Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase

Abstract

We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. Tcells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR Tcells. In addition, ALK CAR Tcells demonstrated rapid and complete antigen-induced loss of receptor from the Tcell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR Tcells and that manipulation of these parameters could allow precise tuning of CAR Tcell activity.