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. T cells 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 T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell 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 T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.

Mechanisms of resistance to CAR T cell therapy

“…For example, with anti-CD22 CAR T cell therapy, a simple quantitative decrease in CD22 cell surface expression or antigen density in the leukaemic population was adequate to evade the CAR T cells, thus enabling leukaemic relapse despite ongoing CD22 positivity, with individual variation seen in the threshold antigen density that conferred relapse or resistance 15 . Notwithstanding, a minimum threshold of antigen expression is likely to be needed for functional and/or preserved CAR T cell activity -a concept that has been realized in preclinical models with targeting of both CD20 in B cell malignancies 117 and ALK in neuroblastoma 118 , in which CAR T cell cytolytic activity and cytokine production were increased with higher levels of antigen expression. Along these lines, preclinical experience with an EGFR-targeted CAR T cell therapy suggests therapeutic potential that relies specifically on the differential antigen density of EGFR in tumours versus nonmalignant tissues to limit on-target, off-tumour toxicities while maintaining anticancer activity 119 .…”

“…One key issue with solid tumours is that the inherent tumour heterogeneity is likely to be a substantial barrier to identifying an optimal target, and relatedly, antigen loss will probably be a key factor precluding curative remissions. Indeed, antigen density (as mentioned above regarding ALK in neuroblastoma cells 118 ) and inherent tumour heterogeneity in antigen expression in solid tumours (for example, mesothelin 154 , HER2 (ref. 155 ) or MUC1 (ref.…”

Programming CAR-T cells to kill cancer

“…In the context of CD22-CARs, a comparison between two scFvs targeting the same epitope but with varying affinities did not reveal a significant impact of affinity on CAR function, whereas an alternative CD22 binder (m971) with lower affinity proved most efficacious, possibly because of better accessibility of the targeted epitope 34,35 . Thus, for a given antigen density on a target cell, there is likely to be an optimal range of scFv affinities and CAR expression levels required for a specific and effective antitumor response [36][37][38] .…”

“…Although targeting TAAs also expressed on normal tissues risks on-target, off-tumour toxicity, evidence from preclinical models suggests that CAR-T cells may not react to low-level antigen expression in healthy tissues 33,[36][37][38] . Instances of human epidermal growth factor receptor 2 (HER2)-CAR off-tumour toxicity 82 have been largely overshadowed by safe trials with a distinct HER2-CAR that demonstrated clinical efficacy in patients with sarcomas 91,92 .…”

Chimeric antigen-receptor T-cell therapy for hematological malignancies and solid tumors: Clinical data to date, current limitations and perspectives

“…Both UniCAR and SplitCAR are titratable ''onswitch'' platforms enabling better control of the timing, dosage and activation of CAR T-cells. Beyond tumor-specificity, both antigen and CAR densities of expression at the membrane are likely to contribute to CAR T-cell activation [58]. Low or fluctuating expressions are unlikely to provide strong enough CAR stimulation.…”