Derek A. Wainwright

Durable therapeutic efficacy utilizing combinatorial blockade against IDO, CTLA-4 and PD-L1 in mice with brain tumors

Purpose: Glioblastoma (GBM) is the most common form of malignant glioma in adults. Although protected by both the blood–brain and blood–tumor barriers, GBMs are actively infiltrated by T cells. Previous work has shown that IDO, CTLA-4, and PD-L1 are dominant molecular participants in the suppression of GBM immunity. This includes IDO-mediated regulatory T-cell (Treg; CD4+CD25+FoxP3+) accumulation, the interaction of T-cell–expressed, CTLA-4, with dendritic cell-expressed, CD80, as well as the interaction of tumor- and/or macrophage-expressed, PD-L1, with T-cell–expressed, PD-1. The individual inhibition of each pathway has been shown to increase survival in the context of experimental GBM. However, the impact of simultaneously targeting all three pathways in brain tumors has been left unanswered. Experimental Design and Results: In this report, we demonstrate that, when dually challenged, IDO-deficient tumors provide a selectively competitive survival advantage against IDO-competent tumors. Next, we provide novel observations regarding tryptophan catabolic enzyme expression, before showing that the therapeutic inhibition of IDO, CTLA-4, and PD-L1 in a mouse model of well-established glioma maximally decreases tumor-infiltrating Tregs, coincident with a significant increase in T-cell–mediated long-term survival. In fact, 100% of mice bearing intracranial tumors were long-term survivors following triple combination therapy. The expression and/or frequency of T cell expressed CD44, CTLA-4, PD-1, and IFN-γ depended on timing after immunotherapeutic administration. Conclusions: Collectively, these data provide strong preclinical evidence that combinatorially targeting immunosuppression in malignant glioma is a strategy that has high potential value for future clinical trials in patients with GBM. Clin Cancer Res; 20(20); 5290–301. ©2014 AACR.

Blood‐Brain Barrier Permeable Gold Nanoparticles: An Efficient Delivery Platform for Enhanced Malignant Glioma Therapy and Imaging

The blood-brain barrier (BBB) remains a formidable obstacle in medicine, preventing efficient penetration of chemotherapeutic and diagnostic agents to malignant gliomas. Here, a transactivator of transcription (TAT) peptide-modified gold nanoparticle platform (TAT-Au NP) with a 5 nm core size is demonstrated to be capable of crossing the BBB efficiently and delivering cargoes such as the anticancer drug doxorubicin (Dox) and Gd(3+) contrast agents to brain tumor tissues. Treatment of mice bearing intracranial glioma xenografts with pH-sensitive Dox-conjugated TAT-Au NPs via a single intravenous administration leads to significant survival benefit when compared to the free Dox. Furthermore, it is demonstrated that TAT-Au NPs are capable of delivering Gd(3+) chelates for enhanced brain tumor imaging with a prolonged retention time of Gd(3+) when compared to the free Gd(3+) chelates. Collectively, these results show promising applications of the TAT-Au NPs for enhanced malignant brain tumor therapy and non-invasive imaging.

IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival

Purpose: Glioblastoma multiforme (GBM) is an aggressive adult brain tumor with a poor prognosis. One hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4+FoxP3+GITR+ regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain tumors and the impact on Treg recruitment. Experimental Design: To determine the clinical relevance of IDO expression in brain tumors, we first correlated patient survival to the level of IDO expression from resected glioma specimens. We also used novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-derived and peripheral IDO expression on Treg recruitment, GITR expression, and long-term survival was determined. Results: Downregulated IDO expression in glioma predicted a significantly better prognosis in patients. Coincidently, both IDO-competent and deficient mice showed a survival advantage bearing IDO-deficient brain tumors, when compared with IDO-competent brain tumors. Moreover, IDO deficiency was associated with a significant decrease in brain-resident Tregs, both in orthotopic and transgenic mouse glioma models. IDO deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term survival advantage conferred by IDO deficiency was lost in T-cell–deficient mice. Conclusions: These clinical and preclinical data confirm that IDO expression increases the recruitment of immunosuppressive Tregs that lead to tumor outgrowth. In contrast, IDO deficiency decreases Treg recruitment and enhances T-cell–mediated tumor rejection. Thus, the data suggest a critical role for IDO-mediated immunosuppression in glioma and support the continued investigation of IDO–Treg interactions in the context of brain tumors. Clin Cancer Res; 18(22); 6110–21. ©2012 AACR.

Fibrin-binding, peptide amphiphile micelles for targeting glioblastoma.

Glioblastoma-targeted drug delivery systems facilitate efficient delivery of chemotherapeutic agents to malignant gliomas, while minimizing systemic toxicity and side effects. Taking advantage of the fibrin deposition that is characteristic of tumors, we constructed spherical, Cy7-labeled, targeting micelles to glioblastoma through the addition of the fibrin-binding pentapeptide, cysteine–arginine–glutamic acid–lysine–alanine, or CREKA. Conjugation of the CREKA peptide to Cy7-micelles increased the average particle size and zeta potential. Upon intravenous administration to GL261 glioma bearing mice, Cy7-micelles passively accumulated at the brain tumor site via the enhanced permeability and retention (EPR) effect, and Cy7-CREKA-micelles displayed enhanced tumor homing via active targeting as early as 1 h after administration, as confirmed via in vivo and ex vivo imaging and immunohistochemistry. Biodistribution of micelles showed an accumulation within the liver and kidneys, leading to micelle elimination via renal clearance and the reticuloendothelial system (RES). Histological evaluation showed no signs of cytotoxicity or tissue damage, confirming the safety and utility of this nanoparticle system for delivery to glioblastoma. Our findings offer strong evidence for the glioblastoma-targeting potential of CREKA-micelles and provide the foundation for CREKA-mediated, targeted therapy of glioma.

CCL2 Produced by the Glioma Microenvironment Is Essential for the Recruitment of Regulatory T Cells and Myeloid-Derived Suppressor Cells

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671-82. ©2016 AACR.

Thymus-derived rather than tumor-induced regulatory T cells predominate in brain tumors

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an average survival time of 15 months. Previously, we and others demonstrated that CD4(+)FoxP3(+) regulatory T cells (Tregs) infiltrate human GBM as well as mouse models that recapitulate malignant brain tumors. However, whether brain tumor-resident Tregs are thymus-derived natural Tregs (nTregs) or induced Tregs (iTregs), by the conversion of conventional CD4(+) T cells, has not been established. To investigate this question, we utilized the i.c. implanted GL261 cell-based orthotopic mouse model, the RasB8 transgenic astrocytoma mouse model, and a human GBM tissue microarray. We demonstrate that Tregs in brain tumors are predominantly thymus derived, since thymectomy, prior to i.c. GL261 cell implantation, significantly decreased the level of Tregs in mice with brain tumors. Accordingly, most Tregs in human GBM and mouse brain tumors expressed the nTreg transcription factor, Helios. Interestingly, a significant effect of the brain tumor microenvironment on Treg lineage programming was observed, based on higher levels of brain tumor-resident Tregs expressing glucocorticoid-induced tumor necrosis factor receptor and CD103 and lower levels of Tregs expressing CD62L and CD45RB compared with peripheral Tregs. Furthermore, there was a higher level of nTregs in brain tumors that expressed the proliferative marker Ki67 compared with iTregs and conventional CD4(+) T cells. Our study demonstrates that future Treg-depleting therapies should aim to selectively target systemic rather than intratumoral nTregs in brain tumor-specific immunotherapeutic strategies.

Molecular Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy

Indoleamine 2, 3-dioxygenase 1 (IDO1), IDO2, and tryptophan 2, 3-dioxygenase (TDO) comprise a family of enzymes that catalyze the first- and rate-limiting step associated with the catabolic conversion of tryptophan (Trp) into kynurenine (Kyn). Through subsequent enzymatic and spontaneous reactions, Kyn is further converted into the energetic substrates, NAD+ and ATP, to fuel cellular metabolic functions. Coincidently, the depletion of Trp and accumulation of Kyn has been demonstrated to induce effector T-cell apoptosis/dysfunction and immunosuppressive regulatory T-cell induction, respectively. Similar to other immune checkpoints, IDO1 and TDO are suggested to be important targets for immunotherapeutic intervention. This is represented by the recent growth of efforts to inhibit the Trp-to-Kyn pathway as a means to control immunosuppression. Inhibitors currently in clinical trials, INCB024360, GDC-0919, indoximod, and an IDO1 peptide-based vaccine, are being evaluated for their efficacy against a wide range of cancers including melanoma, glioblastoma, non–small cell lung, pancreatic, and/or breast cancer, as well as metastatic disease. Despite the rapid development of potent clinical grade inhibitors, strategic questions remain. Here, we review the state of the literature with respect to current therapeutic inhibitors of tryptophan catabolism, evaluation of those efforts preclinically and clinically, compensatory changes that occur with therapeutic targeting, as well as newly recognized signaling features that raise critical questions to the field. Given the rapidly evolving interest in determining how IDO1/TDO, and to an unknown extent, IDO2, can be targeted for increasing cancer immunotherapeutic efficacy, we present a brief but comprehensive analysis that addresses critical questions, while highlighting the mechanics that remain to be explored. Clin Cancer Res; 21(24); 5427–33. ©2015 AACR.

Targeting Tregs in Malignant Brain Cancer: Overcoming IDO

One of the hallmark features of glioblastoma multiforme (GBM), the most common adult primary brain tumor with a very dismal prognosis, is the accumulation of CD4+CD25+Foxp3+ regulatory T cells (Tregs). Regulatory T cells (Tregs) segregate into two primary categories: thymus-derived natural Tregs (nTregs) that develop from the interaction between immature T cells and thymic epithelial stromal cells, and inducible Tregs (iTregs) that arise from the conversion of CD4+FoxP3− T cells into FoxP3 expressing cells. Normally, these Treg subsets complement one another’s actions by maintaining tolerance of self-antigens, thereby suppressing autoimmunity, while also enabling effective immune responses toward non-self-antigens, thus promoting infectious protection. However, Tregs have also been shown to be associated with the promotion of pathological outcomes, including cancer. In the setting of GBM, nTregs appear to be primary players that contribute to immunotherapeutic failure, ultimately leading to tumor progression. Several attempts have been made to therapeutically target these cells with variable levels of success. The blood brain barrier-crossing chemotherapeutics, temozolomide, and cyclophosphamide (CTX), vaccination against the Treg transcriptional regulator, FoxP3, as well as mAbs against Treg-associated cell surface molecules CD25, CTLA-4, and GITR are all different therapeutic approaches under investigation. Contributing to the poor success of past approaches is the expression of indoleamine 2,3-dioxygenase 1 (IDO), a tryptophan catabolizing enzyme overexpressed in GBM, and critically involved in regulating tumor-infiltrating Treg levels. Herein, we review the current literature on Tregs in brain cancer, providing a detailed phenotype, causative mechanisms involved in their pathogenesis, and strategies that have been used to target this population, therapeutically.

IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow-derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response.