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CD19 CAR T-cells with induced secretion of Helicobacter Pylori Neutrophil-Activating Protein (HP-NAP) yields improved anti-tumor activity and reduced immunosuppression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University. (Magnus Essand group)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Magnus Essand group)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Magnus Essand group)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Magnus Essand group)
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-300198OAI: oai:DiVA.org:uu-300198DiVA: diva2:952428
Available from: 2016-08-12 Created: 2016-08-05 Last updated: 2016-09-30
In thesis
1. Improvement of adoptive T-cell therapy for Cancer
Open this publication in new window or tab >>Improvement of adoptive T-cell therapy for Cancer
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancer immunotherapy has recently made remarkable clinical progress. Adoptive transfer of T-cells engineered with a chimeric antigen receptor (CAR) against CD19 has been successful in treatment of B-cell leukemia. Patient’s T-cells are isolated, activated, transduced with a vector encoding the CAR molecule and then expanded before being transferred back to the patient. However some obstacles restrict its success in solid tumors. This thesis explores different aspects to improve CAR T-cells therapy of cancer.

Ex vivo expanded T-cells are usually sensitive to the harsh tumor microenvironment after reinfusion. We developed a novel expansion method for T-cells, named AEP, by using irradiated and preactivated allo-sensitized allogeneic lymphocytes (ASALs) and allogeneic mature dendritic cells (DCs). AEP-expanded T-cells exhibited better survival and cytotoxic efficacy under oxidative and immunosuppressive stress, compared to T-cells expanded with established procedures.

Integrating retro/lentivirus (RV/LV) used for CAR expressions randomly integrate in the T-cell genome and has the potential risk of causing insertional mutagenesis. We developed a non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element (NILV-S/MAR) for T-cells transduction. NILV-S/MAR-engineered CAR T-cells display similar cytotoxicity to LV-engineered CAR T-cells with undetectable level of insertional event, which makes them safer than CAR T-cells used in the clinic today.

CD19-CAR T-cells have so far been successful for B-cell leukemia but less successful for B-cell lymphomas, which present semi-solid structure with an immunosuppressive microenvironment. We have developed CAR T-cells armed with H. pylorineutrophil-activating protein (HP-NAP). HP-NAP is a major virulence factor and plays important role in T-helper type 1 (Th1) polarizing. NAP-CAR T-cells showed the ability to mature DCs, attract innate immune cells and increase secretion of Th1 cytokines and chemokines, which presumably leads to better CAR T-cell therapy for B-cell lymphoma.

Allogeneic-DCs (alloDCs) were used to further alter tumor microenvironment. The premise relies on initiation of an allo-reactive immune response for cytokine and chemokines secretion, as well as stimulation of T-cell response by bringing in tumor-associated antigen. We demonstrated that alloDCs promote migration and activation of immune cells and prolong the survival of tumor-bearing mice by attracting T-cells to tumors and reverse the immune suppressive tumor microenvironment.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1247
Keyword
CAR T-cell therapy; AEP expansion protocol; scaffold matrix attachment region; non-integrating lentivirus; H. pylori Neutrophil-activating protein; allogeneic DCs
National Category
Immunology in the medical area
Research subject
Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-300210 (URN)978-91-554-9661-6 (ISBN)
Public defence
2016-10-06, Rudbecksalen, Dag Hammarskjoldsv 20 Rudbeck laboratory, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2016-09-15 Created: 2016-08-05 Last updated: 2016-09-22
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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