Abstract
Abstract 950
Current treatment regimes for acute myeloid leukemia (AML) achieve complete remissions in only a subset of individuals and most adult patients will relapse within 5-years, emphasizing the need for novel treatment alternatives. One such therapy may be the administration of T cells engineered to express chimeric antigen receptors (CARs) specific for AML-associated antigens. CARs are typically composed of a single chain variable fragment (scFv) from a monoclonal antibody fused to the CD3ζ signaling domain and may contain one or more costimulatory endodomains. When expressed in T cells, CARs redirect T cell specificity to surface antigens on target cells in an MHC-independent manner. The interleukin 3 receptor alpha chain (IL3Rα, CD123) is a cell surface receptor which is aberrantly over-expressed on multiple hematologic malignancies including AML. Previous work has demonstrated that CD123 is not expressed on all CD34+/CD38− hematopoietic stem cells and is restricted to cells of the myeloid lineage, making CD123 an attractive target for CAR T cell therapy. We have therefore generated two novel CD123-specific (CD123R) CARs using scFvs from previously characterized antibodies, designated 26292 and 32716, which bind two distinct epitopes on CD123. Here we demonstrate that T cells expressing CARs derived from either 26292 or 32716 effectively redirect T cell specificity against CD123+ cells. Healthy donor T cells (n=3) engineered to express the CD123R CARs efficiently lysed CD123+ cell lines LCL and KG1a while sparing the CD123− cell line K562 as demonstrated by a 4 hour chromium-51 (51Cr) release assay. Additionally, both of the CD123R CAR T cells produced similar levels of IFN-γ and TNF-α and displayed comparable levels of antigen-dependent proliferation following co-culture with CD123+ cell lines. The potent cytolytic activity and activation of our CD123-targeting T cells was not limited to tumor cell lines. Indeed, CD123R CAR T cells, but not donor-matched CD19-specific (CD19R) CAR T cells, robustly lysed panel of primary AML samples (n=6, 3 – persistent, 1 – relapsed, 2 - untreated) (* p<0.05, ** p<0.001 using the unpaired students' t-test comparing 26292 or 32716 CAR T cells to donor-matched CD19R CAR T cells), and exhibited multiple effector functions for both CD4 and CD8 T cell subsets (ie CD107a degranulation, IFN-γ and TNF-α production, and antigen specific proliferation) when co-cultured with primary AML samples (n=3, 2 – relapsed, 1-persistent). To examine the effect our CD123-specific T cells have on normal and leukemic progenitor cells, we co-cultured CD123R CAR T cells, or donor-matched CD19-targeting T cells, with either CD34-enriched cord blood (CB, n=3) or primary AML samples (n=3, 2 – relapsed, 1 - untreated) for 4 hours (E:T 25:1) prior to plating in semisolid methylcellulose progenitor culture. CD123-targeting T cells did not significantly reduce the number of colony-forming unit granulocyte-macrophage (CFU-GM) or burst-forming unit erythroid (BFU-E) colonies from CB when compared to CD19R CAR T cells. Finally, while CD19-specific T cells had little impact on leukemic colony formation of primary AML samples, CD123-targeting T cells significantly reduced leukemic colony formation in vitro.
Collectively, our data demonstrate that CD123-specifc CARs can be expressed in primary healthy donor T cells, distinguish between CD123+ and CD123− cells, and mediate robust anti-leukemic activity against a panel of poor-risk primary AML patient samples. Importantly, we demonstrate that CD123R CAR T cells have little impact on normal progenitor colony formation while significantly reducing the growth of clonogenic myeloid leukemic progentiors in vitro. Thus, CD123R CAR T cells are a promising candidate for future immunotherapy of AML.
Bhatia:Novartis: Consultancy, Honoraria. Jensen:ZetaRx: Equity Ownership, Patents & Royalties.
