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Preclinical evaluation of immunostimulatory gene therapy for pancreatic cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Angelica Loskog)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Pancreatic cancer is characterized by its desmoplastic tumor microenvironment and the infiltration of immunosuppressive cells. It is a devastating disease where most patients are diagnosed at a late stage and the treatment options are few. The development of new treatments is surly needed. One treatment option explored is the use of immunotherapy with the intent to activate the immune system and change the balance from pro-tumor to anti-tumor. This thesis presents the idea of using oncolytic adenoviruses called LOAd-viruses that are armed with immunostimulatory- and microenvironment-modulating transgenes. For effective treatment of pancreatic cancer, the virus needs to be able to be given in addition to standard therapy, the chemotherapy gemcitabine. In paper I, the immunomodulatory effect of gemcitabine was evaluated in blood from pancreatic cancer patients receiving their first 28-day cycle of treatment with infusions day 1, 8 and 15 followed by a resting period. Gemcitabine reduced the level of immunosup-pressive cells and molecules but the effect did not last throughout the resting period. On the other hand, gemcitabine did not affect the level or proliferative function of effector T cells indicating that gemcitabine could be combined with immunotherapy.

The LOAd700 virus expresses a novel membrane-bound trimerized form of CD40L (TMZ-CD40L). In paper II, LOAd700 showed to be oncolytic in pancreatic cancer cell lines as well as being immunostimulatory as shown by its capacity to activate dendritic cells (DCs), myeloid cells, endothelium, and to promote expansion of antigen-specific T cells. In paper III, LOAd703 armed with both 4-1BBL and TMZ-CD40L was evaluated. LOAd703 gave a more profound effect than LOAd700 on activation of DCs and the virus was also capable of reducing factors in stellate cells connected to the desmo-plastic and immunosuppressive microenvironment. In paper IV, LOAd713 armed with TMZ-CD40L in combination with a single-chain variable fragment against IL-6R was evaluated. The virus could kill pancreatic cancer cells lines through oncolysis and could also reduce factors involved in desmoplasia in stellate cells. Most interestingly, LOAd713 could reduce the up-regulation of PD-1/PD-L1 in DCs after CD40 activation. Taken together, LOAd703 and LOAd713 seem to have interesting features with their combination of immunostimulation and microenvironment modulation. At present, LOAd703 is evaluated in a clinical trial for pancreatic cancer (NCT02705196).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 66 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1379
Keyword [en]
Pancreatic cancer, immunotherapy, oncolytic viruses, adenoviruses, CD40L, 4-1BBL, IL-6, tumor microenvironment
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cancer and Oncology Immunology in the medical area
Research subject
Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-330189ISBN: 978-91-513-0102-0 (print)OAI: oai:DiVA.org:uu-330189DiVA: diva2:1148188
Public defence
2017-12-01, Fåhræussalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-11-08 Created: 2017-10-10 Last updated: 2017-11-08
List of papers
1. Gemcitabine reduces MDSCs, tregs and TGF beta-1 while restoring the teff/treg ratio in patients with pancreatic cancer
Open this publication in new window or tab >>Gemcitabine reduces MDSCs, tregs and TGF beta-1 while restoring the teff/treg ratio in patients with pancreatic cancer
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2016 (English)In: Journal of Translational Medicine, ISSN 1479-5876, E-ISSN 1479-5876, Vol. 14, 282Article in journal (Refereed) Published
Abstract [en]

Background: Cancer immunotherapy can be potentiated by conditioning regimens such as cyclophosphamide, which reduces the level of regulatory T cells (tregs). However, myeloid suppressive cells are still remaining. Accordingly to previous reports, gemcitabine improves immune status of cancer patients. In this study, the role of gemcitabine was further explored to map its immunological target cells and molecules in patients with pancreatic cancer.

Methods: Patient blood was investigated by flow cytometry and cytokine arrays at different time points during gemcitabine treatment.

Results: The patients had elevated myeloid-derived suppressor cells (MDSCs), and Tregs at diagnosis. Myeloid cells were in general decreased by gemcitabine. The granulocytic MDSCs were significantly reduced while monocytic MDSCs were not affected. In vitro, monocytes responding to IL-6 by STAT3 phosphorylation were prevented to respond in gemcitabine medium. However, gemcitabine could not prevent STAT3 phosphorylation in IL-6-treated tumor cell lines. TGF beta-1 was significantly reduced after only one treatment and continued to decrease. At the same time, the effector T cell:Treg ratio was increased and the effector T cells had full proliferative capacity during the gemcitabine cycle. However, after a resting period, the level of suppressor cells and TGF beta-1 had been restored showing the importance of continuous conditioning.

Conclusions: Gemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGF beta-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy.

Keyword
Gemcitabine, Pancreatic cancer, Tregs, MDSCs, TGF beta
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-306753 (URN)10.1186/s12967-016-1037-z (DOI)000384600600002 ()27687804 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2016-11-16 Created: 2016-11-03 Last updated: 2017-10-10Bibliographically approved
2. Activation of myeloid and endothelial cells by CD40L gene therapy supports T-cell expansion and migration into the tumor microenvironment
Open this publication in new window or tab >>Activation of myeloid and endothelial cells by CD40L gene therapy supports T-cell expansion and migration into the tumor microenvironment
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2017 (English)In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 24, no 2, 92-103 p.Article in journal (Refereed) Published
Abstract [en]

CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.

National Category
Other Medical Biotechnology
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-318170 (URN)10.1038/gt.2016.80 (DOI)000394682800006 ()27906162 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2017-10-10Bibliographically approved
3. Shaping the Tumor Stroma and Sparking Immune Activation by CD40 and 4-1BB Signaling Induced by an Armed Oncolytic Virus.
Open this publication in new window or tab >>Shaping the Tumor Stroma and Sparking Immune Activation by CD40 and 4-1BB Signaling Induced by an Armed Oncolytic Virus.
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2017 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265Article in journal (Refereed) Epub ahead of print
Abstract [en]

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma.Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses.Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells.Conclusions: LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. Clin Cancer Res; 1-12. ©2017 AACR.

National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-330158 (URN)10.1158/1078-0432.CCR-17-0285 (DOI)28536305 (PubMedID)
Available from: 2017-09-27 Created: 2017-09-27 Last updated: 2017-10-10
4. An oncolytic virus for pancreatic cancer targeting interleukin-6 signaling and driving CD40-mediated immune activation
Open this publication in new window or tab >>An oncolytic virus for pancreatic cancer targeting interleukin-6 signaling and driving CD40-mediated immune activation
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(English)In: Article in journal (Other academic) Submitted
National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-330161 (URN)
Available from: 2017-09-27 Created: 2017-09-27 Last updated: 2017-10-10

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