Antitumor Cytotoxic Activity Retained in Tolerized T Cells Following Daily Dose Escalation of a T-Cell Engager in Cynomolgus Monkeys to Mitigate Cytokine Release Syndrome.

T-cell engagers demonstrate promise in cancer immunotherapy but often cause severe cytokine release syndrome (CRS). Intrapatient dose escalation can mitigate CRS, but reduced cytokine levels may compromise therapeutic efficacy. We previously reported that a single 1000 μg/kg dose of ERY22, an anti-GPC3/CD3 monkey surrogate T-cell engager, induces life-threatening CRS in cynomolgus monkeys, but that daily dose escalation from 1 to 1000 μg/kg almost completely prevented CRS in the monkeys without premedication with immunosuppressants. This study aimed to investigate whether T cells tolerized in vivo through dose escalation maintain antitumor cytotoxicity. In this study, peripheral blood mononuclear cells (PBMCs) were isolated from animals that were administered daily doses ranging from 1 to 1000 μg/kg and then restimulated with ERY22 ex vivo. Cytokine concentrations in supernatants and cytotoxicity against target cells were examined in ex vivo experiments. We used ATAC-seq to assess chromatin accessibility in T cells responding to daily dose escalation. Under tolerance-induced conditions, ex vivo cytokine release was almost completely inhibited, whereas approximately half of the cytotoxic activity was retained. T-cell chromatin states changed after ERY22 administration. Because the relevancy of the ex vivo cytotoxic assay to clinical effects is unknown, the impact of reduced cytotoxicity through dose escalation on actual cancer treatment efficacy remains unclear. Although this and species differences must be considered, our findings provide valuable insights into mechanisms underlying dose escalation-induced tolerance and may be useful for predicting human responses. A deeper understanding of these shared mechanisms is critical for advancing the clinical development of T-cell engagers.