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. 2018 Nov 5;6(1):116.
doi: 10.1186/s40425-018-0432-9.

CD33/CD3-bispecific T-cell engaging (BiTE®) antibody construct targets monocytic AML myeloid-derived suppressor cells

Regina Jitschin  1 , Domenica Saul  1 , Martina Braun  1 , Sehmus Tohumeken  1 , Simon Völkl  1 , Roman Kischel  2 , Michael Lutteropp  2 , Cedric Dos Santos  3 , Andreas Mackensen  1 , Dimitrios Mougiakakos  4
Affiliations

CD33/CD3-bispecific T-cell engaging (BiTE®) antibody construct targets monocytic AML myeloid-derived suppressor cells

Regina Jitschin et al. J Immunother Cancer. .

Abstract

Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14+ monocytic MDSCs in newly diagnosed AML that co-express CD33 but lack HLA-DR (HLA-DRlo). AML-blasts induce HLA-DRlo cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33+ MDSCs. In fact, T-cells eliminate IDO+CD33+ MDSCs in the presence of AMG 330. Depletion of total CD14+ cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33+ MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy.

Keywords: Acute myeloid leukemia; Bispecific antibodies; Myeloid derived suppressor cells.

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Conflict of interest statement

Ethics approval and consent to participate

All samples were collected upon approval by the local ethics committee (number: 3779/138_17B) and patients’ informed consent.

Consent for publication

Not applicable.

Competing interests

R.K. and M.L. are employed by Amgen Research (Munich) GmbH. C.D.S. is employed by Amgen Inc. R.J., A.M., and D.M. were supported by research funding from Amgen Inc. The remaining authors declare no conflict of interest.

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Figures

Fig. 1
Fig. 1
Primary AML-blasts and AML cell lines promote induction of CD14+ CD33+ IDO+ HLA-DRlo 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 (Zombieneg) CD45hi CD34neg CD117neg (left panels) cell fraction. Frequency of HLA-DRlo cells was assessed in untreated AML patients (n = 13) and healthy controls (HD, n = 37). (b) Correlation of the proportion of HLA-DRlo 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-DRhi and HLA-DRlo 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-DRlo and HLA-DRhi 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
Fig. 2
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+CD3neg 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
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References

    1. Andersen MH. The targeting of immunosuppressive mechanisms in hematological malignancies. Leukemia. 2014;28(9):1784–1792. doi: 10.1038/leu.2014.108. - DOI - PubMed
    1. Pyzer AR, Stroopinsky D, Rajabi H, Washington A, Tagde A, Coll M, et al. MUC1-mediated induction of myeloid-derived suppressor cells in patients with acute myeloid leukemia. Blood. 2017;129(13):1791–1801. doi: 10.1182/blood-2016-07-730614. - DOI - PMC - PubMed
    1. Gleason MK, Ross JA, Warlick ED, Lund TC, Verneris MR, Wiernik A, et al. CD16xCD33 bispecific killer cell engager (BiKE) activates NK cells against primary MDS and MDSC CD33+ targets. Blood. 2014;123(19):3016–3026. doi: 10.1182/blood-2013-10-533398. - DOI - PMC - PubMed
    1. Zhang L, Chen X, Liu X, Kline DE, Teague RM, Gajewski TF, et al. CD40 ligation reverses T cell tolerance in acute myeloid leukemia. J Clin Invest. 2013;123(5):1999–2010. doi: 10.1172/JCI63980. - DOI - PMC - PubMed
    1. Gaiger A, Reese V, Disis ML, Cheever MA. Immunity to WT1 in the animal model and in patients with acute myeloid leukemia. Blood. 2000;96(4):1480–1489. - PubMed

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