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Fms-like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD–positive AML, the prognosis of patients is still poor, and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody-drug conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3-targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9-ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines, and to FLT3-ITD–positive patient-derived xenograft AML cells. In vivo, 20D9-ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Furthermore, 20D9-ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3-ITD–positive AML.

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+and CD123+AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

•FLT3 is a promising target for ADCs in AML therapy.•Combination with midostaurin enhances the effectivity of FLT3-ADC in FLT3-ITD–mutated AML.

Background:Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a “don't eat me” signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity.

Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.