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1.  Enhanced Effector Responses in Activated CD8+ T Cells Deficient in Diacylglycerol Kinases 
Cancer research  2013;73(12):3566-3577.
Recent clinical trials have shown promise in the use of chimeric antigen receptor(CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical utility and improve their efficacy. We hypothesized that, since CAR action requires proteins essential for TCR signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin demonstrated greatly enhanced cytokine production and cytotoxicity when co-cultured with a murine mesothelioma line that stably expresses mesothelin. Additionally, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs.
PMCID: PMC3686869  PMID: 23576561
diacylglycerol kinase; chimeric antigen receptor; T cell receptor; tumor; mesothelin
Clinics in chest medicine  2011;32(4):865-885.
Both advanced stage lung cancer and malignant pleural mesothelioma are associated with a poor prognosis. Although there have been advances in treatment regimens for both diseases, these have had only a modest effect on their progressive course. Gene therapy for thoracic malignancies represents a novel therapeutic approach and has been evaluated in a number of clinical trials over the last two decades. Strategies have included induction of apoptosis, tumor suppressor gene replacement, suicide gene expression, cytokine based therapy, various vaccination approaches, and adoptive transfer of modified immune cells. This review will consider the clinical results, limitations, and future directions of gene therapy trials for thoracic malignancies.
PMCID: PMC3210443  PMID: 22054892
Gene Therapy; Immunotherapy; Lung cancer; Mesothelioma
3.  Overcoming intrinsic inhibitory pathways to augment the antineoplastic activity of adoptively transferred T cells 
Oncoimmunology  2013;2(11):e26492.
Effector T cells become rapidly inactivated after antigen exposure due to extracellular as well as intrinsic signals. We have recently demonstrated that the deletion of diacylglycerol kinases, intrinsic inhibitors of T-cell signaling, enhances the activity of adoptively transferred T cells expressing a chimeric antigen receptor (CAR) specific for a tumor-associated antigen.
PMCID: PMC3897565  PMID: 24490126
Chimeric Antigen Receptor; T cell hypofunction; diacylglycerol kinase; tumor immunosuppression; tumor microenvironment
4.  Immunological effects of the TGFβ-blocking antibody GC1008 in malignant pleural mesothelioma patients 
Oncoimmunology  2013;2(8):e26218.
We evaluated a neutralizing anti-TGFβ antibody (GC1008) in cancer patients with malignant pleura mesothelioma (MPM). The goal of this study was to assess immunoregulatory effects in relation to clinical safety and clinical response. Patients with progressive MPM and 1–2 prior systemic therapies received GC1008 at 3mg/kg IV over 90 min every 21 d as part of an open-label, two-center Phase II trial. Following TGFβ blockade therapy, clinical safety and patient survival were monitored along with the effects of anti-TGFβ antibodies on serum biomarkers and peripheral blood mononuclear cells (PBMC). Although designed as a larger trial, only 13 patients were enrolled when the manufacturer discontinued further development of the antibody for oncology indications. All participants tolerated therapy. Although partial or complete radiographic responses were not observed, three patients showed stable disease at 3 mo. GC1008 had no effect in the expression of NK, CD4+, or CD8+ T cell activating and inhibitory markers, other than a decrease in the expression of 2B4 and DNAM-1 on NK cells. However, serum from 5 patients showed new or enhanced levels of antibodies against MPM tumor lysates as measured by immunoblotting. Patients who produced anti-tumor antibodies had increased median overall survival (OS) (15 vs 7.5 mo, p < 0.03) compared with those who did not. To our knowledge, these data represent the first immune analysis of TGFβ- blockade in human cancer patients.
PMCID: PMC3812201  PMID: 24179709
GC1008; anti-TGFβ antibody; antibody therapy; clinical trial; immunotherapy; malignant mesothelioma
5.  Activation of Mitogen-Activated Protein Kinases by 5, 6-Dimethylxanthenone-4-Acetic Acid (DMXAA) Plays an Important Role in Macrophage Stimulation 
Biochemical pharmacology  2011;82(9):1175-1185.
The small molecule anti-tumor agent, 5, 6-dimethylxanthenone-4-acetic acid (DMXAA, now called Vadimezan) is a potent macrophage and dendritic cell activating agent that, in the murine system, results in the release of large amounts of cytokines and chemokines. The mechanisms by which this release is mediated have not been fully elucidated. The mitogen-activated protein kinase (MAPK) pathways plays an important role in the regulation of proinflammatory cytokines, such as, TNFα, IL-1β, as well as the responses to extracellular stimuli, such as, lipopolysaccharide (LPS). The results of this study demonstrate that DMXAA activates three members of mitogen-activated protein kinase (MAPK) superfamily, namely p38 MAPK, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), and c-Jun N-terminal kinases (JNKs) via a RIP2-independent mechanism in murine macrophages. By using selective inhibitors of MAPKs, this study confirms that both activated p38/MK2 pathways and ERK1/2 MAPK play a significant role in regulation of both TNF-α and IL-6 protein production induced by DMXAA at the post-transcriptional level. Our findings also show that Interferon-γ priming can dramatically augment TNF-α protein secretion induced by DMXAA through enhancing activation of multiple MAPKs pathways at the post-transcriptional level. This study expands current knowledge on mechanisms of how DMXAA acts as a potent anti-tumor agent in murine system and also provides useful information for further study on the mechanism of action of this potential anti-tumor compound in human macrophages.
PMCID: PMC3191304  PMID: 21819972
MAPK; post-transcriptional regulation; TNFα; DMXAA; proinflammatory cytokines
6.  T cells expressing chimeric antigen receptors can cause anaphylaxis in humans 
T cells can be redirected to overcome tolerance to cancer by engineering with integrating vectors to express a chimeric antigen receptor (CAR). In preclinical models, we have previously demonstrated that transfection of T cells with messenger RNA (mRNA) coding for a CAR is an alternative strategy that has antitumor efficacy and the potential to evaluate the on-target off-tumor toxicity of new CAR targets safely due to transient mRNA CAR expression. Here, we report the safety observed in four patients treated with autologous T cells that had been electroporated with mRNA coding for a CAR derived from a murine antibody to human mesothelin. Due to the transient nature of CAR expression on the T cells, subjects in the clinical study were given repeated infusions of the CAR-T cells in order to assess their safety. One subject developed anaphylaxis and cardiac arrest within minutes of completing the 3rd infusion. Although human anti-mouse IgG antibodies have been known to develop with CAR-transduced T cells, they have been thought to have no adverse clinical consequences. This is the first description of clinical anaphylaxis resulting from CAR-modified T cells, most likely through IgE antibodies specific to the CAR. These results indicate that the potential immunogenicity of CARs derived from murine antibodies may be a safety issue for mRNA CARs, especially when administered using an intermittent dosing schedule.
PMCID: PMC3888798  PMID: 24432303
Journal of critical care  2011;27(5):522.e11-522.e17.
Endocan is a proteoglycan expressed by endothelial cells in the lung which may inhibit leukocyte recruitment and thus prevent the development of acute lung injury (ALI). We tested the association of serum endocan levels with subsequent development of ALI after major trauma.
Materials and Methods
Single-center nested case control study within a prospective cohort study of major trauma patients. Using an ELISA test, we measured endocan levels from admission serum in 24 controls (no ALI) and 24 cases (ALI within 5 days of trauma). Multivariable logistic regression was used to test the association of admission serum endocan levels with subsequent ALI.
Patients who developed ALI had lower levels of endocan on admission (mean 3.5 ± 1.4 ng/mL vs. 4.9 ± 2.6 ng/mL in controls, p=0.02). For each 1-unit increase in serum endocan level, the odds ratio for ALI development decreased (0.69, 95% confidence interval (CI): 0.49, 0.97, p=0.03). Lower endocan levels remained associated with a higher incidence of ALI after adjustment for age and illness severity.
Lower levels of serum endocan on admission are associated with subsequent development of ALI in trauma patients. These observations may be explained by endocan-mediated blockade of leukocyte recruitment in the lung.
PMCID: PMC3790584  PMID: 21958978
Trauma; acute respiratory distress syndrome; acute lung injury; biomarkers; endothelium
8.  Thymidylate Synthase and Folyl-polyglutamate synthase (FPGS) Are Not Clinically Useful Markers of Response to Pemetrexed [Pem] in Patients with Malignant Pleural Mesothelioma [MPM] 
Thymidylate synthase (TS) is a potential predictor of outcome after pemetrexed (Pem) in patients with malignant pleural mesothelioma (MPM), and assays measuring TS levels are commercially marketed. The goal of this study was to further evaluate the value of TS and to study another potential biomarker of response, the enzyme, folyl-polyglutamate synthase (FPGS), which activates Pem intracellularly.
Patients and Methods
Levels of TS and FPGS were semi-quantitatively determined immunohistochemically using H-scores on tissue samples from 85 MPM patients receiving Pem as primary therapy. H-score was correlated with radiographic disease control rate (DCR), time to progression (TTP) and overall survival (OS). In addition, expression levels of TS and FPGS in MPM cell lines were determined using immunoblotting and correlated with their sensitivity to Pem-induced cell death.
H-scores from patients with disease control versus progressive disease showed extensive overlap. There were no significant correlations of DCR, TTP, or OS to either TS levels (p = 0.73, 0.93, and 0.59, respectively), FPGS levels (p = 0.95, 0.77 and 0.43 respectively) or the ratio of FPGS/TS using the median scores of each test as cutoffs. There was no correlation between TS or FPGS expression and chemosensitivity of mesothelioma cells to Pem in vitro.
Although previous retrospective data suggest that TS and FPGS expression might be potential markers of Pem efficacy in MPM, our data indicate these markers lack sufficient predictive value in individual patients and should not be used to guide therapeutic decisions in the absence of prospective studies.
PMCID: PMC3601580  PMID: 23486267
9.  Inhibition of p300 impairs Foxp3+ T-regulatory cell function and promotes anti-tumor immunity 
Nature medicine  2013;19(9):1173-1177.
Foxp3+ T-regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity, but can also curtail host immune responses to various types of tumors1,2. Foxp3+ Tregs are therefore considered promising targets to enhance anti-tumor immunity, and efforts are underway to develop approaches for their therapeutic modulation. However, while studies showing that Foxp3+ Treg depletion experimentally can enhance anti-tumor responses provide proof-of-principle, they lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and non-histone proteins3,4. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (Ep300, KAT3B), in Foxp3+ Tregs, increased TCR-induced apoptosis in Tregs, impaired Treg suppressive function and peripheral Treg induction, and limited tumor growth in immunocompetent, but not in immunodeficient, hosts. Our data thereby demonstrate that p300 is important for Foxp3+ Treg function and homeostasis in vivo and in vitro, and identify novel mechanisms by which appropriate small molecule inhibitors can diminish Treg function without overtly impairing T-effector (Teff) cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.
PMCID: PMC3793393  PMID: 23955711
10.  Expression of Interferon-β Enhances Both Efficacy and Safety of Oncolytic Vesicular Stomatitis Virus for Therapy of Mesothelioma 
Cancer research  2009;69(19):7713-7720.
Our pre-clinical and clinical trials using a replication-defective adenoviral vector expressing IFN-β have shown promising results for the treatment of malignant mesothelioma. Based on the hypotheses that a replication-competent Vesicular Stomatitis Virus (VSV) oncolytic vector would transduce more tumor cells in vivo, that co-expression of the immunostimulatory IFN-β gene would enhance the immune-based effector mechanisms associated both with regression of mesotheliomas and with VSV-mediated virotherapy, and that virus-derived IFN-β would add further safety to the VSV platform, we tested the use of IFN-β as a therapeutic transgene expressed from VSV as a novel treatment for mesothelioma. VSV-IFN-β showed significant therapy against AB12 murine mesotheliomas in the context of both local and loco-regional viral delivery. Biologically active IFN-β expressed from VSV added significantly to therapy compared to VSV alone, dependent in part upon host CD8+ T-cell responses. Immune monitoring suggested that these anti-tumor T-cell responses may be due to a generalised T-cell activation rather than the priming of tumor antigen-specific T-cell responses. Finally, IFN-β also added considerable extra safety to the virus by providing protection from off-target viral replication in non-tumor tissues and protected SCID mice from developing lethal neurotoxicity. The enhanced therapeutic index provided by the addition of IFN-β to VSV therefore provides a powerful justification for the development of this virus for future clinical trials.
PMCID: PMC3891512  PMID: 19773437
VSV; interferon-β; mesothelioma; oncolytic virotherapy
11.  Evaluation of an Attenuated Vesicular Stomatitis Virus Vector Expressing Interferon-β for Use in Malignant Pleural Mesothelioma: Heterogeneity in Interferon Responsiveness Defines Potential Efficacy 
Human Gene Therapy  2009;21(1):51-64.
Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSV.IFN-β). We conducted this study to determine the ability of VSV.IFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-β did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-β. In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-β protein and no IFN- or VSV-induced changes in PKR, MxA, and 2′5′-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-β by immunostaining for the presence of p48 protein.
PMCID: PMC2829454  PMID: 19715403
12.  Modifying tumor-associated macrophages 
Oncoimmunology  2013;2(12):e26620.
The therapeutic success of immunotherapy requires specific alterations of the tumor microenvironment and/or the inhibition of tumor-elicited immunosuppression. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment. We have recently shown that modulating TAMs dramatically augments the efficacy of immunotherapy. TAM-activating agents should hence be considered as an addition to immunotherapy in future clinical trials.
PMCID: PMC3902117  PMID: 24498549
tumor associated macrophages; immunotherapy; lung cancer; DMXAA; vaccines
13.  Thrombospondin-1 mediates oncogenic Ras–induced senescence in premalignant lung tumors  
The Journal of Clinical Investigation  2013;123(10):4375-4389.
Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1–/– background. We found that oncogenic Kras–induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.
PMCID: PMC3784530  PMID: 24018559
14.  The Rac1 splice form Rac1b promotes K-ras-induced lung tumorigenesis 
Oncogene  2012;32(7):903-909.
Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. While expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis.
PMCID: PMC3384754  PMID: 22430205
15.  The timing of TGF-β inhibition affects the generation of antigen-specific CD8+ T Cells 
BMC Immunology  2013;14:30.
Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.
BALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.
Inhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).
These studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.
PMCID: PMC3725164  PMID: 23865808
Malignant mesothelioma; Tumor immunology; Immune suppression; TGF-β; CD8+ Cytotoxic T cell
16.  Prevention of Alveolar Destruction and Airspace Enlargement in a Mouse Model of Pulmonary Lymphangioleiomyomatosis (LAM) 
Science translational medicine  2012;4(154):154ra134.
Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic disease characterized by neoplastic growth of atypical smooth muscle–like LAM cells, destruction of lung parenchyma, obstruction of lymphatics, and formation of lung cysts, leading to spontaneous pneumothoraces (lung rupture and collapse) and progressive loss of pulmonary function. The disease is caused by mutational inactivation of the tumor suppressor gene tuberous sclerosis complex 1 (TSC1) or TSC2. By injecting TSC2-null cells into nude mice, we have developed a mouse model of LAM that is characterized by multiple random TSC2-null lung lesions, vascular endothelial growth factor–D expression, lymphangiogenesis, destruction of lung parenchyma, and decreased survival, similar to human LAM. The mice show enlargement of alveolar airspaces that is associated with progressive growth of TSC2-null lesions in the lung, up-regulation of proinflammatory cytokines and matrix metalloproteinases (MMPs) that degrade extracellular matrix, and destruction of elastic fibers. TSC2-null lesions and alveolar destruction were differentially inhibited by the macrolide antibiotic rapamycin (which inhibits TSC2-null lesion growth by a cytostatic mechanism) and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin (which inhibits growth of TSC2-null lesions by a predominantly proapoptotic mechanism). Treatment with simvastatin markedly inhibited MMP-2, MMP-3, and MMP-9 levels in lung and prevented alveolar destruction. The combination of rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction by inhibiting MMP-2, MMP-3, and MMP-9. Our findings demonstrate a mechanistic link between loss of TSC2 and alveolar destruction and suggest that treatment with rapamycin and simvastatin together could benefit patients with LAM by targeting cells with TSC2 dysfunction and preventing airspace enlargement.
PMCID: PMC3684391  PMID: 23035046
17.  Resection of Non-Small Cell Lung Cancers Reverses Tumor-Induced Gene Expression Changes in the Peripheral Immune System 
To characterize the interactions of Non-small Cell Lung Cancer (NSCLC) tumors with the immune system at the level of mRNA and microRNA (miRNA) expression and to define expression signatures that characterize the presence of a malignant tumor vs. a non-malignant nodule.
We have examined the changes of both mRNA and miRNA expression levels in peripheral blood mononuclear cells (PBMC) between paired samples collected from NSCLC patients before and after tumor removal using Illumina gene expression arrays.
We found that malignant tumor removal significantly changes expression of more than 3,000 protein-coding genes, especially genes in pathways associated with suppression of the innate immune response, including NK cell signaling and apoptosis-associated ceramide signaling. Binding sites for the ETS-domain transcription factors ELK1, ELK4 and SPI1 were enriched in promoter regions of genes upregulated in the presence of a tumor. Additional important regulators included five miRNAs expressed at significantly higher levels before tumor removal. Repressed protein-coding targets of those miRNAs included many transcription factors, several involved in immunologically important pathways. While there was a significant overlap in the effects of malignant tumors and benign lung nodules on PBMC gene expression, we identified one gene panel which indicates a tumor or nodule presence and a second panel that can distinguish malignant from non-malignant nodules.
A tumor presence in the lung influences mRNA and miRNA expression in PBMC and this influence is reversed by tumor removal. These results suggest that PBMC gene expression signatures could be used for lung cancer diagnosis.
PMCID: PMC3618688  PMID: 21807633
lung cancer; pbmc; peripheral immune system
18.  Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T Cells Expressing a Mesothelin - Specific Chimeric Antibody Receptor 
Adoptive T cell immunotherapy (ACT) with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors in order to adequately exert therapeutic effects.
Experimental Design
The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM) (a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector and the modified T cells were used to treat established mesothelin-expressing tumors.
CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T cell tumor infiltration by Day 5 compared to mesoCAR T cells. This was associated with significantly increased anti-tumor activity.
CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve anti-tumor efficacy in vivo.
PMCID: PMC3612507  PMID: 21610146
19.  The HDAC inhibitor panobinostat (LBH589) inhibits mesothelioma and lung cancer cells in vitro and in vivo with particular efficacy for small cell lung cancer 
Molecular cancer therapeutics  2009;8(8):2221-2231.
Lung cancer is the leading cause of cancer deaths in the United States. Current therapies are inadequate. Histone deacetylase inhibitors (HDACi) are a recently developed class of anticancer agents that cause increased acetylation of core histones and nonhistone proteins leading to modulation of gene expression and protein activity involved in cancer cell growth and survival pathways. We examined the efficacy of the HDACi panobinostat (LBH589) in a wide range of lung cancers and mesotheliomas. Panobinostat was cytotoxic in almost all 37 cancer cell lines tested. IC50 and LD50 values were in the low nmol/L range (4–470 nmol/L; median, 20 nmol/L). Small cell lung cancer (SCLC) cell lines were among the most sensitive lines, with LD50 values consistently <25 nmol/L. In lung cancer and mesothelioma animal models, panobinostat significantly decreased tumor growth by an average of 62% when compared with vehicle control. Panobinostat was equally effective in immunocompetent and severe combined immunodeficiency mice, indicating that the inhibition of tumor growth by panobinostat was not due to direct immunologic effects. Panobinostat was, however, particularly effective in SCLC xenografts, and the addition of the chemotherapy agent etoposide augmented antitumor effects. Protein analysis of treated tumor biopsies revealed elevated amounts of cell cycle regulators such as p21 and proapoptosis factors, such as caspase 3 and 7 and cleaved poly[ADP-ribose] polymerase, coupled with decreased levels of antiapoptotic factors such as Bcl-2 and Bcl-XL. These studies together suggest that panobinostat may be a useful adjunct in the treatment of thoracic malignancies, especially SCLC.
PMCID: PMC3605895  PMID: 19671764
20.  A Trial of Intrapleural Adenoviral-mediated Interferon-α2b Gene Transfer for Malignant Pleural Mesothelioma 
New therapeutic strategies are needed for malignant pleural mesothelioma (MPM). We conducted a single-center, open-label, nonrandomized, pilot and feasibility trial using two intrapleural doses of an adenoviral vector encoding human IFN-α (Ad.IFN-α2b). Nine subjects were enrolled at two dose levels. The first three subjects had very high pleural and systemic IFN-α concentrations resulting in severe “flu-like” symptoms necessitating dose de-escalation. The next six patients had reduced (but still significant) pleural and serum IFN-α levels, but with tolerable symptoms. Repeated vector administration appeared to prolong IFN-α expression levels. Anti-tumor humoral immune responses against mesothelioma cell lines were seen in seven of the eight subjects evaluated. No clinical responses were seen in the four subjects with advanced disease. However, evidence of disease stability or tumor regression was seen in the remaining five patients, including one dramatic example of partial tumor regression at sites not in contiguity with vector infusion. These data show that Ad.IFN-α2b has potential therapeutic benefit in MPM and that it generates anti-tumor immune responses that may induce anatomic and/or metabolic reductions in distant tumor.
Clinical trial registered with (NCT 01212367).
PMCID: PMC3262033  PMID: 21642245
clinical trials; immunotherapy; gene therapy
21.  Pharmacologic Activation of the Innate Immune System to Prevent Respiratory Viral Infections 
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-β and subsequent high-level induction of IFN-β–dependent proteins, such as myxovirus resistance 1 (Mx1) and 2′,5′-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-β system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and “first responders” in the early stages of viral pandemics or bioterror attacks.
PMCID: PMC3265219  PMID: 21148741
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
22.  Cytoreduction surgery reduces systemic myeloid suppressor cell populations and restores intratumoral immunotherapy effectiveness 
Multiple immunotherapy approaches have improved adaptive anti-tumor immune responses in patients with early stage disease; however, results have been less dramatic when treating patients with late stage disease. These blunted responses are likely due to a host of factors, including changes in the tumor microenvironment and systemic immunosuppressive features, which accompany advanced tumor states. We hypothesized that cytoreductive surgery could control these immunosuppressive networks and restore the potency of immunotherapy in advanced disease scenarios.
To test these hypotheses, two representative intratumoral immunotherapies (an adenoviral vector encoding a suicide gene, AdV-tk, or a type-I interferon, Ad.IFNα) were tested in murine models of lung cancer. Cytoreductive surgery was performed following treatment of advanced tumors. Mechanistic underpinnings were investigated using flow cytometry, in vivo leukocyte depletion methods and in vivo tumor neutralization assays.
AdV-tk and Ad.IFNα were effective in treating early lung cancers, but had little anti-tumor effects in late stage cancers. Interestingly, in late stage scenarios, surgical cytoreduction unmasked the anti-tumor potency of both immunotherapeutic approaches. Immune mechanisms that explained restoration in anti-tumor immune responses included increased CD8 T-cell trafficking and reduced myeloid derived suppressor cell populations.
This study demonstrates that surgical resection combined with immunotherapy may be a rational therapeutic option for patients with advanced stage cancer.
PMCID: PMC3418164  PMID: 22742411
Surgical oncology; Immunotherapy; Cancer; Animal model
23.  ANGPT2 Genetic Variant Is Associated with Trauma-associated Acute Lung Injury and Altered Plasma Angiopoietin-2 Isoform Ratio 
Rationale: Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI.
Objectives: To identify ALI risk variants after major trauma using a large-scale candidate gene approach.
Methods: We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease–centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10−4) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot.
Measurements and Main Results: Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06–1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038).
Conclusions: An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.
PMCID: PMC3114062  PMID: 21257790
acute lung injury; acute respiratory distress syndrome; functional genetic polymorphism; genetic association study
24.  Vascular Endothelial-Targeted Therapy Combined with Cytotoxic Chemotherapy Induces Inflammatory Intratumoral Infiltrates and Inhibits Tumor Relapses after Surgery1 
Neoplasia (New York, N.Y.)  2012;14(4):352-359.
Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.
PMCID: PMC3349261  PMID: 22577350
25.  Peripheral Immune Cell Gene Expression Predicts Survival of Patients with Non-Small Cell Lung Cancer 
PLoS ONE  2012;7(3):e34392.
Prediction of cancer recurrence in patients with non-small cell lung cancer (NSCLC) currently relies on the assessment of clinical characteristics including age, tumor stage, and smoking history. A better prediction of early stage cancer patients with poorer survival and late stage patients with better survival is needed to design patient-tailored treatment protocols. We analyzed gene expression in RNA from peripheral blood mononuclear cells (PBMC) of NSCLC patients to identify signatures predictive of overall patient survival. We find that PBMC gene expression patterns from NSCLC patients, like patterns from tumors, have information predictive of patient outcomes. We identify and validate a 26 gene prognostic panel that is independent of clinical stage. Many additional prognostic genes are specific to myeloid cells and are more highly expressed in patients with shorter survival. We also observe that significant numbers of prognostic genes change expression levels in PBMC collected after tumor resection. These post-surgery gene expression profiles may provide a means to re-evaluate prognosis over time. These studies further suggest that patient outcomes are not solely determined by tumor gene expression profiles but can also be influenced by the immune response as reflected in peripheral immune cells.
PMCID: PMC3315526  PMID: 22479623

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