Publication Date:
2020-11-05
Description:
Introduction: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in the United States, accounting for more than 11,000 deaths each year and with a 5-year overall survival rate of less than 30%. With the exception of Gemtuzumab ozogamycin, an anti-CD33 antibody drug conjugate, the landmark success of immunotherapy in other hematologic malignancies has not translated to AML. Chimeric antigen receptor (CAR) T cell therapy, in which T cells are engineered with redirected tumor specificity, holds promise for the treatment of AML, but optimal antigens for CAR targeting of AML remain to be defined. We identified CD93 as a novel target for AML CAR therapy given high expression on many AML samples and an important role in leukemia stem cell (LSC) biology. Here, we describe anti-leukemic efficacy of CD93 CAR T cells both in vitro and in murine xenograft models. Consistent with predictions based on CD93 expression within the hematopoietic compartment, we demonstrate minimal CAR T cell toxicity to hematopoietic progenitors. However, we identify endothelial cell toxicity as a significant on-target, off-tumor toxicity. We also analyze endothelial expression of other common AML targets including CD123 and CD38 at baseline and in an inflammatory microenvironment and propose a strategy to incorporate endothelial expression considerations into rational design of combinatorial CAR T cells for AML. Results/Methods: CD93 was expressed to some degree on 24/25 (96%) of primary AML specimens, and was homogeneously expressed in 17/25 (68%). T cells were engineered to express second generation CD93 CARs based on the scFv of a humanized monocloncal CD93 antibody (F11) linked to CD28-CD3ζ or 4-1BB-CD3ζ intracellular domains (CD93-28z and CD93-BBz, respectively). CD93-28z and CD93-BBz CAR T cells incubated in vitro with target cells demonstrated AML-specific cytokine production measured by ELISA and cytotoxicity measured by IncucyteTM assay. CD93 CAR T cell treatment of NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML line THP-1 resulted in improved leukemic control in comparison to mock-treated mice. In a patient derived xenograft model of primary AML, CD93 CAR T cell treatment resulted in significantly improved leukemic clearance, T cell expansion, and prolonged survival compared to mock-treated mice. CD93 CAR T cells were incubated with cord-blood derived CD34+ cells to evaluate CD93 CAR recognition of hematopoietic stem cells (HSCs) and other hematopoietic progenitors. In an ELISA, CD93 CAR T cells did not produce cytokines against the bulk CD34+ population, in contrast to a positive control of AML. Additionally, after a 24h co-culture, CD93 CAR T cells did not kill any hematopoietic progenitor cells as assessed by flow cytometry. Furthermore, a methycellulose based colony forming assay confirmed that CD93 CAR T cells do not impact hematopoietic progenitor multipotent functional ability. We analyzed CD93 expression by immunohistochemistry of a tissue microarray of normal tissues. H-scores of all tissues analyzed were
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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