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Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults. The disease course is typically aggressive and despite therapeutic advances only 30% of the patients will be long-term survivors. Emerging evidence suggests that immune evasion in AML favors relapse and could antagonize novel immunotherapeutic concepts [1].

Over the last years, myeloid derived suppressor cells (MDSCs) have been gaining momentum in cancer research as promoters of tumor immune escape. MDSCs represent a heterogeneous population that morphologically resembles monocytes or granulocytes sharing some features: myeloid origin, immature phenotype, and T-cell suppressive activity. Accumulating MDSCs have been described in AML patients [2], in myelodysplasia (MDS) [3], and in murine AML models [4]. In fact, AML-blasts hold the potential to induce MDSCs (from conventional monocytes) by exosomal transfer of MUC-1 [2]. These cells could contribute to immune escape partly explaining why AML-blasts despite expressing antigens recognizable to host T-cells (e.g. WT1) rarely are eradicated by the host’s immune system [5]. Targeting MDSCs in preclinical cancer models has shown efficacy in delaying disease thus suggesting further clinical exploitation [6].

Bispecific T-cell engaging (BiTE®) antibody constructs simultaneously target tumor antigens of interest and the T-cell receptor complex. T-cells can be recruited in an antigen-independent manner [7]. The first BiTE® developed against CD33, which is expressed on the majority of AML-blasts, is AMG 330 (Amgen, Thousand Oaks, CA). Preclinical studies revealed its capacity to recruit and to expand autologous T-cells leading to AML-blasts lysis [8, 9]. In fact, CD33 might have an advantage over other targets (e.g. CD123) since it is also expressed on monocytic MDSCs [10]. In this study we sought out to investigate whether AMG 330 could simultaneously confer two hits by redirecting T-cells against both CD33⁺ AML-blasts and CD33⁺ MDSCs thereby further enhancing anti-leukemic immune activity.

First, CD14⁺CD11b⁺CD33⁺ monocytic cells expressing low levels of HLA-DR (HLA-DR^lo) and resembling one of the most established human MDSC-like phenotype [11] as previously described by us in chronic lymphocytic leukemia (CLL) and malignant melanoma [10, 12] were quantified in the peripheral blood of patients with newly diagnosed AML. A representative flow cytometry (FACS)-based gating strategy is displayed in Fig. 1a, whereby AML-blasts were defined as CD117⁺ and/or CD34⁺ cells during initial AML diagnosis. The proportion of HLA-DR^lo cells among monocytes was significantly increased in AML patients as compared to healthy controls (HD) (28.98 ± 4.19%, n = 13 versus 3.28 ± 0.75%, n = 37) in line with previous observations [2]. In fact, MDSCs can be cytogenetically related to the malignant AML clone as recently reported [2]. Percentage of aberrant monocytes did not correlate (positively) with the frequency of circulating myeloid blasts or (negatively) with the frequency of T-cells in contrast to findings from CLL [10], B-NHL [13], and MDS [3] (Fig. 1b).

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Fig. 1

Primary AML-blasts and AML cell lines promote induction of CD14⁺ CD33⁺ IDO⁺ HLA-DR^lo MDSCs that are targeted by AMG 330. (a) A representative FACS-based analysis of MDSCs within AML patient-derived PBMCs is shown. HLA-DR expression was analyzed in CD14⁺ CD11b⁺ monocytic cells (right panels) among the viable (Zombie^neg) CD45^hi CD34^neg CD117^neg (left panels) cell fraction. Frequency of HLA-DR^lo cells was assessed in untreated AML patients (n = 13) and healthy controls (HD, n = 37). (b) Correlation of the proportion of HLA-DR^lo cells within the CD14⁺ population (n = 13) with frequency of circulating AML-blasts (upper panel) and of CD3⁺ T-cells (lower panel). (c) Expression levels of HLA-DR based on the median fluorescence index (MFI) were measured by FACS on HD-derived purified CD14⁺ monocytes following 5 days of culture in the presence/absence of AML cell lines (OCI-AML3, HL-60, and MOLM-13, n = 5) and primary AML-blasts (n = 9). Monocytes cultured alone are set as 100%. (d) The relative gene expression (mRNA) of indoleamine-2,3-dioxygenase (IDO) in monocytes cultured in the presence/absence of AML cell lines (cAML, n = 6) and primary AML-blasts (pAML, n = 10) was semi-quantified by qPCR. Monocytes cultured alone are set as 1. A representative (for n = 10) histogram of a FACS analysis of IDO expression in monocytes cultured alone (control) or in presence of primary AML-blasts as shown for cells from the patient with the unique patient number 735 (AML⁷³⁵). (e) The dose-dependent suppressive activity of HD-derived monocytes and HD-derived monocytes re-educated by cAML (=induced MDSCs (iMDSCs)) was evaluated in co-cultures with VPD450-labeled autologous T-cells activated using anti-CD2, -CD3, and -CD28 microbeads. T-cell proliferation was assessed based on the VPD450 dye dilution after 5 days by FACS and compared with stimulated T-cells alone (set as 100%). (f) Suppressive activity of FACS-sorted HLA-DR^hi and HLA-DR^lo iMDSCs was separately evaluated in co-culture experiments (n = 4) with autologous T-cells. (g) Cell surface expression of CD33 was semi-quantified by FACS on HLA-DR^lo and HLA-DR^hi CD14⁺ monocytes (n = 10). (h) Representative (for at least three independent experiments) FACS-imaging of cytotoxic CD8⁺ T-cells (red) conjugated with autologous CD14⁺ monocytes (yellow). Nucleic acids were counter-stained with Syto13 (green), actin with phalloidin (grey), and perforin formation additionally visualized (pink). (i) Calcein-labeled monocytes or iMDSCs (n = 10) were co-cultured with pre-stimulated autologous T-cells at a 1:10 ratio for three hours and the release of calcein measured using fluorimetric assay. Bars indicate the standard error of the mean. Abbreviations: *, p < 0.05; **, p < 0.01; ***, p < 0.001

CD14⁺ monocytes isolated from HD were co-cultured for three to five days with AML cell-lines (OCI-AML3, HL-60, and MOLM-13/cAML) or primary AML-blasts (pAML) that were previously labeled with a vital dye for better discriminating both populations in ultra-low attachment surface plates allowing full recovery of monocytes. Presence of AML-blasts led (at day five) to a significant reduction of HLA-DR expression in CD14⁺ monocytes (Fig. 1c). Previous studies as for example in CLL and in patients following allogeneic stem cell transplantation have shown that the monocytic MDSCs can express indoleamine-2,3-dioxygenase (IDO) [10, 14]. In fact, IDO-mediated tryptophan depletion and production of kynurenine can modulate T-cell responses. Furthermore, IDO has been shown to negatively impact efficacy of immune-based therapies such as of T-cells carrying chimeric antigen receptors [15] while high kynurenine concentrations predict an unfavorable outcome in AML patients [16]. We detected a significant IDO upregulation on the gene expression and protein level in monocytes upon contact to cAML- and pAML-blasts (Fig. 1d). As anticipated, the functional assessment of AML-educated monocytes co-cultured at different ratios with activated autologous T-cells revealed a strong T-cell suppressive activity (as compared to non-AML-educated monocytes), which is in line with their MDSC-like phenotype (Fig. 1e) and with previous observations in AML [2], allowing us to denominate them as induced MDSCs (iMDSCs). Next, we separated the HLA-DR^hi and HLA-DR^lo fraction among the bulk of AML-educated monocytes using FACS-based cell sorting and then repeated the T-cell suppression assays. Here, we observed that the HLA-DR^lo subset still holds the strongest T-cell suppressive capacity further confirming the enhanced regulatory features of HLA-DR^lo monocytic iMDSCs (Fig. 1f).

We further assessed whether T-cells can be engaged by AMG 330 to target autologous monocytes and/or T-cell suppressive (AML-educated) iMDSCs. Noticeably, previous studies have shown that AMG 330-mediated lysis is co-determined by the cell surface CD33 levels [8, 9]. The median fluorescence intensity (MFI) of CD33 as assessed by flow cytometry was comparable in both the HLA-DR^lo (=MDSCs) and HLA-DR^hi CD14⁺ subsets (Fig. 1g). Purified CD3⁺ T-cells engaged by AMG 330 were able to form immunological synapses with autologous CD14⁺ cells as revealed by the F-actin and perforin polarization (Fig. 1h) and which is an important determinant for the efficacy T-cell based immune therapies [17]. Specific lysis of calcein-labeled HD-derived monocytes triggered by AMG 330 in presence of autologous T-cells was at comparable levels as for their iMDSC counterparts that had been previously educated by AML cell-lines and despite their elevated IDO expression and their T-cell suppressive activity (Fig. 1d-e, i).

In order to validate the AMG 330-triggered redirection of T-cells towards CD33⁺ AML-blasts AML-PBMC samples from newly diagnosed patients were used for short-term (three to six days) cell cultures in presence of control BiTE® constructs or AMG 330. In line with previous reports [8, 9], AMG 330 treatment resulted in an efficient elimination of CD33⁺ AML-blasts and the concomitant expansion of residual autologous T-cells (Fig. 2a). Most parameters that are indicative for T-cell activation (e.g. CD25 and CD69), cytotoxic activity (e.g. granzyme B and CD107), or cytokine production (e.g. IL2 and IFNγ) as well as the bystander activation of NK-cells (by amongst others abundant pro-inflammatory cytokines) were found upregulated upon AMG 330 application when phenotypically analyzing T- and NK-cells within the AML-PBMCs by FACS (Fig. 2b). The initial T-cell frequency within the PBMCs ranging from 0.16 to 14.30% and/or the initial MDSC levels had both no impact on the assessed levels of T-cell responsiveness (Additional file 1: Tables S1 and S2).

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Fig. 2

AMG 330 triggers T-cell-mediated lysis of AML-blasts that is further enhanced by MDSC depletion. (a) The absolute number of CD33⁺ AML-blasts and CD3⁺ T-cells was quantified in patient-derived AML PBMCs (n = 10) after 6 days of treatment with control BiTE® antibodies (c) or AMG 330. (b) AML-derived PBMCs (n = 12) were treated with control BiTE® antibodies or AMG 330 for three days. The median fluorescence intensity (MFI) of TNFα, granzyme B (grz B), CD107, perforin, CD69, CD137, CD25, CD154, IL2, and IFNγ was assessed by FACS in CD4⁺/CD8⁺ CD3⁺ T-cells and CD56⁺CD3^neg NK-cells as indicated. The cells’ MFI from samples treated with control antibodies was set as 1. (c) CD33 surface antigen quantification was performed for AML-blasts and CD14⁺ monocytes (n = 8). (d) AML-derived PBMCs (n = 5) were treated with 10 pM AMG 330 in the presence or absence of the IDO inhibitor epacadostat (1 μM) and the number of CD33⁺ AML-blasts quantified. The graph displays the individual %al changes in cell numbers in presence of epacadostat. (e) AML PBMCs (n = 7) with/without prior depletion of CD14⁺ cells were treated with AMG 330 for three days. Expansion index and MFI of CD69, CD137, CD25, CD154, IL2, and IFNγ were assessed by FACS in VPD450-labeled CD4⁺/CD8⁺ CD3⁺ T-cells. Samples without depletion of CD14⁺ cells were set as 1. (f) AML-derived PBMCs (n = 5) with/without prior depletion of CD14⁺ cells were treated with AMG 330 for six days. LDH release as a surrogate for cell lysis was measured in the cultures’ supernatants. (g) Calcein-labeled MOLM-13 cells (MOLM) were co-cultured with T-cells alone (upper illustration) or with T-cells together with autologous monocytes or AML-educated iMDSCs (n = 5) +/− AMG 330 (lower illustration). Specific lysis of MOLM-13 cells was assessed after 3 h. (h) Calcein-labeled monocytes or iMDSCs (n = 4) were co-cultured with autologous T-cells and MOLM-13 cells +/− AMG 330. Specific lysis of monocytes/iMDSCs was assessed after 3 h. Bars indicate the standard error of the mean. Abbreviations: *, p < 0.05; **, p < 0.01; ***, p < 0.001

We next hypothesized that CD33⁺IDO⁺ MDSCs might antagonize AMG 330 efficacy by (A) competing over the target antigen CD33 or by (B) suppressing successfully recruited T-cells (by IDO). Using a FACS-based indirect QIFIKIT® immunofluorescence assay we quantified the CD33 cell surface antigen density (which is comparable for monocytes and AML-MDSCs, Fig. 1g) on primary AML-blasts and on CD14⁺ cells and indeed observed higher (potentially competing) CD33 levels on CD14⁺ cells (Fig. 2c). Concomitant blocking of the IDO activity using epacadostat in AML patient-derived PBMCs treated with AMG 330 resulted in a significantly enhanced reduction of the CD33⁺ cell count (Fig. 2d). Depleting all CD14⁺ cells (including monocytic MDSCs) in AML-PBMCs prior to AMG 330 treatment did not have a detectable effect on T-cell activation or their production of cytokines as well as T-cell expansion (Fig. 2e), which has been shown to be highly relevant for the clinical activity of BiTE® antibody constructs [18]. However, removal of all CD14⁺ cells led to an increased AMG 330-mediated lysis (Fig. 2f). For further investigating this phenomenon (in terms of being total CD14⁺ cell- or rather MDSC-driven), we cultured MOLM13 cells (cAML) with HD-derived T-cells and added AMG 330 in the presence of autologous HD-derived CD33⁺ monocytes or CD33⁺ iMDSCs. Co-cultures were performed for three days for preventing a reprogramming of the conventional monocytes into MDSCs that was occurring at day five (Fig. 1c and d). We observed a reduced specific lysis of MOLM13 cells only in co-cultures with iMDSCs (Fig. 2g) suggesting (at least ex vivo) a specifically MDSC-mediated (presumably only transient until MDSCs are eliminated by the redirected T-cells) reduction of AMG 330 efficacy most likely due to the MDSCs’ direct T-cell suppressive activity and not due to competition over the target antigen CD33 (which is found on both conventional monocytes and iMDSCs). At the same time BiTE-triggered lysis of iMDSCs and monocytes as shown in Fig. 1i remained unaffected in presence of MOLM13 AML-cells (Fig. 2h).

Taken together, our preclinical data suggests that AMG 330 could achieve anti-leukemic activity not only through direct engagement of T-cells but also via targeting of CD33⁺ monocytic MDSCs [1]. In accordance with our findings, recent preliminary data indicate that elimination of MDSCs by the bispecific CD33/CD3 T-cell engager AMV564 also restores immune homeostasis in MDS [19]. Furthermore, it remains to be elucidated whether MDSCs impact in vivo (at least temporarily until their AMG 330-triggered elimination) AMG 330 efficacy. In the latter case, MDSC levels could represent a biomarker for the patients’ clinical responsiveness towards an AMG 330-based therapy in analogy to observations from other immunotherapies such as peptide vaccination in renal cancer [20].