Abstract 3992: Allogeneic TCRαβ-deficient CAR T-cells targeting CD123 effectively eliminate myeloid leukemia cells in vitro and in vivo PDX mice
Authors: Guzman Monica L. , Zong Hongliang , Sugita Mayumi , Lara-Martinez Luis A. , Bystrom Laura M. , Cruz Nicole M. , Galetto Roman , Gouble Agnès , Lebuhotel Céline , Bank Alexander , Smith Julianne , Roboz Gail J. 
Published: July 2016

Abstract
Acute myeloid leukemia (AML) is incurable in the majority of patients. While allogeneic stem cell transplantation remains the most effective therapy for AML to date, other types of cellular therapy have not yet been successful in this disease. The success of autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas has encouraged the investigation of CAR technology for the treatment of AML by targeting distinct tumor-specific antigens. We have developed an allogeneic CAR-T cell platform using T-cells from third-party healthy donors to generate T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on blasts, leukemic progenitor and leukemic stem cells from the majority of patients with acute myeloid leukemia (AML).
Transcription Activator-Like Effector Nuclease (TALEN) gene-editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to cause graft versus host disease (GvHD). Using leukemia cell lines in both in vitro and in vivo models, we confirmed that TCR-deficient T-cells expressing an anti-CD123 CAR display significant antitumor activity.
We next evaluated the in vitro cytotoxic activity of TCR KO CD123-CAR T-cells (UCART123) in primary AML (n = 6) samples and normal cells (normal bone and umbilical cord blood; n = 3) using T-cell:AML cell ratios of 5:1, 2:1 and 0.5:1; and T-cell:normal cell ratio of 10:1 and 1:1. Degranulation and IFNγ release assays revealed potent activation of UCART123 cells when exposed to CD123 leukemia cells but not to normal hematopoietic cells. Cytotoxicity of UCART123 cells was observed as early as 4 hours upon initiation of the co-cultures. However, at 24 hours, more than 80% of leukemic cells (blasts, progenitors and stem cells) were eliminated at all ratios tested, whereas only approximately 20% CAR-independent cell death was observed with the TCR-deficient T-cells. Normal hematopoietic cells showed an average of 30% cell death when exposed to either UCART123 or TCR-deficient T-cells, demonstrating the selectivity of CD123 CAR-T cells toward leukemic cells.
Finally, we evaluated the in vivo activity of the CAR-T cells against established patient derived xenografts (PDX) using AML and normal CB CD34+ cells. We found no significant difference between PBS, TCR-deficient (10e6/mouse) and CAR T-cell treatments (10 e6/mouse) in PDX mice transplanted with normal CD34+ CB. Strikingly, 14 days of treatment eliminated most of the leukemic cells from the AML-PDX mice. T-cells were still detected at day 14 after treatment with UCART123 cells (mean 52% CD123 CAR and mean 1.5% TCR-), without evidence of GVHD. Efforts are underway to develop allogeneic CD123 CAR-T cells for clinical trials in AML.
Citation Format: Monica L. Guzman, Hongliang Zong, Mayumi Sugita, Luis A. Lara-Martinez, Laura M. Bystrom, Nicole M. Cruz, Roman Galetto, Agnès Gouble, Céline Lebuhotel, Alexander Bank, Julianne Smith, Gail J. Roboz. Allogeneic TCRαβ-deficient CAR T-cells targeting CD123 effectively eliminate myeloid leukemia cells in vitro and in vivo PDX mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3992.