Enhanced T Cell Function of PTPN2 Deleted CAR-T Cells Comes at a Cost: PTPN2 Knockout CAR-Ts Secrete More Cytokines and Demonstrate Increased Cytotoxicity, but Exhibit More Severe CRS and Icans in a Non-Human Primate Model

Abstract

Background: Despite the success of CD19 CAR-T cells (CAR-Ts) in inducing remission, relapse remains a major issue. Previous studies have shown that upregulation of negative T cell regulators can contribute to CAR-T failure, and that deletion of these regulators can enhance CAR-T efficacy. A potent negative regulator is PTPN2, which inhibits T cell function by modulating T cell receptor and cytokine signaling pathways. While murine models have shown that PTPN2-knockout T cells improve tumor control, these models fail to reliably predict clinical efficacy or toxicity. To improve this predictability, we investigated PTPN2 KO CAR-Ts in primary human cells and in a NHP model of B-cell directed CAR-T therapy. Methods: CRISPR/Cas9-mediated PTPN2 deleted (‘KO‘) human CD19 CAR-Ts were assessed for phenotype/function using cytokine secretion and cytotoxicity assays. PTPN2 KO NHP CD20 CAR-Ts were administered to lymphodepleted NHPs using our established model and a dose escalation with Level 1: 6x10 4, Level 2: 6x10 5, Level 3: 3x10 6, and Level 4: 6x10 6 CAR-T/kg, followed by assessment for CAR-T expansion, B cell aplasia, CRS and ICANS. Results: PTPN2 KO was successfully achieved in human CAR-Ts (83.8 + 5.2% deletion). IFNγ production was significantly higher in PTPN2 KO CD19 CAR-Ts vs control cells (35.1 + 0.97% vs 23.4 + 0.98%, p=0.001). In cytotoxicity assays targeting the B-ALL cell line NALM6, PTPN2 KO CD19 CAR-Ts demonstrated significantly increased killing vs control cells (for example, at E:T ratio 1:1 59.6 + 9.8% vs 42.5 + 12.9% p = 0.03). We evaluated the proliferation, efficacy, and toxicity of PTPN2 KO CD20 CAR-Ts in 5 NHPs. The CAR-T PTPN2 KO rate ranged from 63-91%. During dose escalation, no expansion of CAR-Ts was observed at dose levels 1 and 2 (n=1 for each, Fig 1). However, all animals infused at dose level 3 (n=3, 3x10 6 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 experienced CRS on day +4 that was severe enough to require tocilizumab, with symptoms subsequently improving. On day +6, this animal also developed ICANS, including ataxia and muscle weakness, requiring 1 dose of Dexamethasone (1mg/kg), after which symptoms resolved. One animal received dose level 4 (6x10 6 CAR-Ts/kg). This recipient displayed earlier and higher expansion of CAR-Ts (day 3: 16.7%, day 7: 56.3% CAR-Ts, Fig.1), and developed severe CRS (with elevated CRP, LDH (not shown) and Ferritin, Fig.1) and ICANS that were unresponsive to treatment with 2 doses of tocilizumab and multiple doses of dexamethasone, with this recipient meeting humane euthanasia endpoints. Importantly, previous animals (n=5) receiving 6-12 x 10 6PTPN2 expressing (WT) CAR-Ts/kg, did not develop severe CRS nor ICANS (and did not require Toci or Dex). This suggests that PTPN2 KO CAR-Ts are associated with augmented toxicities. An extensive terminal analysis of the Dose Level 4 animal was performed; pathology demonstrated widespread cerebral edema and CAR-T CNS infiltration. Because the CAR-T infusion contained both PTPN2 KO and PTPN2 WT CAR-Ts, an assessment could be made for enrichment of PTPN2 KO CAR-Ts in the blood and brain parenchyma. Compared to the infused product (63% KO CAR-Ts, Fig.2), there was enrichment for KO CAR-Ts in the blood (72% KO CAR-Ts) and the brain parenchyma (84% KO CAR-Ts, Fig 2). This suggests increased expansion and CNS infiltration of PTPN2 KO versus PTPN2 WT CAR-Ts. Flow cytometry analysis of the PTPN2 KO CAR-Ts from the blood at maximum expansion revealed high expression of the proliferation and activation markers Ki67 and GZMB (84.6%, 74.5% of all CAR-Ts). Of note, at Dose Level 3, neither CAR-T persistence (15 + 2 days vs 20 + 5 days) nor duration of B cell aplasia (41+ 5 days vs 37 + 3 days) were increased with PTPN2 KO CAR-Ts vs historic controls infused with PTPN2 WT CAR-Ts. Conclusion: We demonstrate that PTPN2 KO CAR-T cells possess increased effector function, proliferation, and CNS infiltration versus PTPN2 WT CAR-Ts. However, this enhanced efficacy was associated with increased severity of CRS and ICANS, emphasizing that careful attention to the dose of PTPN2 KO CAR-Ts would be important. These results also highlight the utility of evaluating gene-modified CAR-Ts in clinically relevant animal models, to thoroughly assess their efficacy and toxicity profiles.