Abstract 1534: Analysis of CAR 41-BB versus CD28 co-stimulatory domains exposes emergence of extramedullary disease in acute myeloid leukemia
Authors: Yang Lila , Tarun Samiksha , Chien Christopher D. , Kohler Mark E. , Qin Haiying , Fry Terry J. 
Published: August 2018

Abstract
Phase I CD19 and CD22 targeting Chimeric Antigen Receptor (CAR) T cell trials have shown tremendous results against acute lymphoid leukemia (ALL). Across these trials there were variations in the types of costimulatory molecules included in the CAR constructs and this lead to the discovery that CAR T cells with CD28 result in earlier potency and activation with decreased persistence, while CARs with 4-1BB show increased expansion and persistence. There is great interest in translating these results in ALL trials into other diseases such as acute myeloid leukemia (AML). It is unclear which costimulatory domain in a CAR will be most effective in treating AML. To look at how co-stimulatory domains impact CAR functionality in AML, we developed CD33 targeting CD28 and 4-1BB CARs.
In vitro testing of the constructs revealed that CD33 CD28 CAR consistently produced more IL2 and Interferon-gamma than CD33 4-1BB across multiple AML cell lines. To translate these findings in vivo, xenograft models were injected with Molm 14 AML cells and treated with either CD33 CD28 or CD33 4-1BB CAR T cells. By bioluminescence imaging, CD33 CD28 treated mice had no detectable disease while CD33 4-1BB treated mice were ridden with leukemia. Combined, the in vitro and in vivo results suggest that the co-stimulatory domain does play a critical role in CAR T cell functionality and may improve CAR potency. To confirm the presence of AML in mice detected by bioluminescence, flow cytometry was performed on tissues from mock and CD33 4-1BB treated mice. No leukemia was found in the bone marrow of mock T cell treated mice. In contrast, CD33 4-1BB treated animals were clear of any leukemia in the bone marrow, suggesting the presence of extra medullary disease (EMD). The development of EMD in the less potent CD33 4-1BB CAR treated mice suggests that CAR immune pressure may be potent enough to clear primary sites of leukemia such as the bone marrow, but unable to eliminate disease in secondary tissues that AML can seed. This is not surprising since treatment of AML with chemotherapy often leads to the development of extramedullary disease in the form of chloromas.
To further investigate the effects of these two factors, we moved onto another AML model, THP1, that regularly presents with EMD even in the absence of CAR pressure. With CD33 CD28 against THP1, there was clearance in compartments bone marrow, however CD33 CD28 CAR was not able to prevent the development of EMD. These experiments suggest that although the CD28 costimulatory domain is more potent than 4-1BB in Molm14, the potency of CD28 is still not able to overcome EMD in all models. Using further studies of different AML models, we will continue to tease apart the contribution the impact of immune pressure from CARs, the natural progression of AML models, and effect of CAR potency on leukemia distribution.
Citation Format: Lila Yang, Samiksha Tarun, Christopher D. Chien, Mark E. Kohler, Haiying Qin, Terry J. Fry. Analysis of CAR 41-BB versus CD28 co-stimulatory domains exposes emergence of extramedullary disease in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1534.