IDO1 Inhibition Synergizes with Radiation and PD-1 Blockade to Durably Increase Survival Against Advanced Glioblastoma

Abstract

Purpose: Glioblastoma is the most aggressive primary brain tumor in adults with a median survival of 15-20 months. Numerous approaches and novel therapeutics for treating glioblastoma have been investigated in the setting of phase III clinical trials, including a recent analysis of the immune checkpoint inhibitor, nivolumab (anti-PD-1), which failed to improve recurrent glioblastoma patient survival. However, rather than abandoning immune checkpoint inhibitor treatment for glioblastoma, which has shown promise in other types of cancer, ongoing studies are currently evaluating this therapeutic class when combined with other agents.Experimental Design: Here, we investigated immunocompetent orthotopic mouse models of glioblastoma treated with the potent CNS-penetrating IDO1 enzyme inhibitor, BGB-5777, combined with anti-PD1 mAb, as well as radiotherapy, based on our recent observation that tumor-infiltrating T cells directly increase immunosuppressive IDO1 levels in human glioblastoma, the previously described reinvigoration of immune cell functions after PD-1 blockade, as well as the proinflammatory effects of radiation.Results: Our results demonstrate a durable survival benefit from this novel three-agent treatment, but not for any single- or dual-agent combination. Unexpectedly, treatment efficacy required IDO1 enzyme inhibition in non-glioblastoma cells, rather than tumor cells. Timing of effector T-cell infiltration, animal subject age, and usage of systemic chemotherapy, all directly impacted therapy-mediated survival benefit.Conclusions: These data highlight a novel and clinically relevant immunotherapeutic approach with associated mechanistic considerations that have formed the basis of a newly initiated phase I/II trial for glioblastoma patients. Clin Cancer Res; 24(11); 2559-73. ©2018 AACR.

Figure 1. Biochemical analysis of the IDO1 inhibitor, BGB-5777

A, and B, Kinetic and IC₅₀ measurements of pharmacologic IDO1 enzyme inhibitors. Recombinant human and mouse IDO1 and TDO were expressed in and isolated from E. coli cells. Human IDO1 (50 nM), mouse IDO1 (20 nM), or TDO (100 nM) were then combined with L-tryptophan (0.1mM), L-ascorbic acid (5 mM), methylene Blue (6.25 μM), catalase (0.4 mg/mL), and IDO1 inhibitor or vehicle only (DMSO), in potassium phosphate buffer (50 mM; pH 7.5) and BSA (0.1%). IC₅₀ was calculated based on the remaining enzyme activity in the presence of increasing concentrations of IDO1 inhibitor. K_obs. determination was performed using the same methodology as the IDO1 enzyme activity assay with D-tryptophan, except that 150 nM IDO1 was used and sample absorbance was measured immediately after the initiation of reaction. C, Blood brain penetration of BGB-5777 and INCB03560 was evaluated in 6 to 8 week old male C57BL/6 mice fasted overnight, followed by a single oral dose of BGB-5777 or INCB024360 at 10 mg/kg. The concentration of BGB-5777 or INCB024360 in plasma and brain were determined by the liquid chromatography/mass spectrometry (LC-MS/MS) method.

Figure 2. Overall survival and tumor growth of established murine glioblastoma (GBM) simultaneously treated with an IDO1 enzyme inhibitor, radiation and PD-1 blockade

A, Survival analysis for wild-type C57BL/6 mice intracranially-engrafted 2 × 10⁵ syngeneic unmodified GL261 and receiving mono- or combination-therapy with BGB-5777, whole brain radiation (WBRT) and/or PD-1 mAb beginning at 14 days post-intracranial injection (post-ic.); IgG antibody treatment was included as a control group. B, Wild-type mice were ic.-injected with 2 × 10⁵ GL261 cells expressing luciferase (GL261.luc) and treated with mono- and combination-therapies as described above. Average tumor luminescence was determined using an IVIS spectrum imaging station following intraperitoneal (IP) injection of D-Luciferin (150 mg/kg). C, Representative IVIS spectrum images of treated mice with intracranial GL261.luc at days 9, 16 and 22 post-ic. ****P<0.0001

Figure 3. Maximum survival benefit from trimodal therapy requires IDO1 inhibition in non-tumor cells and select leukocyte engagement

A, Survival analysis of wild-type mice intracranially-injected (ic.) 2 × 10⁵ GL261 cells expressing murine IDO1 cDNA (GL261 mIDO1 O/E) and receiving trimodal therapy, in the presence or absence of CD4-/CD8-T cell-depleting mAb beginning at day 14 post-ic. B, Survival analysis of wild-type and IDO1 knockout (IDO1^−/−) mice ic. 2 × 10⁵ unmodified GL261 tumors and receiving whole brain radiation (WBRT) and PD-1 mAb treatment, in the presence or absence of BGB-5777 IDO1 inhibitor. Survival analysis of wild-type mice after trimodal therapy, with or without addition of CD4-, CD8- or NK1.1-cell depleting antibodies C, beginning at day 5 post-ic. or D, beginning at day 14 post-ic. *P<0.05; **P<0.01; **P<0.001; ****P<0.0001.

Figure 4. Cellular and molecular analysis of unmodified or IDO1-expressing intracranial glioblastoma (GBM) treated with or without trimodal therapy

Wild-type mice were intracranially-injected (ic.) with 2 × 10⁵ unmodified GL261 or with GL261 cells expressing murine IDO1 cDNA (GL261 mIDO1 O/E) and treated with trimodal therapy or IgG antibodies. A, Absolute numbers of intratumoral CD3⁺CD4⁺FoxP3⁺ regulatory T (Treg) and CD3⁺CD8⁺ cytolytic T cells were quantified via flow cytometry (n=5/group). B, Gating strategy and representative proportions of Tregs and cytolytic T cells are shown. C, mRNA expression levels for IDO1, TDO, FoxP3, CTLA-4, PD-L1 and IFNγ in unmodified and mIDO1 O/E GL261 cell lysates. D, Tryptophan (Trp) and kynurenine (Kyn) levels were measured by high performance liquid chromatography (HPLC) in serum and tumors (n=5/group). E, Quantification of BGB-5777 concentration in the same tissue samples analyzed in (D) and determined by liquid chromatography/mass spectrometry (LC-MS/MS). *P<0.05; **P<0.01; ****P<0.0001; ns=not significant

Figure 5. Advanced age decreases the survival benefit of trimodal therapy against glioblastoma (GBM)

A, Survival analysis of young (8 week old) and old (72 week old) wild-type mice intracranially-injected with 2 × 10⁵ unmodified GL261 and treated with BGB-5777, whole brain radiotherapy and PD-1 mAb (trimodal therapy). Young and old mice were intracranially-injected with 2 × 10⁵ unmodified GL261, treated with trimodal therapy beginning at 14 days post-ic., followed by GBM and contralateral brain without tumor (non-tumor) isolation at 3 weeks post-ic. B, Quantitative gene expression analysis for IDO1, IDO2, and TDO2 (n=5/group) was performed. C, Tryptophan (Trp) and kynurenine (Kyn) levels were analyzed (n=5/group). D, Absolute numbers and representative proportions of intratumoral CD3⁺CD4⁺FoxP3⁺ regulatory T (Treg) (n=5/group) were quantified. E, Absolute numbers and representative proportions of CD3⁺CD8⁺ cytolytic T cells were quantified (n=5/group). *P<0.05; **P<0.01; ***P<0.001

Figure 6. Trimodal therapy is effective against multiple glioblastoma (GBM) models, induces strong immunological memory and is limited by systemic temozolomide (TMZ)

A, Survival analysis of wild-type mice intracranially-injected (ic.) 1 × 10⁵ syngeneic CT-2A glioma cells and administered trimodal therapy initiated at 3 days post-ic. B, Wild-type mice were (ic.) 2 × 10⁵ GL261 in the right brain hemisphere and treated with BGB-5777, whole brain radiotherapy and PD-1 mAb. Mice surviving for 120 days were re-challenged with either 2 × 10⁵ unmodified GL261 or 1 × 10⁵ CT-2A cells in the left brain hemisphere. C, Treatment strategy and survival analysis of wild-type mice ic. 2 × 10⁵ unmodified GL261 cells and treated with trimodal therapy, with or without systemic administration of temozolomide (TMZ). D, Model for IDO1 inhibition synergizing with radiotherapy and PD-1 blockade to modulate antitumor immune response against GBM. (i) The GBM is associated with an impaired antitumor immune response due to potently immunosuppressive mechanisms, such as regulatory T cell (Treg) inhibition of antitumor effector T (Teff) cells. (ii) Host treatment with concurrent IDO1 enzyme inhibition, radiotherapy (RT) and systemic PD-1 mAb (iii) limits the ability of immunosuppressive cells to inhibit Teff cell antitumor activities. *P<0.05; ***P<0.001