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Improvement of adoptive T-cell therapy for Cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University. (Magnus Essand group)
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 [en]
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: urn:nbn:se:uu:diva-300210ISBN: 978-91-554-9661-6OAI: oai:DiVA.org:uu-300210DiVA: diva2:953234
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
List of papers
1. Allogeneic lymphocyte-licensed DCs expand T cells withimproved antitumor activity and resistance to oxidative stress andimmunosuppressive factors
Open this publication in new window or tab >>Allogeneic lymphocyte-licensed DCs expand T cells withimproved antitumor activity and resistance to oxidative stress andimmunosuppressive factors
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2014 (English)In: Molecular Therapy Methods & Clinical Development, ISSN 2329-0501, Vol. 1, 14001Article in journal (Refereed) Published
Abstract [en]

Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the “rapid expansion protocol” (REP), which utilizes OKT-3, interleukin (IL)-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs) are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs), together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

Place, publisher, year, edition, pages
Nature Publishing Group, 2014
Keyword
rapid expansion protocol, adoptive T cell transfer, immunosuppression, oxidative stress, immunotherapy, T cell expansion protocol, allogeneic lymphocytes, dendritic cells
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-232848 (URN)10.1038/mtm.2014.1 (DOI)
External cooperation:
Note

De två sista författarna delar sistaförfattarskapet.

Available from: 2014-09-25 Created: 2014-09-25 Last updated: 2016-09-02Bibliographically approved
2. Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal gene transfer
Open this publication in new window or tab >>Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal gene transfer
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2016 (English)In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 8, no 7, 702-711 p.Article in journal (Refereed) Published
Abstract [en]

Chimeric antigen receptor (CAR) T-cell therapy is a new successful treatment for refractory B-cell leukemia. Successful therapeutic outcome depends on long-term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV-S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19(+) target cell recognition as LV-engineered T cells and are as effective in controlling tumor growth in vivo We propose that NILV-S/MAR vectors are superior to current options as they enable long-term transgene expression without the risk of insertional mutagenesis and genotoxicity.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-300197 (URN)10.15252/emmm.201505869 (DOI)27189167 (PubMedID)
External cooperation:
Note

GT och MR delar på andraförfattarskapet.

Available from: 2016-08-05 Created: 2016-08-05 Last updated: 2016-09-02Bibliographically approved
3. CD19 CAR T-cells with induced secretion of Helicobacter Pylori Neutrophil-Activating Protein (HP-NAP) yields improved anti-tumor activity and reduced immunosuppression
Open this publication in new window or tab >>CD19 CAR T-cells with induced secretion of Helicobacter Pylori Neutrophil-Activating Protein (HP-NAP) yields improved anti-tumor activity and reduced immunosuppression
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-300198 (URN)
Available from: 2016-08-12 Created: 2016-08-05 Last updated: 2016-09-02
4. Tumor antigen-loaded allogeneic dendritic cells augment therapeutic effect of adoptively transferred T-cells by altering tumor immune-microenvironment
Open this publication in new window or tab >>Tumor antigen-loaded allogeneic dendritic cells augment therapeutic effect of adoptively transferred T-cells by altering tumor immune-microenvironment
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-300209 (URN)
Available from: 2016-08-12 Created: 2016-08-05 Last updated: 2016-09-02

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