Alan L. Chang

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.

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.

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.

The role of IDO in brain tumor immunotherapy

Malignant glioma comprises the majority of primary brain tumors. Coincidently, most of those malignancies express an inducible tryptophan catabolic enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). While IDO1 is not normally expressed at appreciable levels in the adult central nervous system, it’s rapidly induced and/or upregulated upon inflammatory stimulus. The primary function of IDO1 is associated with conversion of the essential amino acid, tryptophan, into downstream catabolites known as kynurenines. The depletion of tryptophan and/or accumulation of kynurenine has been shown to induce T cell deactivation, apoptosis and/or the induction of immunosuppressive programming via the expression of FoxP3. This understanding has informed immunotherapeutic design for the strategic development of targeted molecular therapeutics that inhibit IDO1 activity. Here, we review the current knowledge of IDO1 in brain tumors, pre-clinical studies targeting this enzymatic pathway, alternative tryptophan catabolic mediators that compensate for IDO1 loss and/or inhibition, as well as proposed clinical strategies and questions that are critical to address for increasing future immunotherapeutic effectiveness in patients with incurable brain cancer.

Dendritic Cell Based Vaccines that Utilize Myeloid Rather than Plasmacytoid Cells Offer a Superior Survival Advantage in Malignant Glioma

Dendritic cells (DCs) are professional APCs that are traditionally divided into two distinct subsets, myeloid DC (mDCs) and plasmacytoid DC (pDCs). pDCs are known for their ability to secrete large amounts of IFN-α. Apart from IFN-α production, pDCs can also process Ag and induce T cell immunity or tolerance. In several solid tumors, pDCs have been shown to play a critical role in promoting tumor immunosuppression. We investigated the role of pDCs in the process of glioma progression in the syngeneic murine model of glioma. We show that glioma-infiltrating pDCs are the major APC in glioma and are deficient in IFN-α secretion (p < 0.05). pDC depletion leads to increased survival of the mice bearing intracranial tumor by decreasing the number of regulatory T cells (Tregs) and by decreasing the suppressive capabilities of Tregs. We subsequently compared the ability of mDCs and pDCs to generate effective antiglioma immunity in a GL261-OVA mouse model of glioma. Our data suggest that mature pDCs and mDCs isolated from naive mice can be effectively activated and loaded with SIINFEKL Ag in vitro. Upon intradermal injection in the hindleg, a fraction of both types of DCs migrate to the brain and lymph nodes. Compared to mice vaccinated with pDC or control mice, mice vaccinated with mDCs generate a robust Th1 type immune response, characterized by high frequency of CD4+T-bet+ T cells and CD8+SIINFEKEL+ T cells. This robust antitumor T cell response results in tumor eradication and long-term survival in 60% of the animals (p < 0.001).

Heme oxygenase-1 protects regulatory T cells from hypoxia-induced cellular stress in an experimental mouse brain tumor model.

Two characteristic features of malignant gliomas (MG) are the presence of hypoxia and accumulation of regulatory T cells (Tregs). Heme-oxygenase-1 (HO1) is a cytoprotective enzyme expressed in high level by Tregs in glioma. In this study, we show that higher HO1 expression in Tregs is associated with increased survival under hypoxic conditions and that HO1 inhibitor, tin protoporphyrin (SnPP), abrogates the survival benefits. Moreover, SnPP preferentially eliminates Tregs and treatment with SnPP of tumor bearing mice significantly increases survival (23 to 31days (p<0.05)). Thus HO1 inhibition provides another alternative way of therapeutically targeting Tregs in MG.

Intratumoral oncolytic adenoviral treatment modulates the glioma microenvironment and facilitates systemic tumor-antigen-specific T cell therapy.

Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor and is associated with poor survival. Virotherapy is a promising candidate for the development of effective, novel treatments for GBM. Recent studies have underscored the potential of virotherapy in enhancing antitumor immunity despite the fact that its mechanisms remain largely unknown. Here, using a syngeneic GBM mouse model, we report that intratumoral virotherapy significantly modulates the tumor microenvironment. We found that intratumoral administration of an oncolytic adenovirus, AdCMVdelta24, decreased tumor-infiltrating CD4+ Foxp3+ regulatory T cells (Tregs) and increased IFNγ-producing CD8+ T cells in treated tumors, even in late stage disease in which a highly immunosuppressive tumor microenvironment is considered to be a significant barrier to immunotherapy. Importantly, intratumoral AdCMVdelta24 treatment augmented systemically transferred tumor-antigen-specific T cell therapy. Furthermore, mechanistic studies showed (1) downregulation of Foxp3 in Tregs that were incubated with media conditioned by virus-infected tumor cells, (2) downregulation of indoleamine 2,3 dioxygenase 1 (IDO) in glioma cells upon infection by AdCMVdelta24, and (3) reprograming of Tregs from an immunosuppressive to a stimulatory state. Taken together, our findings demonstrate the potency of intratumoral oncolytic adenoviral treatment in enhancing antitumor immunity through the regulation of multiple aspects of immune suppression in the context of glioma, supporting further clinical development of oncolytic adenovirus-based immune therapies for malignant brain cancer.

Neural stem cell-mediated delivery of oncolytic adenovirus

The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials. These clinical trials exploring SC‐mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that can provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally‐replicative adenoviruses and delivered to the tumor, whereupon they may evoke pronounced therapeutic efficacy. In this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for SC loading, and evaluate the optimized viral loading on SCs.

Fatty Acid Uptake in T Cell Subsets Using a Quantum Dot Fatty Acid Conjugate

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus, developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein, we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time, we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally, CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets, supporting the role of FA metabolism for Treg function. Furthermore, we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively, our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor, thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo.

Gene Therapy and Virotherapy of Gliomas

Despite many recent advances in the management of gliomas, such as aggressive surgical resection, chemoradiotherapy, antiangiogenic therapy, and molecular targeted therapy, the survival of patients with high-grade neoplasms remains dismal. Gene therapy and oncolytic virotherapy have emerged as highly promising strategies for treatment of malignant brain tumors due to recent progress in understanding of the underlying cancer biology as well as improved techniques for genetic modification of potential therapeutics.