Cd34 pe cy5

NOD/SCID mice were bred and housed at the UHN/Princess Margaret Hospital (PMH; Toronto, Ontario, Canada) animal facility, and all studies were performed in accordance with guidelines approved by the UHN/PMH Animal Care Committee. Eight- to 12-week-old female NOD/SCID mice (10 per cohort) were sublethally irradiated (275 cGy) and interperitoneally injected with anti-CD122 antibody the day before intrafemoral transplantation. Freshly thawed primary AML samples harvested from patients’ peripheral blood were transplanted at cell doses of 5e6/mouse. At day 21, post transplantation, AGS67E and an isotype control ADC were dosed by i.v. injection at 1.5 mg/kg, QW for a total of 4 doses. Mice were sacrificed 7 days after the last treatment to assess the efficacy of AGS67E determined by the human AML engraftment in the injected right femur and non-injected bone marrow (left femur and two tibias). AML outgrowth was evaluated by flow cytometry using the following antibodies: CD45-FITC (BD), CD33-APC (BD), CD34-PE-Cy5 (Beckman Coulter), CD3-ECD (Beckman Coulter), CD38-PE-Cy7 (BD), and AGS67C-Biotin. Secondary detection of biotinylated antibodies utilized streptavidin–PE. Samples were analyzed using an LSRII flow cytometer (BD).

PBMCs from AML patients were isolated and incubated with a cocktail of CD45-FITC (BD), CD33-APC (BD), CD34-PE-Cy5 (Beckman Coulter), CD3-ECD (Beck-man Coulter), CD38-PE-Cy7 (BD), and either AGS67C-Biotin or isotype-Biotin antibodies. Secondary detection for biotinylated antibodies was accomplished with Streptavidin-PE. An LSRII flow cytometer (BD) was used for acquisition. Within the CD45⁺ population, two distinct subpopulations were defined, CD33⁺/CD3⁻/CD20⁻ (myeloid blasts) and CD33⁺/CD3⁻/CD34⁺/CD38⁻ [leukemic stem cells (LSC)]. Analysis was done with FlowJo version 9.5.4. MFIR for each AML sample was calculated by dividing the AGS67C MFI by the isotype MFI.