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1.
Mol Ther Methods Clin Dev ; 21: 670-680, 2021 Jun 11.
Article in English | MEDLINE | ID: mdl-34141822

ABSTRACT

Despite their exceptional capacity for transgene delivery ex vivo, lentiviral (LV) vectors have been slow to demonstrate clinical utility in the context of in vivo applications. Unresolved safety concerns related to broad LV vector tropism have limited LV vectors to ex vivo applications. Here, we report on a novel LV vector-pseudotyping strategy involving envelope glycoproteins of Tupaia paramyxovirus (TPMV) engineered to specifically target human cell-surface receptors. LV vectors pseudotyped with the TPMV hemagglutinin (H) protein bearing the interleukin (IL)-13 ligand in concert with the TPMV fusion (F) protein allowed efficient transduction of cells expressing the human IL-13 receptor alpha 2 (IL-13Rα2). Immunodeficient mice bearing orthotopically implanted human IL-13Rα2 expressing NCI-H1299 non-small cell lung cancer cells were injected intravenously with a single dose of LV vector pseudotyped with the TPMV H-IL-13 glycoprotein. Vector biodistribution was monitored using bioluminescence imaging of firefly luciferase transgene expression, revealing specific transduction of tumor tissue. A quantitative droplet digital PCR (ddPCR) analysis of lung tissue samples revealed a >15-fold increase in the tumor transduction in mice treated with LV vectors displaying IL-13 relative to those without IL-13. Our results show that TPMV envelope glycoproteins can be equipped with ligands to develop targeted LV vectors for in vivo applications.

2.
Mol Ther Methods Clin Dev ; 18: 631-638, 2020 Sep 11.
Article in English | MEDLINE | ID: mdl-32775497

ABSTRACT

The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 107 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 107 TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.

3.
PLoS One ; 12(6): e0177788, 2017.
Article in English | MEDLINE | ID: mdl-28575024

ABSTRACT

Commonly used methods for site-directed DNA mutagenesis require copying the entire target plasmid. These methods allow relatively easy modification of DNA sequences in small plasmids but become less efficient and faithful for large plasmids, necessitating full sequence verification. Introduction of mutations in larger plasmids requires subcloning, a slow and labor-intensive process, especially for multiple mutations. We have developed an efficient DNA mutagenesis technique, UnRestricted Mutagenesis and Cloning (URMAC) that replaces subcloning steps with quick biochemical reactions. URMAC does not suffer from plasmid size constraints and allows simultaneous introduction of multiple mutations. URMAC involves manipulation of only the mutagenesis target site(s), not the entire plasmid being mutagenized, therefore only partial sequence verification is required. Basic URMAC requires two PCR reactions, each followed by a ligation reaction to circularize the product, with an optional third enrichment PCR step followed by a traditional cloning step that requires two restriction sites. Here, we demonstrate URMAC's speed, accuracy, and efficiency through several examples, creating insertions, deletions or substitutions in plasmids ranging from 2.6 kb to 17 kb without subcloning.


Subject(s)
Mutagenesis, Site-Directed , Plasmids , Cloning, Molecular , DNA, Complementary , Dystrophin/genetics , Humans , Open Reading Frames , Polymerase Chain Reaction
4.
Oncotarget ; 7(25): 37762-37772, 2016 Jun 21.
Article in English | MEDLINE | ID: mdl-27192116

ABSTRACT

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.


Subject(s)
Dendritic Cells/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Oximes/pharmacology , Sulfonamides/pharmacology , T-Lymphocytes, Cytotoxic/immunology , Adult , Aged , Cancer Vaccines/pharmacology , Dendritic Cells/drug effects , Female , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Healthy Volunteers , Humans , Interferon-gamma/metabolism , Kynurenine/chemistry , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/chemistry , Lymphocyte Activation/immunology , Male , Middle Aged , Peptides/chemistry , Phenotype , T-Lymphocytes, Cytotoxic/drug effects , Tryptophan/chemistry , Young Adult
5.
Oncotarget ; 7(17): 23498-511, 2016 Apr 26.
Article in English | MEDLINE | ID: mdl-27015557

ABSTRACT

Among breast cancer types, triple-negative breast cancer (TNBC) has the fewest treatment options and the lowest 5-year survival rate. Androgen receptor (AR) inhibition has displayed efficacy against breast cancer preclinically and is currently being examined clinically in AR positive TNBC patients. Androgen deprivation has been shown to induce immunogenic modulation; the alteration of tumor cell phenotype resulting in increased sensitivity to immune-mediated killing. We evaluated the ability of AR inhibition to reduce the growth and improve the immune-mediated killing of breast cancer cells with differing expression of the estrogen receptor and AR. While AR expression was required for the growth inhibitory effects of enzalutamide on breast cancer cells, both enzalutamide and abiraterone improved the sensitivity of breast cancer cells to immune-mediated lysis independent of detectable AR expression. This increase in sensitivity was linked to an increase in cell surface tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression as well as a significant reduction in the expression of osteoprotegerin (OPG). The reduction in OPG was further examined and found to be critical for the increase in sensitivity of AR- TNBC cells to immune-mediated killing. The data presented herein further support the use of AR inhibition therapy in the AR+ TNBC setting. These data, however, also support the consideration of AR inhibition therapy for the treatment of AR- TNBC, especially in combination with cancer immunotherapy, providing a potential novel therapeutic option for select patients.


Subject(s)
Androgen Antagonists/pharmacology , Immunotherapy , Osteoprotegerin/metabolism , Phenylthiohydantoin/analogs & derivatives , Receptors, Androgen/chemistry , T-Lymphocytes, Cytotoxic/immunology , Triple Negative Breast Neoplasms/drug therapy , Apoptosis/drug effects , Benzamides , Biomarkers, Tumor/metabolism , Cell Proliferation/drug effects , Female , Humans , Immunomodulation , Nitriles , Phenylthiohydantoin/pharmacology , Receptors, Androgen/metabolism , Triple Negative Breast Neoplasms/immunology , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Tumor Cells, Cultured
6.
Oncotarget ; 7(13): 16130-45, 2016 Mar 29.
Article in English | MEDLINE | ID: mdl-26910920

ABSTRACT

Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded natural killer (NK) and CD8+ T cells. In examining NK cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1-) phenotype. In 4T1 breast tumor-bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells and CD8+ T cell responders of the innate phenotype, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/immunology , Carcinoma/immunology , Colonic Neoplasms/immunology , Proteins/pharmacology , Animals , Breast Neoplasms/drug therapy , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Carcinoma/drug therapy , Colonic Neoplasms/drug therapy , Female , Immunologic Memory/drug effects , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins
7.
J Immunother Cancer ; 3: 52, 2015.
Article in English | MEDLINE | ID: mdl-26579226

ABSTRACT

BACKGROUND: The angiopoietin/Tie2 pathway is an attractive target for cancer therapy due to its well-known role in regulating angiogenesis. Trebananib, a recombinant peptide-Fc fusion protein, or peptibody, that binds to angiopoietin-1 (Ang1) and Ang2 to block their interaction with the Tie2 receptor, is under active clinical investigation. We investigated whether suppressing the angiopoietin/Tie2 pathway, using the preclinical version of Trebananib (mL4-3 and L1-7(N)), could increase the sensitivity of human tumor cells to immune-mediated lysis through immunogenic modulation, which would make Trebananib a promising candidate for combination with immunotherapy. METHODS: We assessed human carcinoma cells for expression and activation of Ang1 and Ang2 and their receptor tyrosine kinase Tie2. In vitro, we exposed tumor cell lines expressing Tie2 to the peptibodies mL4-3 and L1-7(N), which inhibit the binding of Ang1 and Ang2 to Tie2, and assessed the cells for changes in viability, proliferation, surface phenotype, and sensitivity to attack by antigen-specific cytotoxic T lymphocytes (CTLs). RESULTS: Suppression of the angiopoietin/Tie2 pathway using mL4-3 and L1-7(N) had no effect on the proliferation or viability of tumor cells. However, these inhibitors markedly altered tumor cell phenotype, rendering tumor cells significantly more sensitive to antigen-specific CTL killing. ICAM-1 was shown to be mechanistically involved in these inhibitors' ability to sensitize tumor cells to immune-mediated attack by functional blocking studies. CONCLUSION: Our findings provide a rationale for the combination of agents targeting the angiopoietin/Tie2 pathway with cancer immunotherapies.

8.
Oncotarget ; 6(31): 31344-59, 2015 Oct 13.
Article in English | MEDLINE | ID: mdl-26374823

ABSTRACT

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.


Subject(s)
Adenoviridae/genetics , Adenovirus Vaccines/therapeutic use , Antigens, Neoplasm/immunology , Cancer Vaccines/therapeutic use , Immunotherapy , Neoplasms/therapy , Adenovirus E1 Proteins/genetics , Adenovirus E1 Proteins/immunology , Adenovirus E2 Proteins/genetics , Adenovirus E2 Proteins/immunology , Animals , Antigens, Neoplasm/genetics , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/immunology , Dendritic Cells/immunology , Female , Flow Cytometry , Genetic Vectors/administration & dosage , Humans , Immunization , Interferon-gamma/metabolism , Mice , Mice, Inbred C57BL , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , T-Lymphocytes/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
9.
Oncotarget ; 6(29): 28194-210, 2015 Sep 29.
Article in English | MEDLINE | ID: mdl-26317648

ABSTRACT

Several transcription factors play a role in the alteration of gene expression that occurs during cancer metastasis. Twist expression has been shown to be associated with the hallmarks of the metastatic process, as well as poor prognosis and drug resistance in many tumor types. However, primarily due to their location within the cell and the lack of a hydrophobic groove required for drug attachment, transcription factors such as Twist are difficult to target with conventional therapies. An alternative therapeutic strategy is a vaccine comprised of a Modified vaccinia Ankara (MVA), incorporating the Twist transgene and a TRIad of COstimulatory Molecules (B7-1, ICAM-1, LFA-3; TRICOM). Here we characterize an MVA-TWIST/TRICOM vaccine that induced both CD4+ and CD8+ Twist-specific T-cell responses in vivo. In addition, administration of this vaccine reduced both the primary tumor growth and metastasis in the 4T1 model of metastatic breast cancer. In the TRAMP transgenic model of spontaneous prostate cancer, MVA-TWIST/TRICOM alone significantly improved survival, and when combined with the androgen receptor antagonist enzalutamide, the vaccine further improved survival. These studies thus provide a rationale for the use of active immunotherapy targeting transcription factors involved in the metastatic process and for the combination of cancer vaccines with androgen deprivation.


Subject(s)
Cancer Vaccines/immunology , Mammary Neoplasms, Animal/immunology , Prostatic Neoplasms/immunology , Twist-Related Protein 1/immunology , Vaccinia virus/immunology , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/genetics , Cancer Vaccines/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/immunology , Male , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/prevention & control , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Neoplasm Metastasis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/prevention & control , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis , Treatment Outcome , Tumor Burden/drug effects , Tumor Burden/genetics , Tumor Burden/immunology , Twist-Related Protein 1/genetics , Twist-Related Protein 1/metabolism , Vaccination , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/metabolism , Vaccinia virus/genetics , Vaccinia virus/metabolism
10.
Oncoimmunology ; 4(6): e1009303, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26155431

ABSTRACT

Androgen-deprivation therapy (ADT) induces prostate cancer immunogenic modulation (IM) by reducing human tumor cell expression of anti-apoptotic genes thus facilitating increased sensitivity to immune-mediated lysis. Through its stimulation of IM, ADT has been shown to synergize with active immunotherapy thereby significantly improving overall survival in a mouse model of prostate cancer.

11.
Article in English | MEDLINE | ID: mdl-26005708

ABSTRACT

Combination therapy for the treatment of cancer is becoming increasingly essential as we gain improved understanding of the complexity of cancer progression and the mechanisms by which cancer cells become resistant to single-agent therapy. Recent studies, both clinical and preclinical, have suggested that immunotherapy is a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still needed. A number of recent studies have indicated that antiangiogenic tyrosine kinase inhibitors (TKIs) target multiple components of the tumor microenvironment and are an ideal class of agents for synergizing with cancer immunotherapy. TKIs are well known to modulate tumor endothelial cells, leading to vascular normalization; however, these agents have also been recently shown to decrease tumor compactness and tight junctions, thereby reducing solid tumor pressure and allowing for improved perfusion of collapsed vessels and increased tumor oxygenation. In addition, some TKIs are capable of inducing immunogenic modulation, whereby tumor cells are sensitized to killing by T lymphocytes. Moreover, a number of TKIs have been shown to be involved in immune subset conditioning, increasing the frequency and function of effector immune elements, while decreasing the number and function of immune suppressor cells. The alteration of the immune landscape, direct modification of tumor cells, and improved vascular perfusion leads to improved antitumor efficacy when antiangiogenic TKIs are combined with immunotherapy. Collectively, the data presented in this review support the clinical combination of multi-targeted antiangiogenic TKIs, including but not limited to cabozantinib, sunitinib, and sorafenib, as well as to other antiangiogenic therapies, such as the anti-VEGF antibody bevacizumab, with cancer vaccines for improved treatment of solid tumors.

12.
J Transl Med ; 12: 294, 2014 Nov 13.
Article in English | MEDLINE | ID: mdl-25388653

ABSTRACT

BACKGROUND: Growing awareness of the complexity of carcinogenesis has made multimodal therapies for cancer increasingly compelling and relevant. In recent years, immunotherapy has gained acceptance as an active therapeutic approach to cancer treatment, even though cancer is widely considered an immunosuppressive disease. Combining immunotherapy with targeted agents that have immunomodulatory capabilities could significantly improve its efficacy. METHODS: We evaluated the ability of cabozantinib, a receptor tyrosine kinase inhibitor, to modulate the immune system in vivo as well as alter the phenotype of tumor cells in vitro in order to determine if this inhibitor could act synergistically with a cancer vaccine. RESULTS: Our studies indicated that cabozantinib altered the phenotype of MC38-CEA murine tumor cells, rendering them more sensitive to immune-mediated killing. Cabozantinib also altered the frequency of immune sub-populations in the periphery as well as in the tumor microenvironment, which generated a more permissive immune environment. When cabozantinib was combined with a poxviral-based cancer vaccine targeting a self-antigen, the combination significantly reduced the function of regulatory T cells and increased cytokine production from effector T cells in response to the antigen. These alterations to the immune landscape, along with direct modification of tumor cells, led to markedly improved antitumor efficacy. CONCLUSIONS: These studies support the clinical combination of cabozantinib with immunotherapy for the treatment of cancer.


Subject(s)
Anilides/therapeutic use , Cancer Vaccines/immunology , Cytotoxicity, Immunologic , Neoplasms/drug therapy , Neoplasms/immunology , Pyridines/therapeutic use , Small Molecule Libraries/therapeutic use , Tumor Microenvironment , Anilides/pharmacology , Animals , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Combined Modality Therapy , Cytotoxicity, Immunologic/drug effects , Female , Immunotherapy , Lymphocyte Subsets/drug effects , Lymphocyte Subsets/immunology , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/blood supply , Neoplasms/pathology , Phenotype , Pyridines/pharmacology , Small Molecule Libraries/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Microenvironment/drug effects
13.
Oncotarget ; 5(19): 9335-48, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25344864

ABSTRACT

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT.


Subject(s)
Androgen Antagonists/pharmacology , Neuronal Apoptosis-Inhibitory Protein/genetics , Prostatic Neoplasms, Castration-Resistant/drug therapy , Receptors, Androgen/biosynthesis , T-Lymphocytes/immunology , Androstenes/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Benzamides , Cell Line, Tumor , Disease Progression , Humans , Immunomodulation/drug effects , Immunotherapy , Male , Mice , Neuronal Apoptosis-Inhibitory Protein/biosynthesis , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostate/cytology , Prostatic Neoplasms, Castration-Resistant/immunology , RNA Interference , RNA, Small Interfering , Receptors, Androgen/immunology
14.
Oncoimmunology ; 3: e28643, 2014.
Article in English | MEDLINE | ID: mdl-25097803

ABSTRACT

Tumor cells that survive radiation are more sensitive to T-cell-mediated lysis due to a spectrum of biological adaptations to cellular stress (defined as immunogenic modulation), including enhanced antigen processing and cell-surface presence of calreticulin. This mechanism can be exploited to maximize clinical benefit in patients receiving radiotherapy plus immunotherapy.

15.
Cancer Res ; 74(7): 1945-57, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24520078

ABSTRACT

Numerous reports have now demonstrated that the epithelial-to-mesenchymal transition (EMT) process is involved in solid tumor progression, metastasis, and drug resistance. Several transcription factors have been implicated as drivers of EMT and metastatic progression, including Twist. Overexpression of Twist has been shown to be associated with poor prognosis and drug resistance for many carcinomas and other tumor types. The role of Twist in experimental cancer metastases has been principally studied in the 4T1 mammary tumor model, where silencing of Twist in vitro has been shown to greatly reduce in vivo metastatic spread. Transcription factors such as Twist are generally believed to be "undruggable" because of their nuclear location and lack of a specific groove for tight binding of a small molecule inhibitor. An alternative approach to drug therapy targeting transcription factors driving the metastatic process is T-cell-mediated immunotherapy. A therapeutic vaccine platform that has been previously characterized consists of heat-killed recombinant Saccharomyces cerevisiae (yeast) capable of expressing tumor-associated antigen protein. We report here the construction and characterization of a recombinant yeast expressing the entire Twist protein, which is capable of inducing both CD8(+) and CD4(+) Twist-specific T-cell responses in vivo. Vaccination of mice reduced the size of primary transplanted 4T1 tumors and had an even greater antitumor effect on lung metastases of the same mice, which was dependent on Twist-specific CD8(+) T cells. These studies provide the rationale for vaccine-induced T-cell-mediated therapy of transcription factors involved in driving the metastatic process.


Subject(s)
Neoplasm Metastasis/prevention & control , T-Lymphocytes/immunology , Twist-Related Protein 1/immunology , Vaccination , Animals , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Female , Mice , Mice, Inbred BALB C , Saccharomyces cerevisiae/genetics , Twist-Related Protein 1/genetics , Vaccines, Synthetic/therapeutic use
16.
Oncoimmunology ; 2(12): e26937, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24498561

ABSTRACT

We have recently defined "immunogenic modulation," a mechanism whereby malignant cells that survive anticancer therapy, due to sublethal delivery or development of treatment resistance, become nonetheless more sensitive to killing by cytotoxic T lymphocytes. This mechanism can be exploited to identify which therapies will best synergize with immunotherapy, potentially maximizing patient clinical benefit.

17.
Front Oncol ; 2: 104, 2012.
Article in English | MEDLINE | ID: mdl-22973551

ABSTRACT

Radiation has long been the standard of care for many types of cancer. It is employed to locally eradicate tumor cells as well as alter tumor stroma with either curative or palliative intent. Radiation-induced cell damage is an immunologically active process in which danger signals are released that stimulate immune cells to phagocytose and present locally released tumor-associated antigens (TAAs). Recent studies have indicated that radiotherapy can also alter the phenotype of cancer cells that remain after treatment. These cells upregulate TAAs as well as markers, including major histocompatibility complex and costimulatory molecules, that make them much more immunostimulatory. As our understanding of the immunomodulatory effects of radiation has improved, interest in combining this type of therapy with immune-based therapies for the treatment of cancer has grown. Therapeutic cancer vaccines have been shown to initiate the dynamic process of host immune system activation, culminating in the recognition of host cancer cells as foreign. The environment created after radiotherapy can be exploited by active therapeutic cancer vaccines in order to achieve further, more robust immune system activation. This review highlights preclinical studies that have examined the alteration of the tumor microenvironment with regard to immunostimulatory molecules following different types of radiotherapy, including external beam radiation, radiolabeled monoclonal antibodies, bone-seeking radionuclides, and brachytherapy. We also emphasize how combination therapy with a cancer vaccine can exploit these changes to achieve improved therapeutic benefit. Lastly, we describe how these laboratory findings are translating into clinical benefit for patients undergoing combined radiotherapy and cancer vaccination.

18.
Semin Oncol ; 39(3): 323-39, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22595055

ABSTRACT

Therapeutic cancer vaccines are a unique treatment modality in that they initiate a dynamic process of activating the host immune system, which can then be exploited by concurrent or subsequent therapies. The addition of immunotherapy to standard-of-care cancer therapies has shown evidence of efficacy in preclinical models and in the clinical setting. This review examines the preclinical and clinical interactions between vaccine-mediated tumor-specific immune responses and local radiation, systemic chemotherapy, or select small molecule inhibitors, as well as the potential synergy between these modalities.


Subject(s)
Antineoplastic Agents/administration & dosage , Cancer Vaccines/administration & dosage , Combined Modality Therapy/methods , Drug Therapy/methods , Radiotherapy/methods , Animals , Antineoplastic Agents/immunology , Cancer Vaccines/immunology , Humans , Neoplasms/therapy
19.
J Virol ; 84(15): 7770-81, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20504917

ABSTRACT

Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Cystic Fibrosis/therapy , Epithelial Cells/physiology , Genetic Therapy/methods , Genetic Vectors , Respiratory Syncytial Virus, Human/genetics , Cell Line , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans
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