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T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Pre-existing antibodies that bind polyethylene glycol are present in about 40% of healthy individuals. It is currently unknown if pre-existing anti-polyethylene glycol (PEG) antibodies can alter the bioactivity of pegylated drugs with a single long PEG chain, which represents the majority of newly developed pegylated medicines. Methoxy polyethylene glycol-epoetin beta (PEG-EPO) contains a single 30 kDa PEG chain and is used to treat patients suffering from anemia. We find that the pre-existing human anti-PEG IgM and IgG antibodies from normal donors can bind to PEG-EPO. The prevalence and concentrations of anti-PEG IgM and IgG antibodies were also higher in patients that responded poorly to PEG-EPO. Monoclonal anti-PEG IgM and IgG antibodies at concentrations found in normal donors blocked the biological activity of PEG-EPO to stimulate the production of new erythrocytes in mice and accelerated the clearance of 125I-PEG-EPO, resulting in PEG-EPO accumulation primarily in the liver and spleen. Accelerated clearance by the anti-PEG IgG antibody was mediated by the Fc portion of the antibody. Importantly, infusing higher doses of PEG-EPO could compensate for the inhibitory effects of anti-PEG antibodies, suggesting that pre-existing anti-PEG antibodies can be &ldquo, dosed through.&rdquo, Our study indicates that the bioactivity and therapeutic activity of PEG-EPO may be reduced in patients with elevated levels of pre-existing anti-PEG antibodies. New pegylated medicines with a single long PEG chain may also be affected in patients with high levels of anti-PEG antibodies.