Itacitinib (INCB039110), a JAK1 Inhibitor, Reduces Cytokines Associated with Cytokine Release Syndrome Induced by CAR T-cell Therapy

Abstract

Purpose: T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFNγ and IL6.

Experimental design: Many cytokines implicated in CRS are known to signal through the JAK-STAT pathway. Here we study the effect of blocking JAK pathway signaling on CAR T-cell proliferation, antitumor activity, and cytokine levels in in vitro and in vivo models.

Results: We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an in vivo model, antitumor activity of CD19-CAR T cells adoptively transferred into CD19⁺ tumor-bearing immunodeficient animals was unabated by oral itacitinib treatment.

Conclusions: Together, these data suggest that itacitinib has potential as a prophylactic agent for the prevention of CAR T cell-induced CRS, and a phase II clinical trial of itacitinib for prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366).

Figure 1.. Itacitinib reduces cytokine levels in murine models of acute inflammation.

(A) BALB/c mice were orally dosed with vehicle control, or 60 or 120 mg/kg of itacitinib. One hour later, animals were challenged with Concanavalin-A (ConA), and 120 minutes later sacrificed and plasma collected (prophylactic). (B) BALB/c mice were challenged with ConA, 30 minutes later were orally dosed with vehicle control, or 60 or 120 mg/kg of itacitinib and 2 hours after ConA challenge sacrificed (therapeutic). N = 5 animals per group. MSD analysis was performed to detect the levels of pro-inflammatory cytokines. Data represent mean ± SEM, and P values were calculated by two-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001, **** P < 0.0001. Data are representative of four independent experiments.

Figure 2.. Itacitinib reduces IL-6 production by macrophages.

(A) Macrophages harvested from C57BL-6 mice were treated with increasing doses of itacitinib 1 day in advance of being activated with 5 ng/mL LPS. Twenty-four hours after LPS treatment, supernatant was harvested and cytokine levels measured. (B) C57BL/6 mice were prophylactically orally dosed with vehicle, or 60 or 120 mg/kg of itacitinib b.i.d. for 3 days. Mice then received intraperitoneal injections of LPS (5 μg per animal). Two hours after injection, mice were euthanized and IL-6 levels were measured in the peritoneal lavage. Data represent mean ± SEM, and P values were calculated by two-way ANOVA. *P < 0.05, ***P < 0.001. Data are representative of two independent experiments.

Figure 3.. Itacitinib reduces CAR T-cell cytokine production.

CD19-CAR T-cells were expanded with either itacitinib or the anti–IL-6 receptor tocilizumab. Three days later, they were co-cultured with CD19⁺ lymphoma cells (E:T ratio = 2.5:1) for 6 hours and cytokines were measured in the supernatant by MSD. (A) Experiment scheme. (B) Data represent mean ± SEM, and P values were calculated by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of two independent experiments.

Figure 4.. Itacitinib does not affect PBMC proliferation nor cytolytic activity.

(A) Flow cytometry quantification of T-cell expansion of human PBMCs expanded with DMSO (positive control) or 50, 150, or 1000 nM of itacitinib. T-cells were expanded using anti-CD3/CD28-coated beads, and proliferation was measured every other day by flow cytometry. (B) GD2-CAR T-cells were expanded in the presence of different itacitinib concentrations and proliferation was measured every other day by flow cytometry. (C) Lytic activity of GD2-CAR T-cells against SY5Y neuroblastoma cells (GD-2⁺) was determined by measuring luciferase activity. As an internal control, lytic activity of non-transduced T cells was also measured (background dotted line). (D) EGFR-CAR T-cells were expanded with anti-CD3/CD28 beads for 2 weeks and then restimulated with EGFR beads in the presence of itacitinib (100 or 250 nM). Proliferation was measured every other day by flow cytometry.

Figure 5.. Itacitinib does not impair T-cell antitumor activity in vivo.

C57BL/6 animals were subcutaneously inoculated with an OVA-expressing EG-7 tumor cell line. Starting at day 5, corresponding groups received oral doses of itacitinib or vehicle for 2 weeks, and at day 8 they received an adoptive transfer (A.T.) of naïve OT-1 CD8 T-cells. (A) Experiment scheme. (B) Tumor growth was monitored and compared between groups. N = 10 animals per group. Data represent mean ± SEM, and P values were calculated by two-way ANOVA. * P < 0.05, n.s., not significant. Data are representative of two independent experiments.

Figure 6.. Itacitinib does not affect CD19-CAR T-cell cytolytic activity in vitro.

(A) CD19-CAR T-cells or (B) nontransduced PBMC were expanded with either itacitinib or anti–IL-6 receptor (tocilizumab) at the indicated concentrations. Three days later, they were co-cultured with luciferin expressing CD19⁺ lymphoma cells (E:T ratio = 2.5:1) for 6 hours, luciferase substrate was added and luminescence was recorded by a luminometer. Data represent mean ± SEM, and P values were calculated by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

Figure 7.. Itacitinib does not affect CD19-CAR T-cell antitumor activity in vivo.

Immunodeficient NSG mice were inoculated with 2.5 × 10⁶ CD19⁺ human lymphoma Nalm6 luciferase-expressing Nalm6 cell line. Starting 1 day later, animals received b.i.d. doses of itacitinib or vehicle for 10 days. At day 4, post-tumor injection, corresponding animals received an adoptive transfer of 3 × 10⁶ CD19-CAR T-cells. (A) Experiment scheme. (B) Anesthetized mice were imaged using a Xenogen IVIS Spectrum system to measure bioluminescence once a week. N = 5–10 animals per group.