Midostaurin Reduces Bite® Mediated Cytotoxicity Against Acute Myeloid Leukemia

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 (CD45^DIM/SSC^LOW) 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 CD135^bright (MM6, MFI ratio 15) vs CD135^dim (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.