Loss of anticancer NK cell function in AML patients is associated with fatal disease progression and remains poorly understood. Here, we demonstrate that AML-blasts isolated from patients rapidly inhibit NK cell function and escape NK cell-mediated killing. Transcriptome analysis of NK cells exposed to AML-blasts revealed increased CREM expression and transcriptional activity, indicating enhanced cAMP signalling, confirmed by uniform production of the cAMP-inducing prostanoid PGE2 by all AML-blast isolates from patients. Phosphoproteome analysis disclosed that PGE2 induced a blockade of LCK-ERK signalling that is crucial for NK cell activation, indicating a two-layered escape of AML-blasts with low expression of NK cell-activating ligands and inhibition of NK cell signalling. To evaluate the therapeutic potential to target PGE2 inhibition, we combined Fcg-receptor-mediated activation with the prevention of inhibitory PGE2-signalling. This rescued NK cell function and restored the killing of AML-blasts. Thus, we identify the PGE2-LCK signalling axis as the key barrier for NK cell activation in two-layered immune escape of AML-blasts that can be targeted for immune therapy to reconstitute anti-cancer NK cell immunity in AML patients.

Supplementary Methods. Supplementary Table 1 - Key Resources Including Sources of Biological Samples, Antibodies, and Key Reagents. Supplementary Table 2 -Correlation of FLT3 Expression Levels With Disease Characteristics at Initial Diagnosis. Supplementary Table 3 - Summary of Human Nonimmune Tissues Positive for FLT3 mRNA and/or Protein Expression in At Least One Platform. Supplementary Table 4. Cerebellum FLT3 RNA Analysis. Supplementary Table 5 - Binding Affinities of Experimental FLT3 BiTE® Molecule and AMG 427. Supplementary Table 6 - Cell Line Characterization, Cytotoxicity, and T-Cell Activation (Human). Supplementary Table 7 - Cynomolgus Monkey Pharmacokinetics for Experimental FLT3 BiTE® Molecule. Supplementary Table 8 - Cynomolgus Monkey Pharmacokinetics for AMG 427. Supplementary Table 9 - Cynomolgus Monkey FLT3 Primer and Probe Sequences. Supplementary Table 10 - Patient Characteristics. Supplementary Table 11 - Mass Spectrometry Results. Supplementary Figure 1 - Additional flow cytometry expression analysis for acute myeloid leukemia (AML) and healthy donor (HD) bone marrow (BM). Supplementary Figure 2 - Analysis of FLT3 expression on normal human bone marrow. Supplementary Figure 3 - Analysis of FLT3 transcript and protein in cerebellum revealed truncated transcripts. Supplementary Figure 4 - In vitro characterization of FLT3 BiTE® molecules. Supplementary Figure 5 - Experimental FLT3 BiTE® molecule activity in vivo. Supplementary Figure 6 - FLT3 BiTE® molecule in vitro activity with cynomolgus monkey effector cells. Supplementary Figure 7 - FLT3 expression in cynomolgus monkey blood and bone marrow. Supplementary Figure 8 - AMG 427 pharmacodynamics in cynomolgus monkey. Supplementary Figure 9 - (A) Representative populations of T cells (CD2+) and AML cells (CD33+) over time from a responder (Patient 4; Figure 6A). (B) PD-1 expression on CD3+ cells from a T-cell-dependent cellular cytotoxicity (TDCC) of human pan T cells co-cultured 5:1 with MV4-11 cells for 48 hours assessed by flow cytometry.

Background Immunotherapy of acute myeloid leukemia has experienced considerable advances, however novel target antigens continue to be sought after. To this end, unbiased approaches for surface protein detection are limited and integration with other data types, such as gene expression and somatic mutational burden, are poorly utilized. The Cell Surface Capture technology provides an unbiased, discovery-driven approach to map the surface proteins on cells of interest. Yet, direct utilization of primary patient samples has been limited by the considerable number of viable cells needed. Methods Here, we optimized the Cell Surface Capture protocol to enable direct interrogation of primary patient samples and applied our optimized protocol to a set of samples from patients with acute myeloid leukemia (AML) to generate the AML surfaceome. We then further curated this AML surfaceome to exclude antigens expressed on healthy tissues and integrated mutational burden data from hematologic cancers to further enrich for targets which are likely to be essential to leukemia biology. Finally, we validated our findings in a separate cohort of AML patient samples. Results Our protocol modifications allowed us to double the yield in identified proteins and increased the specificity from 54 to 80.4% compared to previous approaches. Using primary AML patient samples, we were able to identify a total of 621 surface proteins comprising the AML surfaceome. We integrated this data with gene expression and mutational burden data to curate a set of robust putative target antigens. Seventy-six proteins were selected as potential candidates for further investigation of which we validated the most promising novel candidate markers, and identified CD148, ITGA4 and Integrin beta-7 as promising targets in AML. Integrin beta-7 showed the most promising combination of expression in patient AML samples, and low or absent expression on healthy hematopoietic tissue. Conclusion Taken together, we demonstrate the feasibility of a highly optimized surfaceome detection method to interrogate the entire AML surfaceome directly from primary patient samples and integrate this data with gene expression and mutational burden data to achieve a robust, multiomic target identification platform. This approach has the potential to accelerate the unbiased target identification for immunotherapy of AML.

Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).

Bettina Brauchle 1,2,† , Rebecca L. Goldstein 3,† , Christine M. Karbowski 4 , Anja Henn 5 , Chi-Ming Li 3 , Veit L. Bücklein 1,2 , Christina Krupka 1,2 , Michael C. Boyle 4 , Priya Koppikar 4 , Sascha Haubner 1,2 , Joachim Wahl 5 , Christoph Dahlhoff 5 , Tobias Raum 5 , Matthew J. Rardin 3 , Christine Sastri 3 , Dan A. Rock 3 , Michael von Bergwelt-Baildon 1,2,6 , Brendon Frank 3 , Klaus H. Metzeler 2,6 , Ryan Case 3 , Matthias Friedrich 5 , Mercedesz Balazs 3 , Karsten Spiekermann 2,6,7 , Angela Coxon 5 , Marion Subklewe 1,2,6,* , Tara Arvedson 3,*

Abstract Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell–dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo, and ex vivo. FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro, reduced tumor growth and increased survival in AML mouse models in vivo. Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo, including elimination of FLT3-positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.

Background:T cell recruiting antibody constructs represent a novel platform for immunotherapy of acute myeloid leukemia (AML). We previously characterized FLT3 (CD135; FMS‐like tyrosine kinase 3) as a suitable target antigen for treatment of AML based on significantly higher expression on primary AML (pAML) cells in comparison to healthy hematopoietic subpopulations (Lindl ASH 2017).Aims:Here, we evaluated T cell‐redirected target cell lysis mediated by a FLT3 BiTE® antibody construct (FLT3 BiTE®) against AML cells in an ex vivo model for pAML cells, an in vivo NOD‐SCID mouse admixture model and in non human primates (NHP). We hypothesized that combining FLT3 BiTE® with a tyrosine kinase inhibitor (TKI) would enhance target cell lysis. Accordingly, FLT3 expression, mutational status, and TKI‐mediated T‐cell dysfunction were analyzed as relevant factors for FLT3 BiTE®‐mediated cytotoxicity.Methods:FLT3 BiTE®‐mediated cytotoxicity was evaluated by coculture of various FLT3+ AML cell lines with healthy donor (HD) T cells. Cytotoxicity against pAML cells and T‐cell proliferation were tested ex vivo using a long‐term culture system (Krupka Blood 2014) and assessed by flow cytometry. The Effector:Target cell ratio was based on residual T cells within the sample.In vivo, a NOD‐SCID mouse admixture model with human CD3+ T cells and MOLM‐13 cells was used. In NHP, FLT3 BiTE® was administered with step‐dosing at 5, 15, 45, and 100 μg/kg/d in a 16‐day continuous intravenous study. Pharmacodynamics were determined by measuring blood (PB) and bone marrow (BM) FLT3 transcript and soluble FLT3 ligand.TKI‐mediated modulation of T‐cell function was assessed by CD3/CD28‐bead stimulation of CFSE‐labeled HD T cells. Cytotoxicity of 3 TKIs against AML cell lines with different FLT3‐ITD mutational status was evaluated.Last, the combination of TKI with FLT3 BiTE® was tested in cytotoxicity assays using HD T cells with FLT3‐ITD+ AML cell lines or pAML cells.Results:FLT3 BiTE® exhibited cytotoxicity against FLT3+ AML cell lines at picomolar concentrations irrespective of FLT3 mutational status (EC50:1.9 ± 0.7pM, ± SD, n = 6). No cytotoxicity was observed against FLT3− cell lines. pAML cells were lysed (%specific lysis day 9: 43 ± 9%, ± SEM, n = 14) even at low E:T ratios (1:3–1:74) accompanied by strong T‐cell proliferation (fold change CD2+ day 9: 5.9 ± 1.8, ± SEM) independent of FLT3 mutational status. In mice treated intraperitoneally with 200 μg/kg/d FLT3 BiTE®, reduction in tumor volume was observed (tumor volume d15 FLT3 BiTE® vs control BiTE®: 221 vs 1431 mm3, p≤0.0001, n = 10 per group). Furthermore, a decrease in FLT3 transcript levels (d17: 92.1 ± 0.1%, ± SD, n = 3) as well as a time‐dependent increase in circulating soluble FLT3 ligand levels were observed in PB and BM samples from NHP, supporting a FLT3 BiTE®‐mediated decrease in FLT3+cells.Pharmacologically relevant concentrations of TKIs induced cytotoxicity against FLT3‐ITD+ AML cell lines (Table, top) without altering T‐cell proliferation. Higher concentrations of TKI led to a decrease in T‐cell proliferation (Table, middle).Next, we combined TKIs with FLT3 BiTE®, resulting in increased cytotoxicity against FLT3‐ITD+ AML cell lines (p = 0.03, n = 6) as well as pAML cells in 2 FLT3‐ITD+ patients (Table, bottom).Summary/Conclusion:The FLT3 BiTE® antibody construct showed strong cytotoxicity against FLT3+ cells in vitro and ex vivo, mediated antitumor activity and depleted FLT3+ cells in vivo. The addition of clinically relevant concentrations of TKI enhanced FLT3‐BiTE®‐mediated cytotoxicity in FLT3‐ITD+ AML while higher TKI concentrations compromised T‐cell activity.image

The CD33-targeting bispecific T-cell engager (BiTE) AMG 330 proved to be highly efficient in mediating cytolysis of acute myeloid leukemia (AML) cells in vitro and in mouse models. Yet, T-cell activation is correlated with upregulation of programmed cell death-ligand 1 (PD-L1) and other inhibitory checkpoints on AML cells that confer adaptive immune resistance. PD-1 and PD-L1 blocking agents may counteract T-cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a bifunctional checkpoint inhibitory T cell-engaging (CiTE) antibody that combines T-cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade. This is accomplished by fusing the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1, to an αCD3.αCD33 BiTE-like scaffold. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increases T-cell activation (3.3-fold elevation of interferon-γ) and leads to efficient and highly selective cytotoxicity against CD33+PD-L1+ cell lines (50% effective concentration = 2.3-26.9 pM) as well as patient-derived AML cells (n = 8). In a murine xenograft model, the CiTE induces complete AML eradication without initial signs of irAEs as measured by body weight loss. We conclude that our molecule preferentially targets AML cells, whereas high-affinity blockers, such as clinically approved anticancer agents, also address PD-L1+ non-AML cells. By combining the high efficacy of T-cell engagers with immune checkpoint blockade in a single molecule, we expect to minimize irAEs associated with the systemic application of immune checkpoint inhibitors and suggest high therapeutic potential, particularly for patients with relapsed/ refractory AML.

The CD33-targeting bispecific T cell engager (BiTE®) AMG 330 proved to be highly efficient in mediating cytotoxicity of AML cells in vitro and in mouse models (Krupka et al, Blood 2014). Yet, T cell activation is correlated with the upregulation of PD-L1 and other inhibitory checkpoint molecules on AML cells that confer adaptive immune resistance (Krupka et al, Leukemia 2016). PD-1/PD-L1 blocking agents may counteract T cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a checkpoint inhibitory T cell engaging (CiTE) antibody that combines T cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade. CiTE constructs were generated by first fusing a high-affinity CD33 single-chain variable fragment (scFv) to a CD3ε scFv in one polypeptide chain. Next, this single-chain chain was fused to the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1. Antigen binding of CiTE constructs as well as CiTE mediated cytotoxicity of AML cell lines and primary AML cells were done using multiparameter flow cytometry. T cell activation and cytotoxicity assays were complemented by cytometric bead arrays. Murine AML xenograft studies using non-obese diabetic (NOD) scid gamma mice were used for engraftment of primary AML cells and assessment of CiTE mediated cytotoxicity in vivo. CiTE antibody constructs were successfully generated by fusing the bispecific CD33-CD3ε scFv to the endogenous extracellular domain of human PD-1 (PD-1ex). The CiTE was compared to a single chain triplebody (sctb), in which PD-1ex was replaced by a high-affinity PD-L1 scFv. The BiTE-like molecule, PD-1ex.αCD3 and αPD-L1.αCD3, as well as a non-targeting molecule served as controls. When investigating CiTE and sctb as whole molecules, both bound with similar affinities to CD33+PD-L1+ AML cell lines and HD T cells. CiTE- and sctb-induced upregulation of CD69 and CD25 on healthy donor T cells in the presence of MOLM-13-PD-L1 cells. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increased T cell activation (3.3-fold elevation of IFN-γ release) and lead to efficient and highly selective cytotoxicity of CD33+PD-L1+ cells (EC50 = 2.3 pM to 26.9 pM) as well as primary AML patient samples (n=8). CiTE induced preferential lysis of CD33+PD-L1+ cells and had no activity against CD33-PD-L1+ cells. This was supported by the observation that the CiTE molecule was able to selectively induce elimination of CD33+PD-L1+ cells in the presence of PD-L1+ cells. In a murine xenograft model, the CiTE induced complete AML eradication without causing leukemia-unrelated T cell activation or body weight loss. Notably, murine and human PD-L1 bind with similar affinities to PD-1. We conclude that our molecule preferentially targets CD33+PD-L1+ AML cells, whereas high-affinity blocking agents also address PD-L1+ non-AML cells. Based on these findings, we expect to reverse adaptive immune escape mechanisms of T cell recruiting antibody formats and avoid irAEs associated with systemic checkpoint blockade, suggesting efficient therapeutic potential particularly for patients with relapsed or refractory AML. Future studies will need to further examine efficiency and tolerance in advanced in vivo models before applying the CiTE format into a clinical setting. Lindl: Amgen: Research Funding. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy. Subklewe:Gilead: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

A number of agents designed for immunotherapy of Acute Myeloid Leukemia (AML) are in preclinical and early clinical development. Most of them target a single antigen on the surface of AML cells. Here we describe the development and key biological properties of a tri-specific agent, the dual-targeting triplebody SPM-2, with binding sites for target antigens CD33 and CD123, and for CD16 to engage NK cells as cytolytic effectors. Primary blasts of nearly all AML patients carry at least one of these target antigens and the pair is particularly promising for the elimination of blasts and leukemia stem cells (LSCs) from a majority of AML patients by dual-targeting agents. The cytolytic activity of NK cells mediated by SPM-2 was analyzed in vitro for primary leukemic cells from 29 patients with a broad range of AML-subtypes. Blasts from all 29 patients, including patients with genomic alterations associated with an unfavorable genetic subtype, were lysed at nanomolar concentrations of SPM-2. Maximum susceptibility was observed for cells with a combined density of CD33 and CD123 above 10,000 copies/cell. Cell populations enriched for AML-LSCs (CD34pos and CD34pos CD38neg cells) from 2 AML patients carried an increased combined antigen density and were lysed at correspondingly lower concentrations of SPM-2 than unsorted blasts. These initial findings raise the expectation that SPM-2 may also be capable of eliminating AML-LSCs and thus of prolonging survival. In the future, patients with a broad range of AML subtypes may benefit from treatment with SPM-2.

Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML ( n = 356) and normal bone marrow samples ( n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis ( n = 302) and relapse ( n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations ( n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor–ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4+ and CD8+ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.

Acute myeloid leukemia (AML) is a disease with high unmet medical need. Standard induction chemotherapy can lead to complete remission in patients with a favorable genetic risk profile; however, about half of patients are ineligible for this intensive regimen. In those patients with a non-favorable genetic risk profile, the preferred treatment strategy is allogeneic stem cell transfer, but the majority of patients are ineligible due to age and other comorbidities.

The advent of new immunotherapeutic agents in clinical practice has revolutionized cancer treatment in the past decade, both in oncology and hematology. The transfer of the immunotherapeutic concepts to the treatment of acute myeloid leukemia (AML) is hampered by various characteristics of the disease, including non-leukemia-restricted target antigen expression profile, low endogenous immune responses, and intrinsic resistance mechanisms of the leukemic blasts against immune responses. However, considerable progress has been made in this field in the past few years.Within this manuscript, we review the recent developments and the current status of the five currently most prominent immunotherapeutic concepts: (1) antibody-drug conjugates, (2) T cell-recruiting antibody constructs, (3) chimeric antigen receptor (CAR) T cells, (4) checkpoint inhibitors, and (5) dendritic cell vaccination. We focus on the clinical data that has been published so far, both for newly diagnosed and refractory/relapsed AML, but omitting immunotherapeutic concepts in conjunction with hematopoietic stem cell transplantation. Besides, we have included important clinical trials that are currently running or have recently been completed but are still lacking full publication of their results.While each of the concepts has its particular merits and inherent problems, the field of immunotherapy of AML seems to have taken some significant steps forward. Results of currently running trials will reveal the direction of further development including approaches combining two or more of these concepts.

Antibody-based immunotherapy represents a promising strategy to eliminate chemorefractory leukemic cells in acute myeloid leukemia (AML). In this study, we evaluated a novel Fc-engineered antibody against CD157 (MEN1112) for its suitability as immunotherapy in AML. CD157 was expressed in 97% of primary AML patient samples. A significant, albeit lower expression level of CD157 was observed within the compartment of leukemia-initiating cells, which are supposed to be the major source of relapse. In healthy donor bone marrow, CD157 was expressed on CD34+ cells. In ex vivo assays, MEN1112 triggered natural killer (NK) cell-mediated cytotoxicity against AML cell lines and primary AML cells. Compared to its parental analogue, the Fc-engineered antibody exhibited higher antibody dependent cellular cytotoxicity responses. Using NK cells from AML patients, we observed heterogeneous MEN1112-mediated cytotoxicity against AML cells, most likely due to well-documented defects in AML-NK cells and corresponding inter-patient variations in NK cell function. Cytotoxicity could not be correlated to the time after completion of chemotherapy. In summary, we could demonstrate that CD157 is strongly expressed in AML. MEN1112 is a promising antibody construct that showed high cytotoxicity against AML cells and warrants further clinical testing. Due to variability in NK-cell function of AML patients, the time of application during the course of the disease as well as combinatorial strategies might influence treatment results.

T-cell recruiting antibody constructs represent a promising approach to recruit T-cells to tumor cells independent of TCR-specificity. So far, CD33 has been the most commonly targeted antigen in AML. However, its ubiquitous expression pattern within the healthy myeloid hematopoietic system remains a major challenge. To reduce on-target off-leukemia toxicity, alternative AML-associated targets with a more restricted expression pattern are sought after. We evaluated CD135 (FLT3 ; FSM-like tyrosine kinase 3) as a target antigen for T-cell recruiting antibody based immunotherapy in AML. In 159/192 of AML patient samples (83%; MFI ratio of >1.5) a significant expression level was detected by flow cytometry with no differences in expression level at time of initial diagnosis versus relapse (Initial diagnosis: n=161, median MFI ratio 3.0 vs relapse: n=18, median MFI ratio 3.5). Expression level could neither be correlated to FLT3 mutational status (median MFI ratio FLT3-ITD vs FLT3 wild type (WT): 3.7 vs 4.0, n=52 and 173 respectively) nor to FLT3 allelic ratio (median MFI ratio allelic ratio high vs low: 4.1 vs 4.5, n=13 and 19 respectively). Importantly, CD34+/CD38- leukemia initiating cells (LICs) also expressed CD135 at a similar level compared to AML bulk cells (Median MFI ratio LIC: 2.4 vs bulk: 2.6, p=0.1, n=81). Bone marrow progenitor cells (CD45DIM/SSCLOW) cells and CD34+/CD38- expressing hematopoietic precursors from healthy bone marrow expressed CD135 to a significantly lower level compared to AML bulk cells and LICs, respectively (median MFI Ratio: AML bulk vs healthy progenitor cells: 3.2 vs 2.1, p<0.0001; LICs vs HSCs: 3.1 vs 0.7, p=0.0003). Next, we evaluated a CD135xCD3 BiTE® antibody construct (FLT3 BiTE®) for its ability to mediate cytotoxicity against AML cell lines and primary AML cells. Target antigen expression level was relevant for FLT3 BiTE® mediated cytotoxicity as demonstrated by preferential lysis of CD135bright (MM6, MFI ratio 15) vs CD135dim (OCI-AML3, MFI ratio 1.5) AML cell lines. Cytotoxicity against primary AML cells was tested in our long-term culture system using either allogeneic or autologous T cells (Krupka et al, Blood 2014). In the allogeneic set-up, with a fixed E:T ratio of 1:5, the FLT3 BiTE® antibody construct mediated efficient elimination of primary AML cells within 9 days of culture (median % specific lysis 75.9, n=6). This was accompanied by a strong T-cell proliferation (fold change CD2+ T cells: 7.9, n=6). In the autologous system, the E:T ratio was not fixed and influenced by the number of residual T-cells in the respective AML sample. Despite low E:T ratios (1:12-1:74) the FLT3 BiTE® induced high cytotoxicity and T-cell proliferation against primary AML cells within 14 days of culture (median specific lysis: 34.4%; fold change CD2 T cells: 6.6, n=7). We further investigated the influence of Midostaurin on FLT3 BiTE® mediated cytotoxicity against AML cells. Midostaurin (1 µM) drastically decreased FLT3 BiTE® mediated cytotoxicity in 7/8 patient samples (median % cytotoxicity FLT3 BiTE® vs +Midostaurin: 59.0 vs 39.9, p=0.02). This corresponded to a significant decrease in BiTE®- as well as CD3/CD28-mediated T-cell proliferation (median % CD3/CD28 mediated T-cell proliferation FLT3 BiTE® vs +Midostaurin: 56.4 vs 1.46, n=5). It has been reported that Midostaurin inhibits Akt phosphorylation (Kawai, J Bio Sci 2015), a major factor in T-cell proliferation, which might explain the decrease in T-cell proliferation and cytotoxicity. In summary, CD135 was shown to be expressed in the majority of AML patients on AML bulk cells as well as LICs with a restricted expression pattern in the healthy hematopoietic compartment. The FLT3 BiTE® antibody construct mediated strong cytotoxicity against primary AML cells. This correlated to profound T-cell activation and proliferation. The combinatorial approach with Midostaurin abrogated BiTE® mediated cytotoxicity due to a decrease in T-cell proliferation. As T-cell recruiting antibody construct mediated cytotoxicity relies on T-cell function, our data do not support the combinatorial approach with the FLT3 -TKI Midostaurin for targeted therapy of AML.

Background: Despite considerable advances in the field of immunotherapy of acute myeloid leukemia, on-target off-leukemia toxicity remains a challenge. Thus, novel target antigens are sought after. The Cell Surface Capture (CSC) technology enables a discovery-driven approach to detect surface proteins. However, direct processing of primary samples has been limited by the substantial number of viable cells needed for the CSC-workflow. We therefore addressed this issue by improving the technique including the use of our ex vivo culture system. This allowed for an unbiased, direct assessment of primary AML patient samples and thus enabled us to interrogate the AML surfaceome in clinically relevant samples.