. Treatment decisions were often made rapidly based on the patient's intuition and trust for the physician rather than on information, in situations that decrease the patient's decision capacity. The patients emphasized that they are in a desperate situation that makes them willing to accept treatment with low probabilities of being cured.Conclusions: The study raised important issues regarding patients' understanding of precision medicine and challenges concerning how to involve patients in medical decision-making. Although technical advances were viewed positively, the role of the physician as an expert and person-of-trust cannot be replaced. Practice implications: Regardless of patients' preferences for involvement in decision-making, information plays a crucial role for patients' perceived involvement in their care. The concepts related to precision medicine are complex and will imply challenges to patient education.

Introduction: Bexmarilimab, a Clever-1 targeting humanized antibody, is a macrophage checkpoint inhibitor promoting antigen presentation and pro-inflammatory cytokine secretion. Data from the first-in-human clinical Phase I/II study (MATINS; NCT03733990) demonstrate that single agent bexmarilimab is able to ignite an interferon response with survival benefit in 30% of patients with advanced gastric cancer, cutaneous melanoma and cholangiocarcinoma (ASCO 2022). Since Clever-1 knockout mice have been reported to have enhanced T-cell antibody production and bexmarilimab increases peripheral B-cell populations, we investigated B-cell phenotype, clonality and autoantibody formation in bexmarilimab treatmented MATINS patients. Methods: We first performed comprehensive phenotyping of peripheral B-cells from a colorectal carcinoma patient showing partial response (per RECIST v1.1). Autoantibody reactivities were measured in 80 serum samples of bexmarilimab treated patients (37 pre-treatment and 43 post-treatment samples from cycles 3 and 4) and compared to serums from 53 healthy individuals using Oncimmune's SeroTag multiplex technology with an immune-oncology (IO) specific protein array comprised of 1162 antigens. Results: Single-cell sequencing together with BCR sequencing of the responder’s B-cells during cycle 4 revealed an induced and activated B-cell population consisting of four transcriptionally similar clusters expressing IGHM, IGHD, CD23 but not CD43 and one cluster expressing CD79B, PLD4 and MZB1, which was distant from the naïve CCR7 expressing B-cells and IGHA1 and IGHG1 expressing plasma blasts. The expansion of B-cells was not due to clonal expansion as no overlapping BCR clones were identified between B-cells at pre-dose and cycle 4. Autoantibody profiling revealed great inter-individual heterogeneity in the number and targets of induced antibodies among patients treated with bexmarilimab reflecting individual patterns in self- and tumor antigens. However, shared autoantibody changes against targets such as cancer testis antigens (GAGE2), typical autoimmune disease antigens (SNRPC, TPO, TOP1) and antigens related to the induction of innate immunity and interferon responses, e.g. TRIM21 were observed. Importantly, we identified a set of pre-treatment autoantibodies that were associated with longer time to disease progression and were predictive of clinical response (disease control rate [DCR], consisting of CR, PR, SD) and progression-free survival (PFS) on bexmarilimab therapy. Conclusions: Our data implies that bexmarilimab can induce activation and secondary Ig rearrangements in mature B-cells, which has been reported to occur in germinal centres during T-cell dependent antibody responses to increase B-cell diversity and affinity of antigen receptors. B cell diversity and activation was reflected in patient autoantibody production and may enhance cancer immune recognition. Citation Format: Elisa M. Vuorinen, Mari L. Björkman, Reetta Virtakoivu, Juho Jalkanen, Sofia Aakko, Akira Takeda, Petra Budde, Hans-Dieter Zucht, Manuel Brautigam, Behnaz Ahangarianabhari, Petri Bono, Maija Hollmén. Bexmarilimab induces B-cell activation and autoantibody production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2269.