Abstract 2648: Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression
Authors: Majzner Robbie G. , Walker Alec J. , Murgai Meera , Zhang Ling , Long Adrienne H. , Wanhainen Kelsey M. , Orentas Rimas J. , Mackall Crystal L. 
Published: July 2016

Abstract
ALK is overexpressed on the surface of neuroblastoma (NB) and is associated with high risk disease. We developed a second generation CAR based on a monoclonal antibody against ALK. ALK CAR T-cells significantly delay the growth of human NB cell lines in murine xenograft models, but animals eventually succumb to tumors.
In order to understand how target antigen density limits the effectiveness of this CAR, we created a library of NALM-6 B-cell leukemias with variable amounts of ALK expressed on the surface of each clone. In vitro, ALK CAR T-cells lyse high ALK expressing leukemias but have reduced activity against leukemias with low expression of ALK. In co-culture assays, we found that there is a minimum target antigen density required for CAR T-cells to produce appreciable amounts of Th1 cytokines. This threshold of ALK expression is above the expression on human NB lines. There is also a threshold target antigen density at which maximum cytokine production occurs. Above this density, no additional cytokines are produced. In xenograft models, ALK CAR T-Cells are significantly more effective against high ALK expressing leukemia than against low ALK expressing leukemia. To our knowledge, this is the first report of greater in vivo efficacy of CAR T-cells against tumors with higher antigen expression.
The level of surface expression of the CAR on T-cells also alters the function of CAR T-cells. We have identified a phenomenon in which both ALK and CD19 CAR T-cells lose surface expression of CAR over time in culture. Additionally, both the ALK CAR and the CD19 CAR downregulate quickly after T-cells are exposed to antigen. Using a fluorescently tagged CD19 CAR, we demonstrate that CARs are rapidly internalized upon antigen encounter.
We created an assay to understand the interplay of target density and CAR surface expression. We transduced T-cells with different amounts of supernatant to achieve different ALK CAR surface densities and then exposed these T-cells to varying amounts of immobilized protein-L in order to crosslink the CAR. T-cell activation (measured by an NFAT reporter) is greatest when both CAR surface expression and target antigen density are highest. As both variables are decreased, there is a significant loss of T-cell activity. Thus, diminished CAR surface density due to antigen-dependent and independent CAR downmodulation may limit CAR T-cell efficacy, especially in the context of low tumor antigen expression.
In conclusion, we have created a novel CAR targeting ALK that demonstrates in vivo efficacy against NB. Efficacy is limited by low target antigen density on NB and low CAR surface expression. We have identified phenomena in which CAR T-cells lose surface expression of CAR over time and also quickly internalize their receptors in response to antigen. These factors contribute to the efficacy of other CAR T-cells and this data provides important insights into future CAR target selection.
Citation Format: Robbie G. Majzner, Alec J. Walker, Meera Murgai, Ling Zhang, Adrienne H. Long, Kelsey M. Wanhainen, Rimas J. Orentas, Crystal L. Mackall. Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression. [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 2648.