PF217 TARGETING FLT3 IN AML: MODULATION OF FLT3‐BITE® ACTIVITY THROUGH COMBINATION WITH VARIOUS TKI
Authors: Brauchle B. , Goldstein R. , Chi‐Ming L. , Buecklein V. , Krupka C. , Koppikar P. , Haubner S. , Thomas O. , Rock D. , Sastri C. , Cooke K. , von Bergwelt‐Baildon M. , Metzeler K.H. , Spiekermann K. , Arvedson T.L. , Subklewe M. 
Published: June 2019

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