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Purpose: Cancer immunotherapy offers hope of a highly specific nontoxic adjuvant treatment. Heat shock protein peptide complexes (HSPPCs) found in cancer cells carry tumor-specific antigenic proteins and can facilitate adaptive and innate immune responses. Here we show that peptides bound to a 96 kD chaperone protein (HSP-96) from brain tissue containing glioblastoma multiforme (GBM) can be used to safely immunize patients with recurrent GBM.Experimental Design: Multimodality immunomonitoring was completed on 12 patients with recurrent GBM before and after immunization with an autologous HSPPC vaccine derived from surgically resected tumor. Clinical endpoints included safety assessments and overall survival.Results: No adverse events attributable to the vaccine were found. Testing of peripheral blood leukocytes before and after vaccination revealed a significant peripheral immune response specific for the peptides bound to HSP-96, in 11 of the 12 patients treated. Brain biopsies of immune responders after vaccination revealed focal CD4, CD8, and CD56 IFNγ positive cell infiltrates, consistent with tumor site specific immune responses. Immune responders had a median survival of 47 weeks after surgery and vaccination, compared with 16 weeks for the single nonresponder.Conclusions: These data provide the first evidence in humans of individual patient-specific immune responses against autologous tumor derived peptides bound to HSP-96. Clin Cancer Res; 19(1); 205–14. ©2012 AACR.

PDF file - 42K, Figure S2. Glioma-derived soluble factors can further induce B7-H1 expression in non-autologous GBM patient peripheral monocytes, but not in autologous patient monocytes. Quantification of mean fluorescence intensity for B7-H1 staining measured by flow cytometry is shown for monocytes stimulated with IL-10 (10 ng/ml) or glioma-conditioned media (SF1U-CM). In monocytes from healthy control patients, B7-H1 intensity was significantly increased by IL-10, and further increased by conditioned media (* p

PDF file - 22K, Figure S1. Glioma cells induce B7-H1 expression in peripheral monocytes from healthy donors. Monocytes from healthy donors were cultured alone in normal media or in co-culture with U251 glioma cells in contact or through a 0.2μm pore filter. The percentage of B7-H1 expressing cells was determined by flow cytometry. Compared to normal monocytes, cells cultured with glioma cells through a filter (non-contact) have significantly more B7-H1 positive monocytes (* p < 0.005) and cells co-cultured in contact demonstrate a further significant increase in B7-H1 expression (vs. normal, ** p

PDF file, 48K, Overall Survival Characteristics of Treated Patients.

PDF file, 9905K, Representative phenotypic analysis of circulating lymphocytes in a clinical responder (Patient #009).

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PDF file, 17162K, Model of HSPPC-96 mechanism of action.

PDF file, 8781K, Representative phenotypic analysis of circulating lymphocytes in a clinical non-responder (Patient #003).

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PDF file, 29K, Vaccine Administration Schedule and Characteristics of Adverse Events.

Purpose: Gliomas are known to induce local and systemic immunosuppression, inhibiting T-cell–mediated cytotoxic responses to tumor growth. Tumor-associated macrophages are a significant component of the immune infiltrate in gliomas and may express immunosuppressive surface ligands, such as B7-H1.Experimental Design: Tumor and peripheral blood samples from patients with glioblastoma (GBM) were analyzed by flow cytometry to evaluate the expression of B7-H1 in circulating and tumor-infiltrating macrophages. Human monocytes from healthy patients were stimulated with conditioned media from glioma cells to evaluate B7-H1 expression. Production of interleukin (IL)-10 by stimulated monocytes was measured by ELISA, and stimulation with IL-10 alone was evaluated for the ability to induce B7-H1 expression. The effect of inhibiting IL-10 and its receptor on glioma-induced B7-H1 expression in monocytes was evaluated.Results: Circulating monocytes in patients with GBM had significantly increased expression of B7-H1 compared with healthy control patients. Tumor-associated macrophages from matched GBM tissue had even greater B7-H1 expression. Treatment of normal monocytes with glioma-conditioned media could significantly increase B7-H1 expression. Stimulation of monocytes with conditioned media resulted in substantial production of IL-10 and upregulation of the IL-10 receptor. Stimulation of monocytes with IL-10 alone could significantly increase B7-H1 expression, sufficient to induce T-cell apoptosis when cocultured with stimulated monocytes. Inhibition of IL-10 and the IL-10 receptor could knock down the effect of glioma media on B7-H1 by more than 50%.Conclusions: Gliomas can upregulate B7-H1 expression in circulating monocytes and tumor-infiltrative macrophages through modulation of autocrine/paracrine IL-10 signaling, resulting in an immunosuppressive phenotype. Clin Cancer Res; 19(12); 3165–75. ©2013 AACR.

BackgroundHuman immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression is regulated by constitutively active promoters, lentiviral payload gene expression is unregulated, and can result in potentially toxic quantities of proteins. Regulated delivery of lentivirally encoded proteins may allow localized or conditional therapeutic protein expression to support safe delivery of adoptively transferred, genetically modified cells with reduced capacity for systemic toxicities.MethodsIn this study, we engineered human macrophages to express genes regulated by hypoxia responsive elements included in the lentiviral promoter region to drive conditional lentiviral gene expression only under hypoxic conditions. We tested transduced macrophages cultured in hypoxic conditions for the transient induced expression of reporter genes and the secreted cytokine, interleukin-12. Expression of hypoxia-regulated genes was investigated both transcriptionally and translationally, and in the presence of human tumor cells in a slice culture system. Finally, hypoxia-regulated gene expression was evaluated in a subcutaneous humanized-mouse cancer model.ResultsEngineered macrophages were shown to conditionally and tranisently express lentivirally encoded gene protein products, including IL-12 in hypoxic conditions in vitro. On return to normoxic conditions, lentiviral payload expression returned to basal levels. Reporter genes under the control of hypoxia response elements were upregulated under hypoxic conditions in the presence of human colorectal carcinoma cells and in the hypoxic xenograft model of glioblastoma, suggesting utility for systemic engineered cell delivery capable of localized gene delivery in cancer.ConclusionsMacrophages engineered to express hypoxia-regulated payloads have the potential to be administered systemically and conditionally express proteins in tissues with hypoxic conditions. In contrast to immune cells that function or survive poorly in hypoxic conditions, macrophages maintain a proinflammatory phenotype that may support continued gene and protein expression when regulated by conditional hypoxia responsive elements and naturally traffic to hypoxic microenvironments, making them ideal vehicles for therapeutic payloads to hypoxic tissues, such as solid tumors. With the ability to fine-tune delivery of potent proteins in response to endogenous microenvironments, macrophage-based cellular therapies may therefore be designed for different disease settings.

Background Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), have a dismal prognosis, with less than 2% surviving 5 years postdiagnosis. The majority of DIPGs and all DMGs harbor mutations altering the epigenetic regulatory histone tail (H3 K27M). Investigations addressing DMG epigenetics have identified a few promising drugs, including the HDAC inhibitor (HDACi) panobinostat. Here, we use clinically relevant DMG models to identify and validate other effective HDACi and their biomarkers of response. Methods HDAC inhibitors were tested across biopsy-derived treatment-naïve in vitro and in vivo DMG models with biologically relevant radiation resistance. RNA sequencing was performed to define and compare drug efficacy and to map predictive biomarkers of response. Results Quisinostat and romidepsin showed efficacy with low nanomolar half-maximal inhibitory concentration (IC50) values (~50 and ~5 nM, respectively). Comparative transcriptome analyses across quisinostat, romidepsin, and panobinostat showed a greater degree of shared biological effects between quisinostat and panobinostat, and less overlap with romidepsin. However, some transcriptional changes were consistent across all 3 drugs at similar biologically effective doses, such as overexpression of troponin T1 slow skeletal type (TNNT1) and downregulation of collagen type 20 alpha 1 chain (COL20A1), identifying these as potential vulnerabilities or on-target biomarkers in DMG. Quisinostat and romidepsin significantly (P Conclusions Our data highlight the utility of treatment-naïve biopsy-derived models; establishes quisinostat and romidepsin as effective in vivo; illuminates potential mechanisms and/or biomarkers of DMG cell lethality due to HDAC inhibition; and emphasizes the need for brain tumor–penetrant versions of potentially efficacious agents.

BackgroundThough currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies.MethodsUsing lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies.ResultsHere, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins.ConclusionsOur data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

BackgroundTargeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose many barriers to immunotherapies, including therapy half-life and persistence, tumor penetrance, and targeting. Therapeutics delivered systemically may not traffic to the tumor site. If cellular therapies or drugs are able to access the tumor site, or can be delivered directly within the tumor, treatments may not persist for the duration necessary to reduce or eliminate tumor burden. An approach that allows durable and titratable local therapeutic protein delivery could improve antitumor efficacy while minimizing toxicities or unwanted on-target, off-tissue effects.MethodsIn this study, human monocyte-derived macrophages were genetically engineered to secrete a bispecific T cell engager (BiTE) specific to the mutated epidermal growth factor variant III (EGFRvIII) expressed by some GBM tumors. We investigated the ability of lentivirally modified macrophages to secrete a functional BiTE that can bind target tumor antigen and activate T cells. Secreted BiTE protein was assayed in a range of T cell functional assays in vitro and in subcutaneous and intracranial GBM xenograft models. Finally, we tested genetically engineered macrophages (GEMs) secreting BiTE and the proinflammatory cytokine interleukin (IL)-12 to amplify T cell responses in vitro and in vivo.ResultsTransduced human macrophages secreted a lentivirally encoded functional EGFRvIII-targeted BiTE protein capable of inducing T cell activation, proliferation, degranulation, and killing of antigen-specific tumor cells. Furthermore, BiTE secreting macrophages reduced early tumor burden in both subcutaneous and intracranial mouse models of GBM, a response which was enhanced using macrophages that were dual transduced to secrete both the BiTE protein and single chain IL-12, preventing tumor growth in an aggressive GBM model.ConclusionsThe ability of macrophages to infiltrate and persist in solid tumor tissue could overcome many of the obstacles associated with systemic delivery of immunotherapies. We have found that human GEMs can locally and constitutively express one or more therapeutic proteins, which may help recruit T cells and transform the immunosuppressive tumor microenvironment to better support antitumor immunity.

Background Diffuse intrinsic pontine glioma (DIPG) is a uniformly fatal CNS tumor diagnosed in 300 American children per year. Radiation is the only effective treatment and extends overall survival to a median of 11 months. Due to its location in the brainstem, DIPG cannot be surgically resected. Immunotherapy has the ability to target tumor cells specifically; however, little is known about the tumor microenvironment in DIPGs. We sought to characterize infiltrating immune cells and immunosuppressive factor expression in pediatric low- and high-grade gliomas and DIPG. Methods Tumor microarrays were stained for infiltrating immune cells. RNA was isolated from snap-frozen tumor tissue and Nanostring analysis performed. DIPG and glioblastoma cells were co-cultured with healthy donor macrophages, T cells, or natural killer (NK) cells, and flow cytometry and cytotoxicity assays performed to characterize the phenotype and function, respectively, of the immune cells. Results DIPG tumors do not have increased macrophage or T-cell infiltration relative to nontumor control, nor do they overexpress immunosuppressive factors such as programmed death ligand 1 and/or transforming growth factor β1. H3.3-K27M DIPG cells do not repolarize macrophages, but are not effectively targeted by activated allogeneic T cells. NK cells lysed all DIPG cultures. Conclusions DIPG tumors have neither a highly immunosuppressive nor inflammatory microenvironment. Therefore, major considerations for the development of immunotherapy will be the recruitment, activation, and retention of tumor-specific effector immune cells.

Adverse effects and toxicities related to standard treatments for brain tumors significantly reduce patients' quality of life. Although most immunotherapy approaches for solid tumors have not been successful, several early-phase clinical trials are beginning to reveal a potential role for immunotherapy in the treatment of brain tumors. In particular, methods that activate the innate immune system and induce a polyclonal anti-cancer response have demonstrated that brain tumors are susceptible to immune-mediated tumor destruction. Compared with conventional therapies, modulation of the immune system may improve both survivorship and quality of life during and following treatment. Areas covered: An overview of mechanisms of immunotherapy in the context of current treatments for adult and pediatric brain tumors is provided. Results from recent clinical trials will be discussed, focusing on the favorable safety and efficacy profiles of immunotherapeutics. Expert commentary: Although it is too early to judge the long-term safety of immunotherapy for the treatment of patients with brain tumors, early results suggest that these drugs are well-tolerated and may improve survival and quality of life. Importantly, approaches that activate an anti-tumor immune response lay the framework for iterative development of immunotherapies that can reliably treat patients with brain tumors.

Engineered immune cells are an exciting therapeutic modality which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering has been developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed “drugamers”. The fluorescein ligand facilitated stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporated prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustained anti-proliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system was demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporated a small molecule trans-gene activator, CMP8, that switched-on a degron-tagged gene circuit to provide temporal regulation of engineered T cell protein expression. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both anti-tumor and transgene-activating small molecule prodrugs.

In spite of their successes against hematologic malignancies, immunotherapeutic interventions for the treatment of patients with glioblastoma (GBM) have thus far been unsuccessful. This is in part due to the presence of a tumor microenvironment that fosters neoplastic growth and protects the tumor from destruction by the immune system. A novel genetically engineered macrophage-based platform has been developed with the potential to minimize the effects of the suppressive tumor microenvironment and improve innate and adaptive antitumor immune responses. A newly described lentiviral expression system was validated for the generation of transduced monocytes and monocyte-derived macrophages, and transgene expression was shown to be stable over the course of weeks to months, both in vitro and in a mouse xenograft model of GBM. Furthermore, the genetically engineered macrophages (GEMs) neither caused morbidity in animals nor contributed to accelerated tumor growth. The versatility of GEMs is also highlighted by showing that they can be engineered to secrete proteins that either reduce immune suppression, such as the soluble transforming growth factor beta receptor II, or promote immune cell activation, by expressing interleukin 21. There is also the potential to prevent GEM-mediated immune suppression by using the CRISPR system to knock out genes responsible for dysfunction of cytotoxic cells, including interleukin 10 and programmed death-ligand 1. Together, these results suggest that GEMs are an ideal cell type for transforming the tumor microenvironment and enhancing antitumor immunity. Importantly, it is anticipated that these findings will have broad applicability to other types of tumors with microenvironments that currently preclude successful immunotherapeutic approaches.

Background Most glioblastomas recur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an “abscopal effect,” whereby unirradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti–programmed cell death ligand 1 (PD-L1) demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remain unknown. Methods We modified an immune-competent, genetically driven mouse glioma model (forced platelet derived growth factor [PDGF] expression + phosphatase and tensin homolog loss) where a portion of the tumor burden is irradiated (PDGF) and another unirradiated luciferase-expressing tumor (PDGF + luciferase) is used as a readout of the abscopal effect following systemic anti–PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of epidermal growth factor receptor variant III (EGFRvIII) (PDGF + EGFRvIII). Statistical tests were two-sided. Results Following radiation of one lesion, anti–PD-L1 immunotherapy enhanced the abscopal response to the unirradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF + luciferase, n = 8), the abscopal response occurred via anti–PD-L1 driven, extracellular signal-regulated kinase–mediated, bone marrow–derived macrophage phagocytosis of adjacent unirradiated tumor cells, with modest survival implications (median survival 41 days vs radiation alone 37.5 days, P = 0.03). In PDGF-driven gliomas with tumor neoepitope (PDGF + EGFRvIII, n = 8), anti–PD-L1 enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs radiation alone 28 days, P = 0.001). Conclusion Our results indicate that anti–PD-L1 immunotherapy enhances a radiation- induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma.

Adult brain tumors establish an immunosuppressive tumor microenvironment as a modality of immune escape, with several immunotherapies designed to overcome this barrier. However, the relationship between tumor cells and immune cells in pediatric brain tumor patients is not as well-defined. In this study, we sought to determine whether the model of immune escape observed in adult brain tumors is reflected in patients with pediatric brain tumors by evaluating NKG2D ligand expression on tissue microarrays created from patients with a variety of childhood brain tumor diagnoses, and infiltration of Natural Killer and myeloid cells. We noted a disparity between mRNA and protein expression for the 8 known NKG2D ligands. Surprisingly, high-grade gliomas did not have increased NKG2D ligand expression compared to normal adjacent brain tissue, nor did they have significant myeloid or NK cell infiltration. These data suggest that pediatric brain tumors have reduced NK cell-mediated immune surveillance, and a less immunosuppressive tumor microenvironment as compared to their adult counterparts. These data indicate that therapies aimed to improve NK cell trafficking and functions in pediatric brain tumors may have a greater impact on anti-tumor immune responses and patient survival, with fewer obstacles to overcome.

Background Interleukin-10 (IL-10) is an immunosuppressive cytokine that inhibits innate and adaptive antitumor immune responses in the tumor microenvironment (TME). Antibody neutralization of IL-10 in organotypic tumor slice cultures (TSC) in colorectal cancer liver metastases (CRCLM) leads to cancer cell death through. To target IL-10 blockade to the TME, we genetically engineered macrophages (GEM) to produce an IL-10 neutralizing antibody which we hypothesized would reactivate tumor infiltrating immune cells and increase cancer cell death in TSC. Methods We created a lentiviral construct to transduce GEMs with a publicly available neutralizing IL-10 antibody sequence (BT-063). CD14+ monocytes were isolated from healthy donor peripheral blood mononuclear cells and differentiated into macrophages, which were transduced with lentivirus to create control (cGEMs) and anti-IL-10 GEMs (αIL10 GEMs). To test the efficacy of αIL10 GEMs, punch biopsies (6 mm) were obtained from resected CRCLM, sliced to 250 um thickness with a vibratome and placed in individual culture wells. Slices were treated in triplicates with either 20ug/mL of human monoclonal αIL10 (αIL10), 1e5 cGEMs or αIL10 GEMs + 20ug/mL αIL10. GEMs and tumor cells were visualized with a Leica SP8X confocal microscope and apoptosis was measured with SR-FLICA fluorescent poly-caspase assay kit. Supernatant interferon-gamma (IFN-γ) levels were measured with Bioplex immunoassay and IL-10 neutralizing antibody was quantified with an enzyme-linked immunosorbent assay. Results Allogenic GEMs were transduced with BT-063-T2A-CD19t at 31% efficiency. cGEMs and αIL10 GEMs persisted in tumor slice culture for at least 6 days. αIL10 GEM-treated slice cultures contained 6.6ng/mL and 6.5ng/mL of αIL10 antibody after 3 and 6 days, respectively. At day 6 in culture, cancer cell apoptosis was increased in the αIL10 treated groups with 7%, 15%, 25%, 31% and 39% of cells were apoptotic in the no treatment control, cGEM, αIL10, cGEM + αIL10 and αIL10 GEM groups, respectively (One-way ANOVA, p = 0.0004). Elevation in IFN-γ was observed on day 6 in slices treated cGEMs (7.2ng/mL+4.3) and αIL10 GEMs (9.2ng/mL+5) compared to isotype control. Conclusion GEMs transduced with lentivirus encoding IL-10 neutralizing antibody maintain its production and are associated with increased cancer cell apoptosis. Citation Format: Kevin P. Labadie, Shannon A. Kreuser, Katherine J. Brempelis, Xiuyun Jiang, Sara K. Daniel, Courtney A. Crane, Venu G. Pillarisetty. Neutralization of interleukin-10 by genetically engineered macrophages increase cancer cell death in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5545.

Cell-based immunotherapies for cancer, such as chimeric antigen receptor (CAR) T-cells, are effective against certain hematologic malignancies, but less reliably overcome the immunosuppressive tumor microenvironment (TME) of solid tumors to effectively cause cancer remission. Preclinical studies testing interleukin-12 (IL-12), an immune stimulating cytokine that activates T-cells and NK cells, as an anti-tumor agent have been promising. However, clinical trials have demonstrated that IL-12 is toxic if delivered systemically. To achieve local, low-dose delivery of IL-12, we have made genetically engineered macrophages (GEMs) to produce IL-12. In vitro, IL-12 GEMs stably produce IL-12 for at least one month. To evaluate their ability to stimulate an immune response and induce tumor cell death in an intact immune environment, we co-cultured them with tumor slices from both human colorectal cancer liver metastases and pancreatic ductal adenocarcinoma. In both tumor subtypes, the addition of IL-12 GEMs increased tumor cell death and generated an inflammatory response within the tumor slice, as evidenced by an increase in interferon-gamma and C-X-C Motif Chemokine Ligand 9 and 10, when compared to control GEMs. We demonstrate our GEM platform as a promising way to locally and sustainably deliver IL-12 to solid tumors to overcome the immunosuppressive TME.

Recent advances in cellular therapies for patients with cancer, including checkpoint blockade and ex vivo-expanded, tumor-specific T cells, have demonstrated that targeting the immune system is a powerful approach to the elimination of tumor cells. Clinical efforts have also demonstrated limitations, however, including the potential for tumor cell antigenic drift and neoantigen formation, which promote tumor escape and recurrence, as well as rapid onset of T cell exhaustion in vivo. These findings suggest that antigen unrestricted cells, such as natural killer (NK) cells, may be beneficial for use as an alternative to or in combination with T cell based approaches. Although highly effective in lysing transformed cells, to date, few clinical trials have demonstrated antitumor function or persistence of transferred NK cells. Several recent studies describe methods to expand NK cells for adoptive transfer, although the effects of ex vivo expansion are not fully understood. We therefore explored the impact of a clinically validated 12-day expansion protocol using a K562 cell line expressing membrane-bound IL-15 and 4-1BB ligand with high-dose soluble IL-2 on the phenotype and functions of NK cells from healthy donors. Following expansions using this protocol, we found expression of surface proteins that implicate preferential expansion of NK cells that are not fully mature, as is typically associated with highly cytotoxic NK cell subsets. Despite increased expression of markers associated with functional exhaustion in T cells, we found that ex vivo-expanded NK cells retained cytokine production capacity and had enhanced tumor cell cytotoxicity. The preferential expansion of an NK cell subset that is phenotypically immature and functionally pleiotropic suggests that adoptively transferred cells may persist better in vivo when compared with previous methods using this approach. Ex vivo expansion does not quell killer immunoglobulin-like receptor diversity, allowing responsiveness to various factors in vivo that may influence activation and inhibition. Collectively, our data suggest that in addition to robust NK cell expansion that has been described using this method, expanded NK cells may represent an ideal cell therapy that is longer lived, highly potent, and responsive to an array of activating and inhibitory signals.

Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.

BACKGROUND: Because of the potential to specifically eliminate tumor cells while leaving healthy tissue intact, immunotherapy is a particularly appealing treatment option for children with brain tumors. However, little is known about the tumor microenvironment (TME) in pediatric brain tumors, and its effect on the immune system is unclear. We have previously shown that in contrast to adult glioblastoma (GBM), which have extensive accumulation of a variety of suppressive immune cells, such as tumor-associated macrophages (TAMs), aggressive pediatric brain tumors such as medulloblastomas and high grade gliomas (HGG) do not have a similar accumulation of immune cells in the TME. We hypothesize that pediatric brain tumors escape recognition by the immune system as a result of defects in immune trafficking to the TME and/or failure to recognize the tumor. METHODS: Quantitative PCR for immunosuppressive factors was performed on pediatric brain tissue (non-tumor brain, pilocytic astrocytoma, or GBM, n=5 per group). Chemokine concentration in plasma (n=77) obtained at the time of diagnosis or during follow-up treatment was measured by bioplex analysis. Surface protein expression on patient leukocytes (n=40) was evaluated by flow cytometry. RESULTS: Pediatric brain tumors do not exhibit high levels of immunosuppressive factors such as PD-L1 or M-CSF relative to normal brain. Plasma samples from pediatric brain tumor patients have decreased concentrations of chemokines that attract suppressive immune cells to the tumor site relative to control patients without tumor. Furthermore, we did not observe a change in chemokines that attract cytotoxic T and NK cells, supporting the hypothesis that immune cell trafficking and routine surveillance of transformed cells may be impaired in an immature immune system. CONCLUSIONS: These data suggest that in pediatric patients, designing cell therapy products that educate cytotoxic effectors and improve their homing to tumor may be more important than overcoming immunosuppression in the TME.

Chimeric antigen receptor (CAR) T-cell therapy carries the promise of a broadly applicable, targeted, yet molecular pathway-independent, intervention for pediatric central nervous system (CNS) tumors. The BrainChild pipeline at Seattle Children’s utilizes engineered CAR T-cells directed against the surface epitopes HER2, EGFR, B7-H3 and IL13Ralpha2, which are commonly expressed in pediatric CNS tumors including high-grade glioma, diffuse intrinsic pontine glioma, medulloblastoma, and ependymoma. Using in vitro and in vivo preclinical modeling, we have optimized the efficacy and specificity of the CAR constructs. The extracellular target-specific scFv domain for the HER2-specific and EGFR-specific CARs are derived from trastuzumab and an EGFR806 antibody, respectively. Third-generation CAR T-cells with medium-length (HER2-CAR) and short (EGFR806-CAR) spacers coupled to an intracellular 4-1BBζ domain result in complete and durable tumor eradication in mouse xenograft CNS tumor models. We now are poised to begin enrolling Phase 1 clinical trials for children and young adults with recurrent or refractory CNS tumors expressing the respective target epitopes. We will deliver locoregional adoptive therapy with autologous CD4 and CD8 T cells lentivirally transduced with the optimized CAR constructs. Using a dose-escalation regimen, CAR T-cells will be injected on a weekly schedule via indwelling catheters into the tumor resection cavity or the ventricular system. The primary objectives are safety and feasibility, along with secondary and exploratory objectives to define the CAR T-cell distribution in the CSF and peripheral circulation, target epitope expression across tumor populations, progression free and overall survival, and biomarkers of anti-tumor CAR T-cell activity.

Tumor-associated macrophages are a complex and heterogeneous population of cells within the tumor microenvironment. In many tumor types, tumor-associated macrophage contribute toward tumor malignancy and are therefore a therapeutic target of interest. This progress report highlights three major strategies for regulating tumor-associated macrophage, emphasizing the role of biomaterials in these approaches. First, systemic methods for targeting tumor-associated macrophage are summarized and limitations to both passive and active targeting approaches considered. Second, lessons learned from the significant literature on wound healing and macrophage response to implanted biomaterials are discussed with the vision of applying these principles to localized, biomaterials-based modulation of tumor-associated macrophage. Finally, the developing field of engineered macrophages, including genetic engineering and integration with biomaterials or drug delivery systems, is examined. The report includes analysis of major challenges in the field along with exciting opportunities for the future of macrophage-based therapies in oncology.

BackgroundOutcomes for patients with recurrent glioblastoma multiforme (GBM) are poor and may be improved by immunotherapy. We investigated the safety and efficacy of an autologous heat-shock protein peptide complex-96 (HSPPC-96) vaccine for patients with recurrent GBM.MethodsIn this open-label, single-arm, phase II study, adult patients with surgically resectable recurrent GBM were given vaccine after gross total resection. The primary endpoint was overall survival at 6 months. Secondary endpoints included overall survival, progression-free survival, safety, and immune profiling. Outcome analyses were performed in the intention-to-treat and efficacy populations.ResultsBetween October 3, 2007 and October 24, 2011, 41 patients underwent gross total resection of recurrent GBM and received a median of 6 doses of HSPPC-96 vaccine. Following treatment, 90.2% of patients were alive at 6 months (95% confidence interval [CI]: 75.9-96.8) and 29.3% were alive at 12 months (95% CI: 16.6-45.7). Median overall survival was 42.6 weeks (95% CI: 34.7-50.5). Twenty-seven (66%) patients were lymphopenic prior to therapy, and patients with lymphocyte counts below the cohort median demonstrated decreased overall survival (hazard ratio: 4.0; 95% CI: 1.4-11.8; P = .012). There were no treatment-related deaths. There were 37 serious (grades 3-5) adverse events reported, with 17 attributable to surgical resection and a single grade 3 constitutional event related to the vaccine.ConclusionThe HSPPC-96 vaccine is safe and warrants further study of efficacy for the treatment of recurrent GBM. Significant pretreatment lymphopenia may impact the outcomes of immunotherapy and deserves additional investigation.

Purpose: Gliomas are known to induce local and systemic immunosuppression, inhibiting T-cell–mediated cytotoxic responses to tumor growth. Tumor-associated macrophages are a significant component of the immune infiltrate in gliomas and may express immunosuppressive surface ligands, such as B7-H1. Experimental Design: Tumor and peripheral blood samples from patients with glioblastoma (GBM) were analyzed by flow cytometry to evaluate the expression of B7-H1 in circulating and tumor-infiltrating macrophages. Human monocytes from healthy patients were stimulated with conditioned media from glioma cells to evaluate B7-H1 expression. Production of interleukin (IL)-10 by stimulated monocytes was measured by ELISA, and stimulation with IL-10 alone was evaluated for the ability to induce B7-H1 expression. The effect of inhibiting IL-10 and its receptor on glioma-induced B7-H1 expression in monocytes was evaluated. Results: Circulating monocytes in patients with GBM had significantly increased expression of B7-H1 compared with healthy control patients. Tumor-associated macrophages from matched GBM tissue had even greater B7-H1 expression. Treatment of normal monocytes with glioma-conditioned media could significantly increase B7-H1 expression. Stimulation of monocytes with conditioned media resulted in substantial production of IL-10 and upregulation of the IL-10 receptor. Stimulation of monocytes with IL-10 alone could significantly increase B7-H1 expression, sufficient to induce T-cell apoptosis when cocultured with stimulated monocytes. Inhibition of IL-10 and the IL-10 receptor could knock down the effect of glioma media on B7-H1 by more than 50%. Conclusions: Gliomas can upregulate B7-H1 expression in circulating monocytes and tumor-infiltrative macrophages through modulation of autocrine/paracrine IL-10 signaling, resulting in an immunosuppressive phenotype. Clin Cancer Res; 19(12); 3165–75. ©2013 AACR.

Purpose: Cancer immunotherapy offers hope of a highly specific nontoxic adjuvant treatment. Heat shock protein peptide complexes (HSPPCs) found in cancer cells carry tumor-specific antigenic proteins and can facilitate adaptive and innate immune responses. Here we show that peptides bound to a 96 kD chaperone protein (HSP-96) from brain tissue containing glioblastoma multiforme (GBM) can be used to safely immunize patients with recurrent GBM. Experimental Design: Multimodality immunomonitoring was completed on 12 patients with recurrent GBM before and after immunization with an autologous HSPPC vaccine derived from surgically resected tumor. Clinical endpoints included safety assessments and overall survival. Results: No adverse events attributable to the vaccine were found. Testing of peripheral blood leukocytes before and after vaccination revealed a significant peripheral immune response specific for the peptides bound to HSP-96, in 11 of the 12 patients treated. Brain biopsies of immune responders after vaccination revealed focal CD4, CD8, and CD56 IFNγ positive cell infiltrates, consistent with tumor site specific immune responses. Immune responders had a median survival of 47 weeks after surgery and vaccination, compared with 16 weeks for the single nonresponder. Conclusions: These data provide the first evidence in humans of individual patient-specific immune responses against autologous tumor derived peptides bound to HSP-96. Clin Cancer Res; 19(1); 205–14. ©2012 AACR.

Recent years have seen rapid growth in cancer treatments that enhance the anti-tumor activities of the immune system. Collectively known as immunotherapy, modulation of the immune system has shown success treating some hematological malignancies, but has yet to be successfully applied to the treatment of patients with brain tumors.This review highlights mechanistic insights from murine studies and compiled recent clinical trial data, focusing on the most aggressive brain tumor, glioblastoma (GBM). The field has recently accumulated a critical mass of data, and we discuss past treatment failures in the context of newly developed approaches now entering clinical trials. This article provides an overview of the immunotherapeutic armamentarium currently in development for the treatment of patients with GBM, who are in dire need of safe and effective therapies. Expert commentary: Themes that emerge include the importance of mitigating the effects of an immunosuppressive tumor microenvironment and the potential for innate immune cell activation to enhance cytotoxic anti-tumor activity. Consideration of these studies as a collective may inform the design of new immunotherapies, as well as the immune monitoring protocols for patients participating in clinical trials.

In patients with glioma, the tumor microenvironment can significantly impact pro-inflammatory immune cell functions. However, the mechanisms by which this occurs are poorly defined. Because immunosuppressive regulatory T cells (Treg) are over represented in the tumor microenvironment compared with peripheral blood, we hypothesized that the tumor may have an effect on Treg survival, migration, expansion, and/or induction of a regulatory phenotype from non-Treg conventional CD4+ T cells. We defined the impact of soluble factors produced by tumor cells on Treg from healthy patients in vitro to determine mechanisms by which gliomas influence T cell populations. We found that tumor-derived soluble factors allowed for preferential proliferation and increased chemotaxis of Treg, compared with conventional T cells, indicating that these mechanisms may contribute to the increased Treg in the tumor microenvironment. Conventional T cells also exhibited a significantly increased expression of pro-apoptotic transcripts in the presence of tumor-derived factors, indicating that survival of Treg in the tumor site is driven by exposure to soluble factors produced by the tumor. Together, these data suggest that tumor burden may induce increased Treg infiltration, proliferation, and survival, negating productive anti-tumor immune responses in patients treated with immunotherapies. Collectively, our data indicate that several mechanisms of Treg recruitment and retention in the tumor microenvironment exist and may need to be addressed to improve the specificity of immunotherapies seeking to eliminate Treg in patients with glioma.

Immunotherapy approaches for glioblastoma multiforme have been thus far largely unsuccessful, suggesting unappreciated complexity in glioma biology and immunology. The intra-tumoral heterogeneity of these intrinsic brain tumors results in therapies killing only a subset of the tumor cells; therefore, therapeutic success will require achieving and optimizing an “abscopal effect” where tumor cells not specifically targeted are recognized and attacked as bystanders by the immune system. We have modified an immune-competent, genetically-driven mouse glioma model where a portion of the tumor burden is treated and another untreated portion is used as a readout of therapeutic efficacy. We find that following radiation of one lesion, anti-PD-L1 therapy enhances the abscopal response (macrophages and T-cells) to the un-irradiated lesion. In gliomas with few baseline T-cells, the anti-PD-L1-enhanced abscopal response occurs as an anti-PD-L1-driven, macrophage-associated, and ERK-dependent increase in phagocytosis of tumor cells. Our results indicate that combined radiation and anti-PD-L1 therapy for gliomas results in peripherally-derived macrophages being responders in tumors with few baseline T-cells in the microenvironment.

Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal brain tumor diagnosed in approximately 300 children in the US each year. Radiation modestly extends patient survival and provides temporary relief from symptoms, however, surgical resection is impossible due to the tumor’s location in the brainstem, and no chemotherapeutics have shown any efficacy; death generally occurs less than a year after diagnosis. Due to its ability to specifically eliminate tumor cells while leaving healthy tissue intact, immunotherapy could be a major breakthrough in the treatment of DIPG. As a first step towards the rational development of DIPG-specific cellular immunotherapy, we have characterized the immune microenvironment of DIPG tumors using IHC analysis of TMAs and Nanostring transcript analysis of frozen tumor samples. In contrast to pediatric and adult high-grade cortical gliomas (HGGs), DIPGs exhibit minimal infiltration with CD163+ myeloid cells or CD8+ T cells, consistent with their low mutational burden. Furthermore, unlike adult HGGs in particular, DIPG tumors had low levels of classical immunosuppressive factors including PD-L1, TGFβ, and IL-10. To directly examine the effects of DIPG cells on immune cells, we co-cultured primary tumor cells with immune cells from healthy donors. DIPG tumor cells did not polarize macrophages towards a pro-tumor phenotype, while U87 adult GBM cells did. Furthermore, DIPG cells did not slow T cell proliferation or prevent IFNγ production, while U87 cells did. Together, this suggests that although DIPG tumors are not inducing an immune response, adoptively transferred anti-tumor immune cells will not need to overcome an immunosuppressive microenvironment.

The antiapoptotic protein FLIPS is a key suppressor of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homologue (PTEN)–Akt–atrophin-interacting protein 4 (AIP4) pathway regulates FLIPS ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. Here, we report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIPS stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Overexpression of wild-type USP8, but not catalytically inactive USP8, increased FLIPS ubiquitination, decreased FLIPS half-life, decreased FLIPS steady-state levels, and decreased TRAIL resistance, whereas short interfering RNA (siRNA)–mediated suppression of USP8 levels had the opposite effect. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIPS ubiquitination, USP8 seemed to control FLIPS ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIPS E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIPS interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIPS levels by USP8 overexpression was reversed by the introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIPS stability and TRAIL sensitivity. Cancer Res; 70(12); 5046–53. ©2010 AACR.

The central nervous system (CNS) has evolved as an immune-privileged site to protect its vital functions from damaging immune-mediated inflammation. There must be a CNS-adapted system of surveillance that continuously evaluates local changes in the nervous system and communicates to the peripheral immune system during an injury or a disease. Recent advances leading to a better understanding of the CNS disease processes has placed microglia, the CNS-based resident macrophages, at center stage in this system of active surveillance. Evidence points to microglia cells contributing to the immunosuppressive environment of gliomas and actually promoting tumor growth. Microglia accumulation exists in almost every CNS disease process, including CNS tumors. This article discusses the role of microglia in CNS immunity and highlights key advances made in glioma immunology.

B7 homolog 1 (B7-H1) is a recently discovered immunoresistance protein that is regulated posttranscriptionally after PTEN loss in malignant glioma, a deadly form of brain tumor. Here, the impact of gamma-interferon-mediated activation of B7-H1 was investigated in glioblastoma patients with PTEN loss. Lymphocytes and T cells were selected for apoptosis assays after 1 : 1 coculture with autologous glioma cells. Gamma interferon treatment of PTEN-deficient tumors resulted in superinduction of B7-H1 protein that correlated with increased T-cell apoptosis, an effect dependent upon activation of the PI3-kinase pathway. The combination of PTEN loss and gamma-interferon exposure in glioblastoma patients results in an exceptionally immunoresistant phenotype that may negate adaptive immunity through induction of T-cell apoptosis.

The activating receptor NKG2D, expressed by natural killer (NK) cells and CD8(+) T cells, has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8(+) T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients, NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-beta, suggesting that blocking of this cytokine may have therapeutic benefit.

Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIPS, and confers resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIPS protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIPS polyubiquitination. FLIPS half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIPS E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIPS or with the FLIPS-containing death inducing signal complex. Small interfering RNA–mediated suppression of AIP4 levels in PTEN WT cells decreased FLIPS ubiquitination, prolonged FLIPS half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIPS steady-state levels, and suppressed FLIPS ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIPS ubiquitination, FLIPS stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability. [Cancer Res 2009;69(20):7911–6]

Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.

Microglia are the macrophage population residing in the parenchyma of the central nervous system (CNS), and are thought to play critical roles in CNS development, homeostasis and defense against pathogens. Microglia are capable of rapidly responding to microbial pathogens through engagement of their Toll-like receptors (TLRs). We first compared the efficiency of these responses in primary microglia acutely isolated from adult and neonatal mice. While the cytokine and chemokine responses of adult microglia were generally higher than those of neonatal cells stimulated ex vivo through TLRs, the nitric oxide response of neonatal microglia was markedly enhanced relative to the adult cells. We then went on to identify culture conditions such as exposure to M-SCF or GM-CSF that markedly enhanced the nitric oxide response of microglia, particularly those from the adult CNS. Finally, we demonstrate that the differential nitric oxide response of neonatal and adult microglia is not only limited to the mouse, but also extends to rat microglia.

Cancer immunoresistance and immune escape may play important roles in tumor progression and pose obstacles for immunotherapy. Expression of the immunosuppressive protein B7 homolog 1 (B7-H1), also known as programmed death ligand-1 (PD-L1), is increased in many pathological conditions, including cancer. Here we show that expression of the gene encoding B7-H1 increases post transcriptionally in human glioma after loss of phosphatase and tensin homolog (PTEN) and activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway. Tumor specimens from individuals with glioblastoma multiforme (GBM) had levels of B7-H1 protein that correlated with PTEN loss, and tumor-specific T cells lysed human glioma targets expressing wild-type PTEN more effectively than those expressing mutant PTEN. These data identify a previously unrecognized mechanism linking loss of the tumor suppressor PTEN with immunoresistance, mediated in part by B7-H1.