AML tumor cell growth suppression is dependent on affinity to CD3ε and effects of CLL1/CD3H TDB on AML BMMNCs. (A) Dose-dependent survival curves determined by Prism-6 using a nonlinear regression with sigmoidal dose response where X is the log [ ]. Percent survival is calculated by dividing the treatment live events by its untreated control replicate live events, and multiplying by 100. HL60 was performed in a separate experiment from the other cell lines (data not shown for CLL1/CD3H). (B) EOL-1 dose-dependent survival and percent of CD8⁺CD69⁺CD25⁺ effector cells. One of 3 experiments, each using a different blood donor. Similar results were observed for other AML cell lines. (C-D) CD8⁺ T cells were purified from peripheral blood of a healthy human donor (ALLCells, Alameda, CA) for donor 1 and a Genentech donor for donor 2 by Ficoll density gradient centrifugation and a human CD8⁺ T cell isolation kit from Miltenyi (130-096-495). Patient AML bone marrow was obtained from the Stanford Cancer Center and purified by Ficoll centrifugation. The AML blasts were preincubated for ∼6 hours with hIgG (EU = 0.07/mg) to reduce nonspecific binding to FcγRs. The E:T ratio was 3:1 (150 000 CD8 T cells to 50 000 blasts). Medium used for the assay was RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, Pen-Strep, 1X cytokine bullet (IL-3, IL-6, SCF, Flt3; StemCell Technologies, Vancouver, Canada), 0.1 μg/mL GM-CSF and 0.1 μg/mL G-CSF. The test articles were NT/CD3H and CLL1/CD3H diluted serially in threefold steps. The assay was set up in triplicate with either a 20-hour (donor 2; blue lines) or 40-hour (donor 1; red lines) exposure to test article. (C) Dot plot shows SSC/CD45 profile of AML patient donor 1 and donor 2. Histogram shows positive staining of CD11b^–/CD34⁺ AML blasts for human CD33 (red) and CLL-1 (blue) compared with isotype control antibody. (D) Allogeneic CD8⁺ effector T cells in the presence of threefold serially diluted CLL1/CD3H shows concentration-dependent killing of AML blasts with an EC₅₀ ∼0.45 ng/mL for donor 1 compared with the NT/CD3H TDB. Dotted line (NT/CD3H) and solid line (CLL1/CD3H). Donor 1 phenotype was consistent with AML LSCs—CD45⁺/CD34⁺/CD33⁺/CLL-1⁺/CD38^– (99%) with <1% CD38⁺. Donor 2 phenotype was consistent with AML progenitors—CD45⁺/CD34⁺/CD33⁺/CLL-1⁺/CD38⁺ (∼97%).