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An infection-enhanced oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Magnus Essand)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
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2013 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 21, no 11, 2008-2018 p.Article in journal (Refereed) Published
Abstract [en]

Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is a major virulence factor involved in H. pylori infection. HP-NAP can mediate anti-tumor effects by recruiting neutrophils and inducing Th1-type differentiation in the tumor microenvironment. It therefore holds strong potential as a therapeutic gene. Here, we armed a replication-selective, infection-enhanced adenovirus with secretory HP-NAP, Ad5PTDf35-[Δ24-sNAP], and evaluated its therapeutic efficacy against neuroendocrine tumors. We observed that it could specifically infect and eradicate a wide range of tumor cells lines from different origin in vitro. Insertion of secretory HP-NAP did not affect the stability or replicative capacity of the virus and infected tumor cells could efficiently secrete HP-NAP. Intratumoral administration of the virus in nude mice xenografted with neuroendocrine tumors improved median survival. Evidence of biological HP-NAP activity was observed 24 hours after treatment with neutrophil infiltration in tumors and an increase of proinflammatory cytokines such as TNF-α and MIP2-α in the systemic circulation. Furthermore, evidence of Th1-type immune polarization was observed as a result of increase in IL-12/23 p40 cytokine concentrations 72 hours post-virus administration. Our observations suggest that HP-NAP can serve as a potent immunomodulator in promoting anti-tumor immune response in the tumor microenvironment and enhance the therapeutic effect of oncolytic adenovirus.

Place, publisher, year, edition, pages
2013. Vol. 21, no 11, 2008-2018 p.
Keyword [en]
Helicobacter pylori, Neutrophil Activating Protein, adenovirus, cancer therapy
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine; Clinical Virology; Medical Virology; Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-203649DOI: 10.1038/mt.2013.153ISI: 000326937000007PubMedID: 23817216OAI: oai:DiVA.org:uu-203649DiVA: diva2:637203
Funder
Swedish Cancer Society, 10‐0105Swedish Cancer Society, 10‐0552Swedish Research Council, K2013‐55X‐22191‐01‐3
Note

De två (2) sista författarna delar sistaförfattarskapet.

Open access under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License (CC BY-NC-ND). 2013.

Supported funds:

The Swedish Children Cancer Foundation(PROJ10/027), Gunnar Nilsson’s Cancer Foundation, Marcus and Marianne Wallenberg’sFoundation

Available from: 2013-07-16 Created: 2013-07-16 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Adenovirus for Cancer Therapy: With a Focus on its Surface Modification
Open this publication in new window or tab >>Adenovirus for Cancer Therapy: With a Focus on its Surface Modification
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 914
Keyword
Adenovirus, cancer, therapy, neuroblastoma, neuroendocrine, modification, Tat, PTD, cell penetrating peptide, Helicobacter pylori, NAP
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Cancer and Oncology Microbiology in the medical area
Research subject
Biology with specialization in Molecular Biology; Clinical Virology; Medical Virology; Molecular Medicine; Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-203662 (URN)978-91-554-8700-3 (ISBN)
Public defence
2013-09-06, Rudbecksalen, The Rudbeck Laboratory C11, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-08-16 Created: 2013-07-17 Last updated: 2014-01-07
2. Cancer Immunotherapy: Evolving Oncolytic viruses and CAR T-cells
Open this publication in new window or tab >>Cancer Immunotherapy: Evolving Oncolytic viruses and CAR T-cells
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 77 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1258
Keyword
Oncolytic virus, Adenovirus, Semliki Forest virus, Cancer immunology, Chimeric antigen receptor T-cells
National Category
Immunology in the medical area
Research subject
Immunology; Oncology; Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-302891 (URN)978-91-554-9705-7 (ISBN)
Public defence
2016-11-21, Rudbecksalen, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-10-31 Created: 2016-09-12 Last updated: 2016-11-02

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Ramachandran, MohanrajYu, DiWanders, AlkwinEssand, Magnus

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