Abstract 2760: Hypophosphatemia due to increased effector cell metabolic activity is associated with neurotoxicity symptoms in anti-CD19 CAR T cell therapy
Authors: Tang Jack Pengfei , Peters Cole W. , Quiros Crystal , Wang Xiaoyan , Nowicki Theodore S. 
Published: June 2022

Abstract
Background: A significant complication of CAR T cell therapy is immune effector cell-associated neurotoxicity syndrome (ICANS), which presents as aphasia, confusion, weakness, somnolence, seizures, and coma. This is similar to the neurological manifestations of serum hypophosphatemia, which typically results from sudden increased metabolic demand for phosphorylated intermediates, as in the setting of refeeding syndrome. Given these similarities, we investigated whether CAR T cell effector metabolic activity is associated with increased extracellular phosphate consumption, and the association of serum hypophosphatemia with ICANS.
Methods: Human CAR T cells expressing anti-CD19scFV linked to either 4-1BB-CD28-CD3z or CD28-CD3z signaling domains were co-cultured with CD19+/firefly luciferase+ Raji cells. Media phosphorus consumption and cell cytotoxicity were assessed throughout co-culture. Single cell cytokine polyfunctionality of CAR T cells was measured via IsoPlexis platform. Oxidative phosphorylation and glycolytic activity of CAR T cells were measured via Agilent Seahorse assay. In parallel, we retrospectively analyzed 77 patients treated with CD28 or 41BB anti-CD19 CAR T cell therapy. A linear mixed effects model was generated from this dataset to assess the temporal relationship between longitudinal serum phosphorus measurements and ICANS/CARTOX-10 scores (clinical measures of CAR T neurotoxicity).
Results: When co-cultured with Raji cells, 4-1BBz consumed 75.9% more phosphate (p=0.0019) and CD28z consumed 69.9% more phosphate (p=0.0048) compared to mock CAR T cells. Phosphate consumption was associated with increased Raji cell killing (4-1BBz: 97.0%, p=0.0011; CD28z: 89.5%, p=0.0015), increased single cell cytokine polyfunctionality, and increased cellular metabolic demand (oxidative and glycolytic ATP production rate). Of 77 patients treated with CD19 CAR T cells, 23 developed ICANS. A linear mixed effects model predicted a 0.29mg/dL decrease in serum phosphorus for each grade increment in ICANS score (p&lt;0.0001) and a 0.11mg/dL decrease in serum phosphorus for each point of decrease in CARTOX-10 (p=0.0025). Importantly, this model predicts neurotoxicity only when drops in serum phosphorus bring patients into significant hypophosphatemia (&lt;2.7mg/dL). The strength of the relationship is also dependent on time, with serum phosphorus decreases occurring shortly after cell infusion having stronger correlation with ICANS/CARTOX-10 scores.
Conclusions: Our data suggest the increased metabolic demand of adoptively transferred CAR T cells, as a consequence of their cytokinetic polyfunctionality and antigen directed cytotoxic activity, may drive a state of hypophosphatemia, which is associated with ICANS in CAR T cell therapy. Early phosphate repletion may help mitigate the neurologic toxicity of CAR T cell therapy.
Citation Format: Jack Pengfei Tang, Cole W. Peters, Crystal Quiros, Xiaoyan Wang, Theodore S. Nowicki. Hypophosphatemia due to increased effector cell metabolic activity is associated with neurotoxicity symptoms in anti-CD19 CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2760.