TARGETING FLT3 IN AML: MODULATION OF FLT3-BITE® ACTIVITY THROUGH COMBINATION WITH VARIOUS TKI
Author(s): ,
Bettina Brauchle
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;Laboratory for Translational Cancer Immunology,Gene Center of the LMU Munich,Munich,Germany
,
Rebecca Goldstein
Affiliations:
Department of Oncology Research,Amgen Inc.,South San Francisco,United States
,
Li Chi-Ming
Affiliations:
Department of Oncology Research,Amgen Inc.,South San Francisco,United States
,
Veit Buecklein
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;Laboratory for Translational Cancer Immunology,Gene Center of the LMU Munich,Munich,Germany
,
Christina Krupka
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;Laboratory for Translational Cancer Immunology,Gene Center of the LMU Munich,Munich,Germany
,
Priya Koppikar
Affiliations:
Department of Oncology Research,Amgen Inc.,Thousand Oaks,United States
,
Sascha Haubner
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;Laboratory for Translational Cancer Immunology,Gene Center of the LMU Munich,Munich,Germany
,
Oliver Thomas
Affiliations:
AMGEN Research Munich GmbH,Munich,Germany
,
Dan Rock
Affiliations:
Department of Oncology Research,Amgen Inc.,South San Francisco,United States
,
Christine Sastri
Affiliations:
Department of Oncology Research,Amgen Inc.,Thousand Oaks,United States
,
Keegan Cooke
Affiliations:
Department of Oncology Research,Amgen Inc.,Thousand Oaks,United States
,
Michael von Bergwelt-Baildon
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany
,
Klaus H Metzeler
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ),Heidelberg,Germany
,
Karsten Spiekermann
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;Experimental Leukemia and Lymphoma Research (ELLF),Department of Medicine III, University Hospital LMU Munich,Munich,Germany;German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ),Heidelberg,Germany
,
Tara L Arvedson
Affiliations:
Department of Oncology Research,Amgen Inc.,South San Francisco,United States
Marion Subklewe
Affiliations:
Department of Medicine III,University Hospital LMU Munich,Munich,Germany;Laboratory for Translational Cancer Immunology,Gene Center of the LMU Munich,Munich,Germany;German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ),Heidelberg,Germany
EHA Library. Brauchle B. Jun 14, 2019; 266007; PF217
Bettina Brauchle
Bettina Brauchle
Contributions
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Abstract

Abstract: PF217

Type: Poster Presentation

Presentation during EHA24: On Friday, June 14, 2019 from 17:30 - 19:00

Location: Poster area

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 FLT3AML 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 CD3T 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-ITDAML cell lines or pAML cells.

Results

FLT3 BiTE® exhibited cytotoxicity against FLT3AML cell lines at picomolar concentrations irrespective of FLT3 mutational status (EC50:1.9±0.7pM, ±SD, n=6). No cytotoxicity was observed against FLT3cell 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 CD2day 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-ITDAML 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-ITDAML cell lines (p=0.03, n=6) as well as pAML cells in 2 FLT3-ITDpatients (Table, bottom).

Conclusion

The FLT3 BiTE® antibody construct showed strong cytotoxicity against FLT3cells in vitro and ex vivo, mediated antitumor activity and depleted FLT3cells in vivo. The addition of clinically relevant concentrations of TKI enhanced FLT3-BiTE®-mediated cytotoxicity in FLT3-ITDAML while higher TKI concentrations compromised T-cell activity.

Session topic: 3. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): Acute myeloid leukemia, FLT3, Immunotherapy, Tyrosine kinase inhibitor

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