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  • br Introduction Acute myeloid leukemia AML remains a hematol


    Introduction Acute myeloid leukemia (AML) remains a hematologic malignancy with poor outcome and a high risk for relapse believed to be caused by the survival of leukemic stem cells (LSCs), which are responsible for disease repopulation following treatment [1]. This important implication of LSCs was determined in in vivo assays that allowed engraftment of primary AML specimens into the bone marrow (BM) and spleen of immunocompromised mice. In the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse, researchers determined that LSCs have the ability to self-renew and the capacity to proliferate and differentiate into diverse leukemic cells [2,3]. Engraftment in the NOD/SCID mouse has also been shown to correlate with poor response to chemotherapy and overall poor patient survival [4,5] making it a clinically predictive in vivo model. Based on the importance of LSCs, novel approaches that target these cells are being developed and converging with the NOD/SCID mouse assay [6–9] in efforts to advance desperately needed therapies into the clinic. We have developed a novel radioimmunotherapy (RIT) strategy targeting CD123 overexpressed on LSCs that we are evaluating in the NOD/SCID mouse AML model. Indium-111 (111In) labeled to CSL360 (CSL Limited, Parkville, Australia), a chimeric IgG1 specific for CD123, was used to deliver Auger electron irradiation to LSCs. 111In emits nanometer–micrometer range Auger electrons, which are highly toxic when delivered in close proximity to the DNA [10]. We previously observed that the modification of anti-CD123 an American idol with peptides [CGYGPKKKRKVGG] that harbor a nuclear localization sequence (NLS; underlined) is able to deliver Auger electrons to the nucleus of CD123+ AML cells and cause significant cytotoxicity in vitro[11]. In addition to Auger electrons, 111In emits γ-photons used for imaging and we previously reported that 111In-NLS-CSL360 accumulated specifically at sites of AML engraftment in the BM and spleen of NOD/SCID mice, permitting disease visualization by microSPECT/CT [12]. In order to investigate the potential of 111In-NLS-CSL360 as a RIT agent for AML, we describe here the results of initial studies to examine its effects on LSCs in engrafted NOD/SCID mice and the utility of this mouse model.
    Materials and methods Radioimmunoconjugates were prepared as previously reported [12]. Primary AML specimens were collected under a protocol (01–0573-C) approved by the Research Ethics Board of the University Health Network (UHN). Sublethally irradiated (200cGy) NOD/SCID mice were intravenously (i.v.) inoculated within 12h with 5×106 cells from BM specimens from AML patients (specimens 090295 and 080179). All animal studies were conducted under a protocol (864.5) approved by the Animal Care Committee at the UHN following Canadian Council on Animal Care (CCAC) guidelines. Three RIT studies were performed. In the first study, groups of 5 mice with nearly complete leukemic repopulation of the BM at 8 weeks post-engraftment with specimen 090295 were injected intravenously with 16.7±1.3MBq of 111In-NLS-CSL360 or 18.3±0.8MBq of irrelevant 111In-NLS-isotype control chimeric IgG1 (111In-NLS-chIgG1) and microSPECT/CT imaging of AML engraftment was performed as reported [12]. The mass of radioimmunoconjugates injected was 20–27μg. The effect on human (h)CD45+/CD123+ leukemia cells in the BM was measured following imaging at 72h post-treatment by flow cytometry [12]. The repopulating capacity of BM cells was studied by inoculating 3.75×104 viable donor cells from treated mice into groups of 5 recipient NOD/SCID mice. After 8 weeks, the BM from recipient mice was analyzed for hCD45+ cells, hCD45+/CD34+ progenitor cells and hCD45+/CD34+/CD38- stem cells. In a second study, groups of 5–7 mice with leukemia engrafted at one week post-inoculation with specimen 080179 were treated with 111In-NLS-CSL360 (4.5±0.2MBq), 111In-CSL360 (4.9 ± 0.1 MBq), 111In-NLS-chIgG1 (4.4 ± 0.1 MBq) or received no treatment. The mass of radioimmunoconjugates injected was 13–15μg. The BM was analyzed at 8 weeks for leukemic engraftment. In the third study, groups of 4–5 NOD/SCID mice were inoculated with 2.5×105 CD34+/CD38−/CD123+ sorted cells from specimen 080179. At 48h post-inoculation, mice were treated with 111In-NLS-CSL360 (2.3±0.0MBq), 111In-CSL360 (2.9±0.2MBq), 111In-NLS-IgG1 (2.2±0.0MBq), or received no treatment. The mass of radioimmunoconjugates administered was 5–10μg. The survival up to 180 days was determined. At the time of death or at the completion of the study, the spleen/body weight ratio was measured.