Steroids Abrogate BiTE^® Antibody Construct-Mediated Cytotoxicity in Primary AML Cells

Antibody based immunotherapy represents a promising strategy to eliminate chemoresistant cells in acute myeloid leukemia (AML). Clinical experience in acute lymphoblastic leukemia (ALL) has shown a clear correlation of leukemic burden and the occurrence of a cytokine release syndrome (CRS) during treatment with blinatumomab (CD19/CD3 BiTE^®). A cytoreductive phase prior to or in combination with antibody therapy might be beneficial to reduce the severity of adverse events like CRS. The latter is often treated with steroids (dexamethasone, DEX) or less commonly, with the IL-6R antibody tocilizumab (TOC). As T-cell proliferation and function are of crucial importance for BiTE^® activity (Zugmaier 2015), the effect of the drugs on effector cell function will dictate clinical response to therapy. In this study, we evaluated the influence of cytoreductive- (azacythidine, AZA; decitabine, DEC), and immunmodulatory (DEX and TOC) drugs on antibody-mediated cytotoxicity and T-cell proliferation. A CD33/CD3 BiTE^® antibody construct (AMG 330) served as model T-cell recruiting antibody in this study. To address this question we set up the following experimental approaches: AML cells were cocultured with healthy donor (HD) T cells for up to 14 days ex vivo. T cells were either incubated with the specific drug for 3 days prior to coculture or the drugs were simultaneously added to AML-T cell cultures. Drug concentrations were chosen based on published serum concentrations in AML patients and their ex vivo stability in culture, validated by mass spectrometry. BiTE^® mediated cytotoxicity and T-cell proliferation were assessed by flow cytometry. Preincubation of T cells with AZA and DEC impaired antibody mediated cytotoxicity of HL60 cells in a concentration dependent manner (% lysis control (ctrl) vs AZA at 1, 5, 10 µM: 99.9 vs 99.2 vs 52.1 vs 28.7, n=7; ctrl vs DEC at 0.2, 2, 5 µM: 98.4 vs 71.3 vs 60.0 vs 50.0, n=3). Similarly, T-cell proliferation was also markedly decreased (fold change (FC) T cells ctrl vs AZA at 1, 5, 10 µM: 2.9 vs 2.8 vs 1.5 vs 0.7; ctrl vs DEC at 0.2, 2, 5 µM: 3.8 vs 3.0 vs 2.3 vs 1.2). For DEX it was shown that incubation of T cells with steroids prior to cocultures had no negative effect on BiTE^® mediated cytotoxicity (Brandl 2007). However, as steroids are often used simultaneously with T-cell recruiting immunotherapies, we tested the influence of DEX in combination with AMG 330. The addition of DEX to primary AML-T cell cultures (75 ng/ml) significantly impaired AMG 330 mediated cytotoxicity (% lysis AMG 330 vs AMG 330+DEX day (d) 6: 95.9 vs 47.5, n=9). This correlated to a markedly reduced T-cell proliferation (FC T cells AMG 330 vs AMG 330+DEX d6: 11.2 vs 1.2, n=9). Correspondingly, secretion of IFNγ was also decreased (n=3). Upon discontinuation of DEX an increase in AMG 330 mediated cytotoxicity was observed. Nevertheless, cytotoxicity was still considerably lower compared to control cultures (%lysis AMG 330 vs AMG 330+DEX d9: 95.6 vs 77.0). In contrast to DEX, TOC (110 µg/ml) had no negative effect on T-cell proliferation (FC T cells d6: AMG 330 vs AMG 330+TOC: 42.3 vs 36.9, n=4). Similarly, secretion of IFNγ was not affected through the simultaneous addition of TOC to primary AMG 330 cultures (pg/ml AMG 330 vs AMG 330+TOC d6: 543.9 vs 345.8 n=2). Importantly, drugs might not only interfere with effector cell function but also modulate target antigen expression. As we have previously demonstrated that antigen expression levels influence BiTE^® mediated cytotoxicity (Krupka 2016), we analysed the effect of the drugs on CD33 expression. None of the drugs induced a significant up- or downregulation of CD33 on AML celllines as detected by flow cytometry. Hence our data support the notion that these drugs do not modulate antigen expression dependent lysis kinectics. We conclude, that drugs given prior or concomitant to BiTE^® therapy have the potential to reduce T-cell proliferation and cytotoxicity. In particular, we observed a negative impact of AZA and DEC when given prior to AML-T cell cocultures. Importantly, even short exposure to DEX led to a significanly reduced T-cell responsiveness. Our data suggest the careful evaluation of concomitant drugs in T-cell recruiting antibody therapies and support the restrictive use of steroids in patients receiving BiTE^® antibody therapy. For management of severe CRS, TOC could be considered as a targeted biologic therapy that preserves BiTE^®-dependent T cell function.