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Sinaga DS, Ho SL, Lu CA, Yu SM, Huang LF. Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells. PLANT METHODS 2021; 17:99. [PMID: 34560901 PMCID: PMC8464127 DOI: 10.1186/s13007-021-00799-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Accepted: 09/12/2021] [Indexed: 05/04/2023]
Abstract
BACKGROUND Transgenic plant suspension cells show economic potential for the production of valuable bioproducts. The sugar starvation-inducible rice αAmy3 promoter, together with its signal peptide, is widely applied to produce recombinant proteins in rice suspension cells. The OsMYBS2 transcription factor was shown recently to reduce activation of the αAmy3 promoter by competing for the binding site of the TA box of the αAmy3 promoter with the potent OsMYBS1 activator. In this study, rice suspension cells were genetically engineered to silence OsMYBS2 to enhance the production of recombinant proteins. RESULTS The mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene was controlled by the αAmy3 promoter and expressed in OsMYBS2-silenced transgenic rice suspension cells. Transcript levels of the endogenous αAmy3 and the transgene mGM-CSF were increased in the OsMYBS2-silenced suspension cells. The highest yield of recombinant mGM-CSF protein attained in the OsMYBS2-silenced transgenic suspension cells was 69.8 µg/mL, which is 2.5-fold that of non-silenced control cells. The yield of recombinant mGM-CSF was further increased to 118.8 µg/mL in cultured cells derived from homozygous F5 seeds, which was 5.1 times higher than that of the control suspension cell line. CONCLUSIONS Our results demonstrate that knockdown of the transcription factor gene OsMYBS2 increased the activity of the αAmy3 promoter and improved the yield of recombinant proteins secreted in rice cell suspension cultures.
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Affiliation(s)
- Desyanti Saulina Sinaga
- Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan City, 320, Taiwan, ROC
- Department of Life Sciences, National Central University, Taoyuan City, 320, Taiwan, ROC
| | - Shin-Lon Ho
- Department of Agronomy, National Chiayi University, Chiayi City, 600, Taiwan, ROC
| | - Chung-An Lu
- Department of Life Sciences, National Central University, Taoyuan City, 320, Taiwan, ROC
| | - Su-May Yu
- Institute of Molecular Biology, Academia Sinica, Nankang, Taipei City, 115, Taiwan, ROC
| | - Li-Fen Huang
- Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan City, 320, Taiwan, ROC.
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Sanmartín I, Sendra L, Moret I, Herrero MJ, Aliño SF. Multicompartmental Lipopolyplex as Vehicle for Antigens and Genes Delivery in Vaccine Formulations. Pharmaceutics 2021; 13:pharmaceutics13020281. [PMID: 33669785 PMCID: PMC7922173 DOI: 10.3390/pharmaceutics13020281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2020] [Revised: 02/17/2021] [Accepted: 02/18/2021] [Indexed: 11/16/2022] Open
Abstract
Vector design and its characterization is an area of great interest in current vaccine research. In this article, we have formulated and characterized a multicompartmental lipopolyplex, which associates multiple liposomes and polyplexes in the same complex. These particles allow the simultaneous delivery of lipid or water-soluble antigens associated with genes to the same cell, in much higher amounts than conventional lipopolyplexes. The vector characterization and optimization were carried out using liposomes with entrapped carboxyfluorescein and adapted electrophoretic assays. Two types of lipopolyplexes (containing hydrophilic or lipophilic antigens) were employed to evaluate their interest in vaccination. The lipopolyplex loaded with an extract of water-soluble melanoma proteins proved to efficiently induce humoral response in murine melanoma model, increasing the levels of IgM and IgG. The specificity of the immune response induced by the lipopolyplex was demonstrated in mice with the lipopolyplex containing the GD3 ganglioside lipid antigen, abundant in melanoma cells. The levels of anti-GD3 IgG increased markedly without modifying the expression of humoral antibodies against other gangliosides.
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Affiliation(s)
- Isaías Sanmartín
- Faculty of Veterinary and Experimental Sciences, Universidad Católica de Valencia, 46001 Valencia, Spain;
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
| | - Luis Sendra
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
| | - Inés Moret
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Inflammatory Bowel Disease Research Group, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
| | - María José Herrero
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
| | - Salvador F. Aliño
- Pharmacology Department, Faculty of Medicine, Universidad de Valencia, 46010 Valencia, Spain; (L.S.); (I.M.); (M.J.H.)
- Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
- Clinical Pharmacology Unit, Hospital Universitario y Politécnico La Fe, 46026 Valencia, Spain
- Correspondence: ; Tel.: +34-963-864-972
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3
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Ustyanovska Avtenyuk N, Visser N, Bremer E, Wiersma VR. The Neutrophil: The Underdog That Packs a Punch in the Fight against Cancer. Int J Mol Sci 2020; 21:E7820. [PMID: 33105656 PMCID: PMC7659937 DOI: 10.3390/ijms21217820] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Revised: 10/19/2020] [Accepted: 10/19/2020] [Indexed: 02/07/2023] Open
Abstract
The advent of immunotherapy has had a major impact on the outcome and overall survival in many types of cancer. Current immunotherapeutic strategies typically aim to (re)activate anticancer T cell immunity, although the targeting of macrophage-mediated anticancer innate immunity has also emerged in recent years. Neutrophils, although comprising ≈ 60% of all white blood cells in the circulation, are still largely overlooked in this respect. Nevertheless, neutrophils have evident anticancer activity and can induce phagocytosis, trogocytosis, as well as the direct cytotoxic elimination of cancer cells. Furthermore, therapeutic tumor-targeting monoclonal antibodies trigger anticancer immune responses through all innate Fc-receptor expressing cells, including neutrophils. Indeed, the depletion of neutrophils strongly reduced the efficacy of monoclonal antibody treatment and increased tumor progression in various preclinical studies. In addition, the infusion of neutrophils in murine cancer models reduced tumor progression. However, evidence on the anticancer effects of neutrophils is fragmentary and mostly obtained in in vitro assays or murine models with reports on anticancer neutrophil activity in humans lagging behind. In this review, we aim to give an overview of the available knowledge of anticancer activity by neutrophils. Furthermore, we will describe strategies being explored for the therapeutic activation of anticancer neutrophil activity.
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Affiliation(s)
| | | | - Edwin Bremer
- Department of Hematology, Cancer Research Center Groningen, University Medical Center Groningen (UMCG), University of Groningen, Hanzeplein 1/DA13, 9713 GZ Groningen, The Netherlands; (N.U.A.); (N.V.)
| | - Valerie R. Wiersma
- Department of Hematology, Cancer Research Center Groningen, University Medical Center Groningen (UMCG), University of Groningen, Hanzeplein 1/DA13, 9713 GZ Groningen, The Netherlands; (N.U.A.); (N.V.)
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Miguel A, Herrero MJ, Sendra L, Botella R, Algás R, Sánchez M, Aliño SF. Comparative antitumor effect among GM-CSF, IL-12 and GM-CSF+IL-12 genetically modified tumor cell vaccines. Cancer Gene Ther 2013; 20:576-81. [PMID: 23969885 DOI: 10.1038/cgt.2013.54] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2013] [Accepted: 07/03/2013] [Indexed: 02/06/2023]
Abstract
Genetically modified cells have been shown to be one of the most effective cancer vaccine strategies. An evaluation is made of the efficacy of both preventive and therapeutic antitumor vaccines against murine melanoma, using C57BL/6 mice and irradiated B16 tumor cells expressing granulocyte and macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12) or both. Tumor was transplanted by the injection of wild-type B16 cells. Tumor growth and survival were measured to evaluate the efficacy of vaccination. Specific humoral response and immunoglobulin G (IgG) switch were evaluated measuring total IgG and IgG1 and IgG2a subtypes against tumor membrane proteins of B16 cells. In preventive vaccination, all treated groups showed delayed tumor growth. In addition, the group vaccinated to express only GM-CSF achieved 100% animal survival (P<0.005). Vaccination with GM-CSF+IL-12-producing B16 cells yielded lesser results (60% survival, P<0.005). Furthermore, all surviving animals remained disease-free after second tumor implantation 1 year later. The therapeutic vaccination strategies resulted in significantly delayed tumor growth, mainly using B16 cells producing GM-CSF+IL-12 cytokines, with 70% tumor growth inhibition (P<0.001)-although none of the animals reached overall survival. The results obtained suggest that the GM-CSF+IL-12 combination only increases the efficacy of therapeutic vaccines. No differences in classical regulatory T cells were found among the different groups.
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Affiliation(s)
- A Miguel
- Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Valencia, Spain
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5
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Comparative antitumor effect of preventive versus therapeutic vaccines employing B16 melanoma cells genetically modified to express GM-CSF and B7.2 in a murine model. Toxins (Basel) 2012. [PMID: 23202306 PMCID: PMC3509698 DOI: 10.3390/toxins4111058] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Cancer vaccines have always been a subject of gene therapy research. One of the most successful approaches has been working with genetically modified tumor cells. In this study, we describe our approach to achieving an immune response against a murine melanoma model, employing B16 tumor cells expressing GM-CSF and B7.2. Wild B16 cells were injected in C57BL6 mice to cause the tumor. Irradiated B16 cells transfected with GM-CSF, B7.2, or both, were processed as a preventive and therapeutic vaccination. Tumor volumes were measured and survival curves were obtained. Blood samples were taken from mice, and IgGs of each treatment group were also measured. The regulatory T cells (Treg) of selected groups were quantified using counts of images taken by confocal microscopy. Results: one hundred percent survival was achieved by preventive vaccination with the group of cells transfected with p2F_GM-CSF. Therapeutic vaccination achieved initial inhibition of tumor growth but did not secure overall survival of the animals. Classical Treg cells did not vary among the different groups in this therapeutic vaccination model.
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6
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van den Engel NK, Rüttinger D, Rusan M, Kammerer R, Zimmermann W, Hatz RA, Winter H. Combination immunotherapy and active-specific tumor cell vaccination augments anti-cancer immunity in a mouse model of gastric cancer. J Transl Med 2011; 9:140. [PMID: 21859450 PMCID: PMC3169470 DOI: 10.1186/1479-5876-9-140] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2011] [Accepted: 08/22/2011] [Indexed: 12/22/2022] Open
Abstract
Background Active-specific immunotherapy used as an adjuvant therapeutic strategy is rather unexplored for cancers with poorly characterized tumor antigens like gastric cancer. The aim of this study was to augment a therapeutic immune response to a low immunogenic tumor cell line derived from a spontaneous gastric tumor of a CEA424-SV40 large T antigen (CEA424-SV40 TAg) transgenic mouse. Methods Mice were treated with a lymphodepleting dose of cyclophosphamide prior to reconstitution with syngeneic spleen cells and vaccination with a whole tumor cell vaccine combined with GM-CSF (a treatment strategy abbreviated as LRAST). Anti-tumor activity to subcutaneous tumor challenge was examined in a prophylactic as well as a therapeutic setting and compared to corresponding controls. Results LRAST enhances tumor-specific T cell responses and efficiently inhibits growth of subsequent transplanted tumor cells. In addition, LRAST tended to slow down growth of established tumors. The improved anti-tumor immune response was accompanied by a transient decrease in the frequency and absolute number of CD4+CD25+FoxP3+ T cells (Tregs). Conclusions Our data support the concept that whole tumor cell vaccination in a lymphodepleted and reconstituted host in combination with GM-CSF induces therapeutic tumor-specific T cells. However, the long-term efficacy of the treatment may be dampened by the recurrence of Tregs. Strategies to counteract suppressive immune mechanisms are required to further evaluate this therapeutic vaccination protocol.
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Affiliation(s)
- Natasja K van den Engel
- Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany.
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Driessens G, Nuttin L, Gras A, Maetens J, Mievis S, Schoore M, Velu T, Tenenbaum L, Préat V, Bruyns C. Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery. Cancer Immunol Immunother 2011; 60:273-81. [PMID: 21076828 PMCID: PMC11029469 DOI: 10.1007/s00262-010-0941-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2010] [Accepted: 10/26/2010] [Indexed: 01/01/2023]
Abstract
Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an "all in vivo" strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful "all in vivo" vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.
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Affiliation(s)
- Gregory Driessens
- Interdisciplinary Research Institute (IRIBHM), Université Libre de Bruxelles, Faculty of Medicine, route de Lennik 808, Brussels, Belgium.
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8
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Seo SH, Han HD, Noh KH, Kim TW, Son SW. Chitosan hydrogel containing GMCSF and a cancer drug exerts synergistic anti-tumor effects via the induction of CD8+ T cell-mediated anti-tumor immunity. Clin Exp Metastasis 2008; 26:179-87. [PMID: 19082918 DOI: 10.1007/s10585-008-9228-5] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2008] [Accepted: 12/02/2008] [Indexed: 12/22/2022]
Abstract
Cancer treatments consisting of a combination of chemotherapy and immunotherapy have been vigorously exploited to further improve the efficacy of cancer therapies. In this study, we utilized a chitosan hydrogel (CH) system loaded with GMCSF and a cancer drug as a chemo-immunotherapeutic agent in an effort to assess the effects on tumor growth in mice using TC-1 cervical tumor cells, which express the tumor-specific antigen, HPV-16 E7. The growth of TC-1 tumors was significantly reduced in mice treated with a CH harboring a cancer drug (doxorubicin (DOX), cisplatin (CDDP), or cyclophosphamide (CTX)) and GMCSF (CH-a cancer drug + GMCSF), as compared to other groups that were treated with CH containing only a cancer drug(CH-a cancer drug) or GMCSF(CH-GMCSF). Among the cancer drugs, CTX exerted the most potent anti-tumor effects. Interestingly, the intra-tumoral injection of CH-a cancer drug + GMCSF induced a significant E7-specific CD8(+) T cell immune response as compared to CH-GMCSF or CH-a cancer drug. This enhancement of tumor antigen-specific CD8(+) T cell immunity was associated principally with the anti-tumor effects induced by CH-CTX + GMCSF, as demonstrated by antibody depletion. Collectively, the aforementioned results indicate that co-treatment of tumors with a combination of GMCSF and a cancer drug incorporated into a CH system results in synergistic anti-tumor effects, which occur via the induction of a tumor antigen-specific CD8(+) T cell-mediated anti-tumor immunity. This study demonstrates the use of a biodegradable hydrogel system for the co-delivery of an immunoadjuvant and an anti-cancer drug for successful chemo-immunotherapy.
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Affiliation(s)
- Soo Hong Seo
- Department of Dermatology, Korea University Ansan Hospital, Korea University College of Medicine, Gojan 1-dong, Danwon-gu, Ansan-si, Gyeonggi-do, South Korea
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9
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Lin JM, Li B, Rimmer E, VanRoey M, Jooss K. Enhancement of the anti-tumor efficacy of a GM-CSF-secreting tumor cell immunotherapy in preclinical models by cytosine arabinoside. Exp Hematol 2008; 36:319-28. [PMID: 18279719 DOI: 10.1016/j.exphem.2007.11.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2007] [Revised: 11/08/2007] [Accepted: 11/13/2007] [Indexed: 11/28/2022]
Abstract
OBJECTIVE Acute myeloid leukemia (AML) is a highly malignant neoplasm responsible for nearly 10,000 cancer-related deaths annually in the United States. Treatment options for elderly patients with AML remain limited. Standard regimens using cytarabine (cytosine arabinoside [AraC]), a nucleotide analogue, result in significant toxicity with poor overall response. Combination of a cytotoxic chemotherapy and tumor-specific immunotherapy has the potential to improve overall efficacy by inducing an anti-tumor immune response against minimal residual disease. The studies reported here were performed to evaluate the therapeutic benefit of combining a granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy with AraC treatment. MATERIALS AND METHODS C57Bl/6 mice were challenged with C1498-luc cells intravenously and evaluated by in vivo imaging throughout the study to monitor the systemic progression of the tumor. Individual animals were euthanized when in vivo total photon counts exceeded 5 x 10(8) and/or when they were in poor clinical condition. Cytotoxicity assay was performed to evaluate effector function and flow cytometry was used for phenotyping of splenocytes from experimental animals. RESULTS Administration of GM-CSF-secreting tumor cell immunotherapy during AraC -induced cytopenia enhanced the anti-tumor efficacy of the immunotherapy, resulting in prolonged survival. AraC treatment did not negatively impact antigen-specific T-cell activation elicited by the immunotherapy and surviving animals treated with the combination demonstrated strong tumor-specific memory responses. CONCLUSION GM-CSF-secreting tumor cell immunotherapy in combination with AraC prolongs survival of tumor-bearing mice, with a median survival time of 61 days observed in mice treated with AraC alone and 90% of mice treated with the combination therapy still alive by day 150.
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Affiliation(s)
- Jian Min Lin
- Department of Preclinical Oncology, Cell Genesys Inc, South San Francisco, CA 94080, USA
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10
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Abstract
Cancer vaccines have always been in the scope of gene therapy research. One of the most successful approaches has been working with genetically modified tumor cells. However, to become a clinical reality, tumor cells must suffer a long and risky process from the extraction from the patient to the reimplantation as a vaccine. In this work, we explain our group’s approach to reduce the cell number required to achieve an immune response against a melanoma murine model, employing bead-selected B16 tumor cells expressing GM-CSF and B7.2.
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Affiliation(s)
- Mj Herrero
- Gene Therapy Group, Dpto. Farmacologia, Fac. Medicina, Univ. Valencia, Valencia, España
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11
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Simmons AD, Li B, Gonzalez-Edick M, Lin C, Moskalenko M, Du T, Creson J, VanRoey MJ, Jooss K. GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action. Cancer Immunol Immunother 2007; 56:1653-65. [PMID: 17410360 PMCID: PMC11029840 DOI: 10.1007/s00262-007-0315-2] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2006] [Accepted: 03/08/2007] [Indexed: 01/22/2023]
Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy.
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Affiliation(s)
- Andrew D Simmons
- Cell Genesys Inc., 500 Forbes Blvd., South San Francisco, CA, 94080, USA,
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12
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Young JG, Green NK, Mautner V, Searle PF, Young LS, James ND. Combining gene and immunotherapy for prostate cancer. Prostate Cancer Prostatic Dis 2007; 11:187-93. [PMID: 17726452 DOI: 10.1038/sj.pcan.4501008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in pre-clinical studies and is currently being assessed in phase I and II clinical trials in prostate cancer. Enhanced cell killing by apparent immune-mediated mechanisms has been shown in pancreatic and colorectal cancer models, by co-expressing murine granulocyte macrophage colony-stimulating factor (GM-CSF) with NR in a single replication deficient adenoviral vector. This consists of the CMV immediate early promotor driving expression of NR, with an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF). To examine if similar enhancement of tumour cell killing could be produced in prostate cancer, the TRAMP model was chosen. Results illustrate that the combination of suicide gene therapy using NR and CB1954, with cytokine stimulation with mGM-CSF gives an improved response compared with either modality alone. The mechanism of this improved response is however likely to be non-immune based as it lacks a memory effect.
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Affiliation(s)
- J G Young
- Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, UK
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13
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Abstract
There is currently great interest in molecular therapies to treat various diseases, and this has prompted extensive efforts to achieve target-specific and controlled delivery of bioactive macromolecules (for example, proteins, antibodies, DNA and small interfering RNA) through the design of smart drug carriers. By contrast, the influence of the microenvironment in which the target cell resides and the effect it might have on the success of biomacromolecular therapies has been under-appreciated. The extracellular matrix (ECM) component of the cellular niche may be particularly important, as many diseases and injury disrupt the normal ECM architecture, the cell adhesion to ECM, and the subsequent cellular activities. This Review will discuss the importance of the ECM and the ECM-cell interactions on the cell response to bioactive macromolecules, and suggest how this information could lead to new criteria for the design of novel drug delivery systems.
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Affiliation(s)
- Hyun Joon Kong
- Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA
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Hamada K, Desaki J, Nakagawa K, Zhang T, Shirakawa T, Gotoh A, Tagawa M. Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy. Mol Ther 2007; 15:1121-8. [PMID: 17387337 DOI: 10.1038/sj.mt.6300128] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
Although replication-competent viruses have been developed to treat cancers, their cytotoxic effects are insufficient, as infection is inhibited by the generation of neutralizing antibodies. To address this limitation, we developed a carrier cell system to deliver a replication-competent adenovirus. Carrier cells infected with replication-competent adenovirus were incubated with target cancer cells in a high titer of anti-adenovirus antibody. Carrier cells were injected into syngeneic subcutaneous tumors after immunization with adenovirus. Carrier cell-derived cell fragments containing viral particles were engulfed by proliferative target cancer cells. This engulfment-mediated transfer of adenovirus was not inhibited by the anti-adenovirus antibody and enabled repetitive infection. After the induction of anti-adenoviral cytotoxic T-lymphocyte (CTL) responses by immunization with adenovirus, administration of carrier cells infected with a replication-competent adenovirus induced complete tumor regression. Adenovirus-GM-CSF augmented the anti-tumor effect of carrier cells by increasing anti-adenoviral and anti-tumoral CTL responses and decreased the number of injections of carrier cells required to induce complete tumor regression. This novel carrier cell-mediated viral transfection system might prove useful in a variety of cancer therapies.
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Affiliation(s)
- Katsuyuki Hamada
- Department of Obstetrics and Gynecology, School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, Japan.
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15
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Herrero MJ, Botella R, Dasí F, Algás R, Sánchez M, Aliño SF. Antigens and Cytokine Genes in Antitumor Vaccines. Ann N Y Acad Sci 2006; 1091:412-24. [PMID: 17341632 DOI: 10.1196/annals.1378.084] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Studies against cancer, including clinical trials, have shown that a correct activation of the immune system can lead to tumor rejection whereas incorrect signaling results in no positive effects or even anergy. We have worked assuming that two signals, GM-CSF (granulocyte and macrophage colony-stimulating factor) and tumor antigens are necessary to mediate an antitumor effective response. To study which is the ideal temporal sequence for their administration, we have used a murine model of antimelanoma vaccine employing whole B16 tumor cells or their membrane protein antigens (TMPs) in combination with gm-csf transfer before or after the antigen delivery. Our results show that: (i) When gm-csf tisular transfection is performed before TMP delivery, a tumor growth inhibition is observed, but with a limit effect when administering high antigen doses; in contrast, when signals are inverted, the limited effect is lost and greater antitumor efficacy is obtained. (ii) A similar behavior, but with stronger positive results, is observed employing gm-csf transfection and whole tumor cells as antigens. While negative results are obtained with gm-csf before cells, the best results (total survival of treated mice) are obtained when GM-CSF is administered in transfected cells. We conclude that optimal antitumoral response can be obtained when the antigen signal is given before (or simultaneous with) GM-CSF production, while the inversion of the signals could result in the undesired inhibition or anergy of the immune response.
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MESH Headings
- Animals
- Antigens, Neoplasm/administration & dosage
- Antigens, Neoplasm/genetics
- Antigens, Neoplasm/immunology
- Antigens, Neoplasm/physiology
- Cancer Vaccines/administration & dosage
- Cancer Vaccines/genetics
- Cancer Vaccines/immunology
- Cell Line, Tumor
- Cytokines/genetics
- Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage
- Granulocyte-Macrophage Colony-Stimulating Factor/genetics
- Granulocyte-Macrophage Colony-Stimulating Factor/immunology
- Granulocyte-Macrophage Colony-Stimulating Factor/physiology
- Immunotherapy, Adoptive
- Melanoma, Experimental/genetics
- Melanoma, Experimental/immunology
- Melanoma, Experimental/prevention & control
- Mice
- Mice, Inbred C57BL
- Neoplasm Transplantation/immunology
- Signal Transduction/genetics
- Signal Transduction/immunology
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Affiliation(s)
- María José Herrero
- Gene Therapy Group, Department of Pharmacology, Faculty of Medicine, University of Valencia, Av. Blasco Ibáñez 15, 46010 Valencia, Spain.
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16
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Dammeyer P, Jaramillo MC, Pipes BL, Badowski MS, Tsang TC, Harris DT. Heat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor. Int J Hyperthermia 2006; 22:407-19. [PMID: 16891243 DOI: 10.1080/02656730600765312] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
Abstract
PURPOSE In cytokine immunotherapy of cancer it is critical to deliver sufficiently high local cytokine concentrations in order to reach the therapeutic threshold needed for clinical efficacy. Simultaneously, for optimal clinical safety adverse effects caused by high systemic cytokine levels must be minimized. One of the most promising anti-cancer therapeutic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), has elicited anti-tumour immune responses in animal studies and clinical trials. However, the clinical efficacy has been limited, with local GM-CSF levels being therapeutically insufficient and systemic toxicity being a limiting factor. METHODS To address these problems we have developed a novel GM-CSF expression vector, pAD-HotAmp-GM-CSF, which can provide high levels of GM-CSF expression, and induction of cytokine expression to limited tissue areas. This expression system combines inducible and amplifying elements in a single multi-genic construct. The first transcriptional unit contains the inducible element, the heat shock protein 70B (HSP70B) promoter that regulates expression of the transcription-activating factor tat. RESULTS Upon the binding of tat to the second promoter, the HIV2 long terminal repeat amplifies downstream gene expression of the therapeutic cytokine GM-CSF. Moderate hyperthermia at 42 degrees C for 30 min induced GM-CSF expression in pAD-HotAmp-GM-CSF that was over 2.5- and 2.8-fold higher than levels reached with HSP70B promoter alone and the prototypical human cytomegalovirus promoter. CONCLUSIONS Thus, the inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system for regulated and enhanced GM-CSF expression, which enables both greater efficacy and safety in cytokine immunotherapy of cancer.
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Affiliation(s)
- Pascal Dammeyer
- Department of Microbiology and Immunology, University of Arizona, Tucson, AZ, USA.
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17
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Choi BS, Sondel PM, Hank JA, Schalch H, Gan J, King DM, Kendra K, Mahvi D, Lee LY, Kim K, Albertini MR. Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma. Cancer Immunol Immunother 2006; 55:761-74. [PMID: 16187086 PMCID: PMC11030027 DOI: 10.1007/s00262-005-0069-7] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2005] [Accepted: 07/28/2005] [Indexed: 10/25/2022]
Abstract
PURPOSE We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. EXPERIMENTAL DESIGN Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5 x 10(6) U/m(2)/day, 26 patients) or Chiron IL-2 (4.5 x 10(6) U/m(2)/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2-7.5 mg/m(2)/day) and R24 (1-10 mg/m(2)/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. RESULTS When given in combination in this study, the MTD for ch14.18 was 5 mg/m(2)/day and the MTD for R24 was 5 mg/m(2)/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m(2)/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. CONCLUSIONS Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies.
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Affiliation(s)
- Brian S. Choi
- Department of Medicine, University of Wisconsin, Madison, WI USA
| | - Paul M. Sondel
- Department of Human Oncology, University of Wisconsin, Madison, WI USA
- Department of Pediatrics, University of Wisconsin, Madison, WI USA
- Department of Genetics, University of Wisconsin, Madison, WI USA
| | - Jacquelyn A. Hank
- Department of Human Oncology, University of Wisconsin, Madison, WI USA
| | - Heidi Schalch
- Department of Medicine, University of Wisconsin, Madison, WI USA
| | - Jacek Gan
- Department of Human Oncology, University of Wisconsin, Madison, WI USA
| | - David M. King
- Department of Medicine, University of Wisconsin, Madison, WI USA
| | - Kari Kendra
- Department of Hematology and Oncology, Ohio State University, Columbus, OH USA
| | - David Mahvi
- Department of Surgery, University of Wisconsin, Madison, WI USA
| | - Li-Yin Lee
- Department of Biostatistics, University of Wisconsin, Madison, WI USA
| | - KyungMann Kim
- Department of Biostatistics, University of Wisconsin, Madison, WI USA
| | - Mark R. Albertini
- Department of Medicine, University of Wisconsin, Madison, WI USA
- K4/414 Clinical Science Center, University of Wisconsin, 600 Highland Avenue, Madison, WI 53792 USA
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18
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Driessens G, Hamdane M, Cool V, Velu T, Bruyns C. Highly Successful Therapeutic Vaccinations Combining Dendritic Cells and Tumor Cells Secreting Granulocyte Macrophage Colony-stimulating Factor. Cancer Res 2004; 64:8435-42. [PMID: 15548715 DOI: 10.1158/0008-5472.can-04-0774] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-gamma production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.
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Affiliation(s)
- Gregory Driessens
- Interdisciplinary Research Institute (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles, Bruxelles, Belgium
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19
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Zheng S, Xiao ZX, Pan YL, Han MY, Dong Q. Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy. World J Gastroenterol 2003; 9:951-5. [PMID: 12717836 PMCID: PMC4611403 DOI: 10.3748/wjg.v9.i5.951] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene.
METHODS: Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated.
RESULTS: Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-γ, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice.
CONCLUSION: The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy.
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Affiliation(s)
- Shu Zheng
- Cancer Institute, Zhejiang University, Hangzhou 310009, Zhejiang Province, China.
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20
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Green NK, McNeish IA, Doshi R, Searle PF, Kerr DJ, Young LS. Immune enhancement of nitroreductase-induced cytotoxicity: studies using a bicistronic adenovirus vector. Int J Cancer 2003; 104:104-12. [PMID: 12532426 DOI: 10.1002/ijc.10916] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials. It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR. We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed. The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model. These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence.
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MESH Headings
- Adenocarcinoma/genetics
- Adenocarcinoma/immunology
- Adenocarcinoma/therapy
- Adenoviruses, Human/genetics
- Adjuvants, Immunologic/therapeutic use
- Animals
- Antineoplastic Agents, Alkylating/pharmacokinetics
- Antineoplastic Agents, Alkylating/therapeutic use
- Aziridines/pharmacokinetics
- Aziridines/pharmacology
- Aziridines/therapeutic use
- Bystander Effect
- Colorectal Neoplasms/genetics
- Colorectal Neoplasms/immunology
- Colorectal Neoplasms/therapy
- Combined Modality Therapy
- Cytomegalovirus/genetics
- Defective Viruses/genetics
- Drug Resistance, Neoplasm/genetics
- Escherichia coli Proteins/genetics
- Escherichia coli Proteins/metabolism
- Female
- Genes
- Genes, Synthetic
- Genetic Therapy
- Genetic Vectors/genetics
- Genetic Vectors/therapeutic use
- Granulocyte-Macrophage Colony-Stimulating Factor/genetics
- Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use
- Humans
- Immunotherapy
- Mesothelioma/enzymology
- Mesothelioma/pathology
- Mice
- Mice, Inbred BALB C
- Neoplasm Transplantation
- Nitroreductases/genetics
- Nitroreductases/metabolism
- Prodrugs/pharmacokinetics
- Prodrugs/pharmacology
- Prodrugs/therapeutic use
- Specific Pathogen-Free Organisms
- Tumor Cells, Cultured/drug effects
- Tumor Cells, Cultured/transplantation
- Tumor Cells, Cultured/virology
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Affiliation(s)
- Nicola K Green
- Department of Clinical Pharmacology, University of Oxford, Radcliffe Infirmary, Oxford, United Kingdom.
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21
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Guo SY, Gu QL, Zhu ZG, Hong HQ, Lin YZ. TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer. World J Gastroenterol 2003; 9:233-7. [PMID: 12532437 PMCID: PMC4611317 DOI: 10.3748/wjg.v9.i2.233] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Cancer gene therapy has received more and more attentions in the recent decade. Various systems of gene therapy for cancer have been developed. One of the most promising choices is the suicide gene. The product of thymidine kinase (TK) gene can convert ganciclovir (GCV) to phosphorylated GCV, which inhibits the synthesis of cell DNA, and then induces the cells to death. Cytokines play an important role in anti-tumor immunity. This experiment was designed to combine the TK gene and mIL-2/mGM-CSF genes to treat gastric cancer, and was expected to produce a marked anti-tumor effect.
METHODS: TK gene was constructed into the retroviral vector pLxSN, and the mIL-2 and mGM-CSF genes were inserted into the eukaryotic expressing vector pIRES. The gastric cancer cells were transfected by retroviral serum that was harvested from the package cells. In vitro study, the transfected gastric cancer cells were maintained in the GCV- contained medium, to assay the cell killing effect and bystander effect. In vivo experiment, retroviral serum and cytokines plasmid were transfected into tumor-bearing mice, to observe the changes of tumor volumes and survival of the mice.
RESULTS: In vitro experiment, 20% TK gene transduced cells could cause 70%-80% of total cells to death. In vivo results showed that there was no treatment effect in control group and TK/GCV could inhibit the tumor growth. The strongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis of the cancer in the treated groups.
CONCLUSION: TK/GCV can kill tumor cells and inhibit the tumor growth in vivo. IL-2 and GM-CSF strongly enhance the anti-tumor effect. Through the retrovirus and liposome methods, the suicide gene and cytokine genes are all expressed in the tissues.
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Affiliation(s)
- Shan-Yu Guo
- Department of Surgery, the Affiliated Shanghai Ninth People's Hospital, Shanghai Second Medical University, Shanghai 200011, China.
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22
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Wang L, Qi X, Sun Y, Liang L, Ju D. Adenovirus-mediated combined P16 gene and GM-CSF gene therapy for the treatment of established tumor and induction of antitumor immunity. Cancer Gene Ther 2002; 9:819-24. [PMID: 12224022 DOI: 10.1038/sj.cgt.7700502] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2002] [Indexed: 11/09/2022]
Abstract
Antitumor effects of combined transfer of P16 and cytokine genes were investigated in this study. The adenovirus harboring the P16 gene (AdP16) and murine granulocyte-macrophage colony-stimulating factor gene (AdGM-CSF) were utilized for the treatment of established tumors. The mice were inoculated subcutaneously with Renca cells and, 6 days later, received an intratumoral injection of AdP16 in the presence or absence of AdGM-CSF. The results demonstrated that tumor-bearing mice treated with AdP16 in combination with AdGM-CSF showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdP16, AdGM-CSF, adenovirus expressing beta-galactosidase, or phosphate-buffered saline alone (P<.01). The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD(4)(+) and CD(8)(+) T cells infiltrating the tumor after combined therapy. After combined therapy, the expression of MHC-1 (H-2K(d)) and Fas molecules on freshly isolated tumor cells increased greatly. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy (P<.01). Our results demonstrated that combined therapy with P16 and GM-CSF genes can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.
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Affiliation(s)
- Linhui Wang
- The Department of Urology, Changhai Hospital, The Second Military Medical University, Shanghai, China.
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23
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Mahvi DM, Shi FS, Yang NS, Weber S, Hank J, Albertini M, Schiller J, Schalch H, Larson M, Pharo L, Gan J, Heisey D, Warner T, Sondel PM. Immunization by particle-mediated transfer of the granulocyte-macrophage colony-stimulating factor gene into autologous tumor cells in melanoma or sarcoma patients: report of a phase I/IB study. Hum Gene Ther 2002; 13:1711-21. [PMID: 12396624 DOI: 10.1089/104303402760293556] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
The primary objective of this phase I study was to determine the safety of an autologous tumor vaccine given by intradermal injection of lethally irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transfected autologous melanoma and sarcoma cells. Secondary objectives included validation of the gene delivery technology (particle-mediated gene transfer), determining the host immune response to the tumor after vaccination, and monitoring patients for evidence of antitumor response. Sixteen patients were treated with either of two different doses of GM-CSF-treated tumor cells. One patient received treatment with both doses of tumor cells. No treatment-related local or systemic toxicity was noted in any patient. Patients administered 100% treated cells (i.e., with a preparation of tumor cells that had all been exposed to GM-CSF DNA transfection) had a more extensive lymphocytic infiltrate at the vaccine site than did patients given 10% treated cells (a preparation of tumor cells in which 10% had been exposed to GM-CSF transfection) or nontreated tumor. The generation of a systemic immune response to autologous tumor by a delayed-type hypersensitivity response to the intradermal placement of nontransfected tumor cells was noted in one patient. One patient had a transient partial response of metastatic tumor sites. The entire procedure, from tumor removal to vaccine placement, was accomplished in less than 6 hr in all patients. Four of 17 patient tumor preparations produced greater than 3.0 ng of GM-CSF per 10(6) cells per 24 hr in vitro. The one patient with greater than 30 ng of GM-CSF per 10(6) cells per 24 hr in vitro had positive DTH, a significant histologic inflammatory response, and clinically stable disease. This technique of gene transfer was safe and feasible, but resulted in clinically relevant levels of gene expression in only a minority of patients.
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Affiliation(s)
- D M Mahvi
- Department of Surgery, University of Wisconsin School of Medicine, Madison, WI 53792, USA.
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24
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Jabłońska E, Kiluk M, Markiewicz W, Jabłoński J. Priming effects of GM-CSF, IFN-gamma and TNF-alpha on human neutrophil inflammatory cytokine production. Melanoma Res 2002; 12:123-8. [PMID: 11930108 DOI: 10.1097/00008390-200204000-00004] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Identifying and evaluating the priming agents for cytokine release by neutrophils might be helpful in controlling the innate immune response of the host. In the present study we examined the role of granulocyte/macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) as priming agents for interleukin (IL)-1beta, IL-6 and TNF-alpha production by stimulated neutrophils from control subjects and malignant melanoma patients. When the cells from controls and patients were preincubated with primer agents, opsonized zymosan-stimulated inflammatory cytokine production was enhanced. The major neutrophil-priming factor for IL-6 secretion by polymorphonuclear leukocytes (PMNs) in the control and patient groups was TNF-alpha. However, GM-CSF and IFN-gamma are also significant primers. GM-CSF priming was critical for the release of TNF-alpha from PMNs in control and melanoma patients. The ability of GM-CSF, IFN-gamma and TNF-alpha to serve as effective priming agents for inflammatory mediator production by PMNs revealed a new role for these cytokines in the innate immune response of the melanoma-bearing host.
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Affiliation(s)
- E Jabłońska
- Department of Immunopathology, Kilinski 1, Medical Academy of Biauulystok, 15-230 Biauulystok 8, Poland.
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25
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Smilowitz HM, Coderre JA, Nawrocky MM, Tu W, Pinkerton A, Jahng GH, Gebbers N, Slatkin DN. The combination of X-ray-mediated radiosurgery and gene-mediated immunoprophylaxis for advanced intracerebral gliosarcomas in rats. J Neurooncol 2002; 57:9-18. [PMID: 12125969 DOI: 10.1023/a:1015709406449] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Rats with advanced, imminently lethal, approximately 4 mm diameter, left-sided intracerebral 9L gliosarcoma (9LGS), a well characterized malignant tumor with some similarities to human high-grade astrocytomas, were used as a therapy model 14 days post-implantation of 10(4) cells. Such tumor-bearing rats die within two weeks (median, 6 days) thereafter if untreated. However, if these tumors are exposed on day 14 to 12-25 Gy of an electron-equilibrated 6 MV photon beam (radiosurgery), survival is extended about 5-6 fold to a median of 34 days, but long-term survival (> 1 year) is increased only to approximately 18%. Multiple subcutaneous inoculations of radiation-disabled 9LGS cells post-radiosurgery (immunoprophylaxis) extended lifespan and long-term (> 1 year) survival minimally (median, 37 days; 25%, respectively). In sharp contrast, radiosurgery followed by multiple subcutaneous inoculations of radiation-disabled 9LGS cells that had been transfected with granulocyte macrophage colony stimulating factor (GMCSF), a cytokine with demonstrated immune-enhancing properties (i.e. gene-mediated immunoprophylaxis, GMIMPR) increased long-term survival to approximately 67%. To our knowledge, these results are the first to show that the combination of photon radiosurgery and GMIMPR is effective for an advanced, imminently lethal brain tumor in a mammal. These data raise the possibility that GMIMPR following radiation therapy might prove effective for the treatment of some human malignant gliomas.
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Affiliation(s)
- H M Smilowitz
- Department of Pharmacology, University of Connecticut Health Center, Farmington 06030, USA.
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26
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Shirahata Y, Ohkohchi N, Itagak H, Satomi S. New technique for gene transfection using laser irradiation. J Investig Med 2001; 49:184-90. [PMID: 11288759 DOI: 10.2310/6650.2001.34045] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
BACKGROUND We have developed a gene transfection system using laser beams. The principle of this procedure is that a small hole is made in a cell membrane by pulse laser irradiation, and a gene contained in a medium is transferred into the cytoplasm through the hole. This hole disappears immediately with the application of laser irradiation of the appropriate power. METHODS A pulse-wave Nd:YAG laser with a wavelength of 355 nm was used to make a hole in a cell membrane. To trap a cell, a continuous-wave Nd:YAG laser with a wavelength of 1015 nm was used. Plasmids that encode the enhanced green fluorescent protein (EGFP) gene were contained in a medium and transferred to HuH-7 and NIH/3T3 cells with pulse laser irradiation. We evaluated transfection efficiency on the basis of the number of cells that expressed EGFP. Stimulatory protein 2 cells in suspension were fixed using a trapping laser and the neomycin-resistance gene was transfected by pulse laser irradiation. We examined cell proliferation in the selection medium. RESULTS Cells that expressed EGFP were recognized in the group that was irradiated by pulse laser. No cells expressed EGFP without irradiation. Transfection efficiency was approximately 10% at a plasmid concentration of 10.0 microg/mL. At concentrations greater than 20 microg/mL, the transfection rate reached a plateau. We also successfully transfected neomycin-resistance genes to cells floating in suspension after fixation that was achieved with trapping laser irradiation. CONCLUSIONS This method enables us to transfect targeted cells, ie, cells in suspension as well as attached cells, with a simple technique that does not involve harmful vectors. The present method is very useful for gene transfection in cellular biotechnology.
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Affiliation(s)
- Y Shirahata
- Division of Advanced Surgical Science and Technology, Graduate School of Medicine, Tohoku University, Sendai, Japan.
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27
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Oshikawa K, Rakhmilevich AL, Shi F, Sondel PM, Yang N, Mahvi DM. Interleukin 12 gene transfer into skin distant from the tumor site elicits antimetastatic effects equivalent to local gene transfer. Hum Gene Ther 2001; 12:149-60. [PMID: 11177552 DOI: 10.1089/104303401750061212] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
We have reported that particle-mediated interleukin 12 (IL-12) gene transfer into the skin overlying the local tumor inhibits systemic metastases. To further characterize this effect, we compared the antitumor and antimetastatic effects of IL-12 cDNA delivered at the local tumor site versus at a site distant from the primary tumor, in a spontaneous metastasis model of LLC-F5 tumor. Local IL-12 gene delivery into the skin overlying the intradermal tumor (local IL-12 treatment) on days 7, 9, and 11 after tumor implantation resulted in the most suppression of the growth of the primary LLC-F5 tumor, whereas IL-12 gene transfer into the skin distant from the tumor (distant IL-12 treatment) was less effective. In contrast, both local IL-12 and distant IL-12 treatment, followed by tumor excision, inhibited lung metastases to a similar extent, resulting in significantly extended survival of test mice. The results of in vivo studies using depleting anti-asialo GM1 antibody and anti-CD4/anti-CD8 monoclonal antibodies, or neutralizing anti-interferon gamma (IFN-gamma) monoclonal antibody demonstrated that natural killer (NK) cells, CD8(+) T cells, and IFN-gamma contributed to the antimetastatic effects in both treatment groups. Furthermore, the levels of mRNA expression of vascular endothelial growth factor and matrix methalloproteinase 9 at the tumor microenvironment were suppressed after both local and distant IL-12 treatment. These results suggest that the current particle-mediated IL-12 gene delivery in the spontaneous LLC-F5 metastasis model can confer antimetastatic activities, irrespective of the gene transfection site, via a combination of several mechanisms involving CD8(+) T cells, NK cells, IFN-gamma, and antiangiogenesis.
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Affiliation(s)
- K Oshikawa
- Department of Pulmonary Medicine, Jichi Medical School, 3311, Minamikawachi, Tochigi, 329-0498, Japan
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Maheshwari A, Mahato RI, McGregor J, Han SO, Samlowski WE, Park JS, Kim SW. Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment. Mol Ther 2000; 2:121-30. [PMID: 10947939 DOI: 10.1006/mthe.2000.0105] [Citation(s) in RCA: 76] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-l-glycolic acid] (PAGA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PAGA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-¿4, 5-dimethylthiazol-2-yl¿-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PAGA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.
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Affiliation(s)
- A Maheshwari
- Center for Controlled Chemical Delivery, University of Utah, Salt Lake City, Utah 84112-5820, USA
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Strong TV. Gene therapy for carcinoma of the breast: Genetic immunotherapy. Breast Cancer Res 2000; 2:15-21. [PMID: 11250688 PMCID: PMC521209 DOI: 10.1186/bcr24] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/1999] [Accepted: 11/18/1999] [Indexed: 11/14/2022] Open
Abstract
Advances in gene transfer technology have greatly expanded the opportunities for developing immunotherapy strategies for breast carcinoma. Genetic immunotherapy approaches include the transfer of genes encoding cytokines and costimulatory molecules to modulate immune function, as well as genetic immunization strategies which rely on the delivery of cloned tumor antigens. Improved gene transfer vectors, coupled with a better understanding of the processes that are necessary to elicit an immune response and an expanding number of target breast tumor antigens, have led to renewed enthusiasm that effective immunotherapy may be achieved. It is likely that immunotherapeutic interventions will find their greatest clinical application as adjuvants to traditional first-line therapies, targeting micrometastatic disease and thereby reducing the risk of cancer recurrence.
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Affiliation(s)
- T V Strong
- Gene Therapy Cewnter, Division of Heamtology-Oncology, University of Alabama at Birmingham, Birmingham, AL 35294-3300, USA.
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