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The Multiple Faces of Genetically-Modified T Cells: Potential Applications in Therapy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this PhD thesis the potential of T-cells as therapy for disease are explored. The applications of genetically modified T-cells for treatment of cancer and autoimmune disease; the functionality and optimal activation of T-cells are discussed.

Successful treatment of cancer with T-cell receptor (TCR)-modified T-cells was first reported in 2006, and is based on recognition of a specific peptide by the TCR in the context of the MHC molecule. As antigen presentation in tumors is often defective and to avoid MHC-restriction, chimeric antigen receptors (CAR) molecules containing an antibody part for recognition of cell surface antigens and TCR and co-receptor signaling domains have been developed. Activated T-cells mount an efficient immune response resulting in the killing of the cancer cell and initiating T-cell proliferation. The rationale for using genetically modified T-cells instead of isolating tumor infiltrating lymphocytes from the tumor and expanding them (TIL therapy) is that it is often very difficult to obtain viable lymphocytes that are able to expand enough in order to use them for therapy.

This thesis explores the possibility of using prostate-specific antigens to target T-cells towards prostate cancer. The prostate has many unique tissue antigens but most patients with metastatic prostate cancer have undergone prostatectomy and consequently have “prostate antigen” expression only in cancer cells. We targeted the prostate antigens TARP and PSCA with a HLA-A2 restricted TCR and a CAR respectively. In both cases the tumor-specific T-cells were able to generate potent proliferative and cytotoxic responses in vitro. The PSCA CAR-modified T-cells delayed subcutaneous tumor growth in vivo. It is evident from our in vivo experiments that the PSCA CAR T-cells were unable to completely cure the mice. Therefore, we aimed to improve the quality of the transferred T-cells and their resistance to the immunosuppressive tumor microenvironment. Stimulation with allogeneic lymphocyte-licensed DCs improved the resistance to oxidative stress and antitumor activity of the T-cells.

We further investigated the potential of genetically modified regulatory T-cells (Tregs) to suppress effector cells in an antigen-specific manner. Using a strong TCR we hypothesize that the phenotype of the TCR-transduced Tregs may be affected by antigen activation of those cells. We found that the engineered Tregs produced cytokines consistent with Th1, Th2 and Treg phenotypes.

Place, publisher, year, edition, pages
Uppsala: Uppsala universitet, 2014. , 78 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1032
Keyword [en]
cancer immunotherapy, genetically engineered T cells, chimeric antigen receptor, T cell receptor, antigen-specific T cells, immunotherapy
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Immunology; Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-232850ISBN: 978-91-554-9050-8 (print)OAI: oai:DiVA.org:uu-232850DiVA: diva2:750688
Public defence
2014-11-15, Rudbecksalen, Dag Hammarsjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-10-24 Created: 2014-09-25 Last updated: 2015-01-23
List of papers
1. T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells
Open this publication in new window or tab >>T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells
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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 39, 15877-15881 p.Article in journal (Refereed) Published
Abstract [en]

To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2–restricted T-cell recptor γ-chain alternate reading-frame protein (TARP)4–13 epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2+ prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP4–13 epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-181199 (URN)10.1073/pnas.1209042109 (DOI)000309604500071 ()
Available from: 2012-09-19 Created: 2012-09-19 Last updated: 2017-12-07Bibliographically approved
2. Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice
Open this publication in new window or tab >>Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice
2014 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 14, 30- p.Article in journal (Refereed) Published
Abstract [en]

Background:

Adoptive transfer of T cells genetically engineered with a chimeric antigen receptor (CAR) has successfully been used to treat both chronic and acute lymphocytic leukemia as well as other hematological cancers. Experimental therapy with CAR-engineered T cells has also shown promising results on solid tumors. The prostate stem cell antigen (PSCA) is a protein expressed on the surface of prostate epithelial cells as well as in primary and metastatic prostate cancer cells and therefore a promising target for immunotherapy of prostate cancer.

Methods:

We developed a third-generation CAR against PSCA including the CD28, OX-40 and CD3 zeta signaling domains. T cells were transduced with a lentivirus encoding the PSCA-CAR and evaluated for cytokine production (paired Student's t-test), proliferation (paired Student's t-test), CD107a expression (paired Student's t-test) and target cell killing in vitro and tumor growth and survival in vivo (Log-rank test comparing Kaplan-Meier survival curves).

Results:

PSCA-CAR T cells exhibit specific interferon (IFN)-gamma and interleukin (IL)-2 secretion and specific proliferation in response to PSCA-expressing target cells. Furthermore, the PSCA-CAR-engineered T cells efficiently kill PSCA-expressing tumor cells in vitro and systemic treatment with PSCA-CAR-engineered T cells significantly delays subcutaneous tumor growth and prolongs survival of mice.

Conclusions:

Our data confirms that PSCA-CAR T cells may be developed for treatment of prostate cancer.

Keyword
CAR T cells, PSCA, Genetic engineering, Prostate cancer, Adoptive transfer
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-218943 (URN)10.1186/1471-2407-14-30 (DOI)000330050700001 ()
Available from: 2014-02-27 Created: 2014-02-20 Last updated: 2017-12-05Bibliographically approved
3. 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
4. Genetic engineering of T regulatory cells with an MHC class I-restricted TCR for controlled peptide-specific activation
Open this publication in new window or tab >>Genetic engineering of T regulatory cells with an MHC class I-restricted TCR for controlled peptide-specific activation
(English)Manuscript (preprint) (Other academic)
Keyword
regulatory T cells, genetic engineering, T cell receptor
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Immunology; Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-232849 (URN)
Available from: 2014-09-25 Created: 2014-09-25 Last updated: 2015-01-23

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