A Novel Combination of Anti-CD3 x Anti-CD123 Monoclonal Antibodies to Redirect Activated T Cells to Target Acute Myelogenous Leukemia

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy of the bone marrow (BM) and blood, characterized by clonal expansion of neoplastic myeloid precursor cells, and impairment of normal hematopoiesis. AML accounts for the largest number of annual deaths from leukemias among adults in the United States. AML blasts as well as leukemic stem cells express high levels of interlekin-3 (IL-3) receptor-a(CD123). Our previous studies show the anti-CD3 activated T cells (ATC) can be redirected with bispecific antibodies (BiAb) to CD20 for non-Hodgkin's lymphoma after stem cell transplantation (SCT), Her2/ neu for breast cancer, EGFR for pancreatic cancer and GD2 for neuroblastoma. In this study, we investigated whether ATC armed with anti-CD3 x anti-CD123 bispecific antibody (CD123Bi) can target CD123 expressing targets. Methods: ATC were produced by stimulating peripheral blood mononuclear cells (PBMC) with anti-CD3 in the presence of IL-2. Bispecific antibody was produced by chemical hetero-conjugation of anti-CD3 (OKT3) with anti-CD123 monoclonal antibodies. We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD123Bi armed ATC using ⁵¹Cr release assay. Cytokines and chemokine release during CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. Results: All four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) showed 70-90% CD123 expression and 10-70% CD123 expression on leukemic blasts. Arming dose titration ranging from 0.1 to 100 ng CD123Bi/10⁶ ATC showed saturation at 5 ng dose, the mean specific cytotoxicity 20% plateaued at arming doses of 5, 50 and 100 ng/10⁶ ATC at an effector to target ratio (E/T) of 10:1. ATC from 4 normal donors at 50 ng arming dose at E/T of 25:1 showed mean specific cytotoxicity of 22 and 40% directed at TF-1 and NoMo-1 cell lines, respectively. The cytotoxicity was also confirmed by analysis of IFNγ and TNFα release during cytotoxic activity directed at AML cell lines by CD123Bi armed ATC. Conclusions: ATC armed with CD123Bi at 50 ng/10⁶ cells showed comparable cytotoxicity directed at either high or low leukemic blast populations. Since most of the CD33 positive cells were also CD123+, it is likely that CD123Bi armed ATC will be able to target CD33 positive leukemia stem cells. This approach may provide a non-toxic strategy to target leukemic blasts and leukemic stem cells.