Enhanced Proliferation and Effector Function in PTPN2 Knockout CAR-T Cells Associated with Toxicities in a Non-Human Primate Model of CD20 CAR-T Cell Therapy

Relapse following CD19 CAR-T cell (CAR-T) therapy has been linked to the upregulation of negative regulators of T cell function. Recent studies showed that inhibition of the negative T cell regulator PTPN2 using a small molecule inhibitor greatly improves T cell mediated tumor clearance in murine models. We now assessed the function, proliferation, persistence and toxicity of PTPN2 KO CAR-Ts in primary human CD19 CAR-Ts and in a clinical relevant model of Non-Human Primate (NHP) CD20 CAR-Ts. Human PTPN2 CRISPR/Cas9 KO CD19 CAR-Ts showed enhanced in vitro effector function as evidenced by increased IFNg production (35.1 + 0.97% vs 23.4 + 0.98%, p=0.001) and cytotoxicity against NALM6 B-ALL leukemia cells (59.6 + 9.8% vs 42.5 + 12.9% p = 0.03) when compared to control CD19 CAR-T. Furthermore, we assessed proliferation, persistence, and toxicity of PTPN2 KO CD20 CAR-Ts through a dose escalation study in 5 NHPs with Dose Level 1: 6x104, Dose Level 2: 6x105, Dose Level 3: 3x106, and Dose Level 4: 6x106 CAR-T/kg. Dose Level 1 and 2 showed no measurable expansion of CAR-Ts while all animals infused at dose level 3 (n=3, 3x106 CAR-Ts/kg) demonstrated CAR-T expansion (max expansion 18.6 + 6.8% CAR-Ts) and B cell aplasia, and all animals developed laboratory and clinical signs of CRS and ICANS (Fig 1). One animal at Dose Level 3 required treatment with Tocilizumab and Dexamethasone due to severe CRS and ICANS, which eventually resolved. A single NHP received the highest Dose Level (Level 4, 6x106 CAR-Ts/kg), exhibiting earlier and higher CAR-T expansion (Fig.1), severe CRS, and ICANS unresponsive to treatment with Tocilizumab and repeated doses of Dexamethasone, necessitating euthanasia. Importantly, previous animals receiving PTPN2-expressing (WT) CAR-Ts at higher doses (12x106) did not experience severe CRS or ICANS, suggesting that PTPN2 knockout CAR-Ts are associated with augmented effector function resulting in increased toxicity. Detailed analysis of the Dose Level 4 NHP revealed widespread cerebral edema and CAR-T cell infiltration in the central nervous system (CNS). Notably, there was an enrichment of PTPN2 knockout CAR-Ts in the blood (72%) and brain parenchyma (84%) when compared to the infused product (63%), indicating preferential expansion and CNS infiltration of PTPN2 KO CAR-Ts (Fig.2). Flow analysis conducted at the time of euthanasia revealed enrichment of CD8+ CAR-Ts (Fig.3) characterized by elevated expression levels of the proliferative and cytotoxic molecules Ki67 and GZMB. Extended CAR-T persistence was not seen in PTPN2 KO animals. We demonstrate that PTPN2 KO CAR-Ts possess increased effector function, proliferation, and CNS infiltration. Augmented effector function was associated with greater toxicities, highlighting the utility of evaluating gene-modified CAR-Ts in clinically relevant animal models.