A CD117-Amanitin Antibody Drug Conjugate (ADC) Effectively Depletes Human and Non-Human Primate Hematopoietic Stem and Progenitor Cells (HSPCs): targeted Non-Genotoxic Conditioning for Bone Marrow Transplant

Current regimens for patient preparation, or conditioning, prior to bone marrow transplantation (BMT) limit the use of this curative procedure due to regimen-related mortality and morbidities. To address this, we developed a CD117-ADC to specifically deplete recipient HSPCs. CD117 is an ideal target for an ADC-based approach to patient preparation as it is selectively expressed on HSPCs. In addition, CD117 is frequently overexpressed on in acute myelogenous leukemia (AML). Thus, a CD117-ADC may enable curative treatment while potentially improving disease control prior to and following transplant. We developed a potent CD117 antibody conjugated to amanitin (AM), an RNA polymerase inhibitor, that depletes primary human CD34+ cells in vitro with an IC50 of 21pM (Fig. 1A). The ADC was designed to have a fast half-life, allowing the HSPC ablating ADC to clear prior to stem cell transplant. Humanized NSG mice treated with the anti-CD117-AM had >95% depletion of human HSPCs in bone marrow following a single dose of 0.1 mg/kg (Fig. 1B). The specificity of anti-CD117-AM was confirmed by the persistence of peripheral human lymphocytes, indicating the ability to maintain adaptive immunity. In addition, a single dose of anti-CD117-AM extended survival of NSG mice challenged with CD117+ patient derived xenografts (PDX), demonstrating potent anti-tumor effects. We treated NHPs with the cross-reactive anti-CD117-AM to determine if the ADC could deplete HSPCs in immune-competent animals. Dose-dependent decreases in phenotypic HSPCs and colony-forming units (CFUs) were observed in the bone marrow 7 days after dosing, with >95% HSPC depletion after a single dose of 0.3 mg/kg (Fig. 1C, 1D). Depletion was target- and AM-dependent, as the antibody alone and isotype-AM had no effect. In the periphery a transient decrease in reticulocytes was observed at day 4 with a neutrophil nadir at day 18. WBC and lymphocytes were within normal range throughout. Transient off-target effects consisted of reversible thrombocytopenia (Day 4-8) and reversible and mild liver enzyme levels (Day 4-10). Fractionated dosing achieved similar efficacy with reduced off-target effects. As expected, the fast half-life anti-CD117-AM was rapidly cleared (t 1/2 15-18 h), providing appropriate pharmacokinetics for BMT. In summary, anti-CD117-AM can selectively deplete HSPCs in humanized NSG mice and NHP. In addition, the ADC exhibited potent anti-tumor activity in AML PDX models. This strategy could preserve the adaptive immune system with delayed onset of neutrophil nadir, potentially shortening the period of neutropenia during BMT. Targeted non-genotoxic conditioning with ADCs could provide a significant improvement to current approaches to patient preparation with an acceptable safety profile prior to BMT and gene therapies.