FLT3-directed antibody-drug conjugates to target leukemic stem cells in acute myeloid leukemia

Able, Marina (2025): FLT3-directed antibody-drug conjugates to target leukemic stem cells in acute myeloid leukemia. Dissertation, LMU München: Medizinische Fakultät

Abstract

A large part of acute myeloid leukemia (AML) patients is either refractory to induction therapy or relapses after treatment stop leading to poor prognosis especially in patients that are ineligible for hematopoietic stem cell transplantation. While fast-proliferating blasts are eradicated by chemotherapy, quiescent and drug resistant leukemic stem cells (LSCs) persist and fuel disease re-occurrence. Fms-like receptor tyrosine kinase 3 (FLT3) is overexpressed on LSCs and antibody-drug conjugates (ADCs) provide one elegant way to target this receptor and specifically deliver a toxin that is capable to eliminate those cells. In this project, two FLT3-targeting ADCs were developed either incorporating a microtubule-toxin or a DNA-damaging drug. Primary goal was to investigate if those ADCs can eradicate LSCs and which payload to choose for this purpose. To this end, the previously generated chimeric FLT3 monoclonal antibody (mAb) 20D9 was humanized and out of several candidates 20D9h3-mAb was selected due to high and specific binding to wildtype and mutant FLT3, fast internalization and good producibility. I show here that while microtubule-toxins have a largely reduced toxicity on cell lines in proliferation arrest, the activity of the DNA-damaging drug DUBA is sustained in this setting which may indicate its better suitability for LSC targeting. Drugs from both classes were conjugated to 20D9h3-mAb resulting in 20D9h3-DUBA and 20D9h3-MMAF. Both ADCs effectively eliminated FLT3-positive cell lines via inhibition of proliferation and apoptosis induction. While 20D9h3-MMAF arrests cells in G2/M, 20D9h3-DUBA halts the cell cycle in G1/S and triggers DNA damage repair via ATR-CHK1 pathway. The efficacy of either ADC towards leukemic stem and progenitor cells was analyzed in-depth in vitro/ex vivo using AML patient-derived xenograft (PDX) and primary cells. 20D9h3-DUBA was highly effective in preventing colony growth in colony-forming unit (CFU) and long-term culture initiating cell (LTC-IC) assays as well as leukemic outgrowth in NSG mice even at concentrations as low as 0.025 µg/ml. It could be shown that its anti-LSC activity is mediated via both FLT3 and FcγRI, which is targeted by the IgG1 antibodies’ constant part. Despite the common notion that microtubule-toxins are not suitable to eliminate LSCs, also 20D9h3-MMAF treatment of AML-393 or -388 PDX cells prevented engraftment in 4/5 and 5/5 mice, respectively. However, in colony assays 20D9h3-MMAF had a reduced activity compared to 20D9h3-DUBA. Either ADC only marginally affected healthy CD34-positive bone marrow cells at the relevant doses which highlights the suitability of FLT3 as an LSC target. In contrast to the stable P5-conjugated 20D9h3-MMAF, 20D9h3-DUBA employs a maleimide-linker and a hydrophobic linker payload which comes with several limitations. Those include ADC aggregate formation and instability in vivo due to thiol-exchange reaction and rodent-specific carboxylesterase 1c (CES1c) cleavage. Despite those short-comings, 2x3 mg/kg intravenous dosage of either ADC led to strong and durable leukemia reductions in AML-388 PDX mouse models. Lastly, combinations of 20D9h3-DUBA with inhibitors of ATR and BCL-2 were tested, which both synergistically enhanced the ADC’s activity and could be especially promising for certain patient subsets e.g., p53 mutated patients. Overall, it was shown that FLT3-ADCs can be used for the targeted elimination of LSCs and that both DUBA and MMAF are possible payloads for this purpose. Further improvements of the linker of the DUBA-ADC and the drug loading of the MMAF-ADC will eventually allow the selection of a candidate to enter pre-clinical evaluations.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: AML, Leukemic stem cells, Targeted therapy, Antibody-drug conjugates
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 26. Februar 2025
• 1. Bericht­erstatter:in: Spiekermann, Karsten