IMMUNOBIOLOGY T cells expressing CD 123-speci fi c chimeric antigen receptors exhibit speci fi c cytolytic effector functions and antitumor effects against human acute myeloid leukemia

Acutemyeloid leukemia (AML) is a disease characterized by the rapid proliferation of immature myeloid cells in the bone marrow resulting in dysfunctional hematopoiesis. Although standard induction chemotherapy can induce complete remissions, many patients eventually relapse and succumb to the disease. Therefore, the development of novel therapeutics for AML is crucial. Recent advances in the immunophenotyping of AML cells have revealed several AML-associated cell surface antigens that may act as targets for future therapies. Indeed, preclinical investigations using antibodies targeting CD44, CD47, T-cell immunoglobulin mucin-3 (TIM-3), and the interleukin 3 receptor a chain (CD123) for the treatment of AML have been described and have demonstrated promising antileukemic activity in murine models. Additionally, 2 phase 1 trials for CD123-specific therapeutics have been completed, with both drugs displaying good safety profiles (ClinicalTrials.gov ID #NCT00401739 and #NCT00397579). Unfortunately, these CD123-targeting drugs had limited efficacy, suggesting that alternative and more potent therapies targeting CD123 may be required to observe antileukemic activity. A possibly more potent alternative therapy for the treatment of AML is the use of T cells expressing chimeric antigen receptors (CARs) that redirect T-cell specificity toward cell surface tumorassociated antigens in a major histocompatibility complex– independent manner. In most cases, CARs consist of a single-chain variable fragment (scFv) from a monoclonal antibody fused to the signaling domain of CD3z and may contain a costimulatory endodomain. Several groups have developed CARs targeting various antigens for the treatment of B-cell malignancies, and many have gone on to evaluate CAR-expressing T cells in phase 1 clinical trials. In contrast, CAR-engineered T cells for the treatment of AML remain scarce. Here, we describe the generation of 2 novel CD123-targeting CARs using scFvs from previously described recombinant immunotoxins, 26292 and 32716, which bind distinct epitopes and have similar binding affinities for CD123. We hypothesized that T cells expressing CARs derived from either 26292 or 32716 would effectively redirect T-cell specificity against CD123-expressing cells. Using a standard 4-hour chromium-51 (Cr) release assay, healthy donor T cells engineered to express the CD123 CARs efficiently lysedCD123 cell lines and primaryAMLpatient samples. Additionally, both of the CD123 CAR T cells activated multiple effector functions following coculture with CD123 cell lines and primary AML patient samples. Further, CD123-targeting T cells did not ablate colony-forming unit granulocyte-macrophage