ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1258

Cancer Immunotherapy

Evolving Oncolytic viruses and CAR T-cells

MOHANRAJ RAMACHANDRAN

ISSN 1651-6206ISBN 978-91-554-9705-7urn:nbn:se:uu:diva-302891

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, DagHammarskjöldsväg 20, Uppsala, Monday, 21 November 2016 at 09:15 for the degree ofDoctor of Philosophy (Faculty of Medicine). The examination will be conducted in English.Faculty examiner: Professor Richard Vile (Mayo Clinic, Rochester, Minnesota, USA).

AbstractRamachandran, M. 2016. Cancer Immunotherapy. Evolving Oncolytic viruses and CART-cells. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty ofMedicine 1258. 77 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9705-7.

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedsideand being approved as drugs. Cancer immunotherapy harnesses the power of patient’s ownimmune system to fight cancer. Approaches are diverse and include antibodies, therapeuticvaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses andimmune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responseshave been observed for certain cancers with checkpoint antibodies and chimeric antigenreceptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to becombined for broader effective treatment responses because cancers evolve to escape immunerecognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24])was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expressionof HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatorycytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activatedendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically maturedand 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-cellactivation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CART-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus withnatural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA targetsequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthyCNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while itretained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneicneuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated withtype-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combiningimmunotherapeutic strategies with HP-NAP is a promising approach to combat cancers andSFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.

Keywords: Oncolytic virus, Adenovirus, Semliki Forest virus, Cancer immunology, Chimericantigen receptor T-cells

Mohanraj Ramachandran, Science for Life Laboratory, SciLifeLab, Box 256, UppsalaUniversity, SE-75105 Uppsala, Sweden. Department of Immunology, Genetics and Pathology,Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden.

© Mohanraj Ramachandran 2016

ISSN 1651-6206ISBN 978-91-554-9705-7urn:nbn:se:uu:diva-302891 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-302891)

Dreams are not those which come while we are sleeping, but dreams are those when you don’t sleep before fulfilling them.

— Dr. A.P.J. Abdul Kalam

ேகடில் விழுச்ெசல்வம் கல்வி ெயாருவற்கு

மாடல்ல மற்ைற யைவ.

—திருவள்ளுவர்

Dedicated

-To my family

List of original papers

This thesis is based on the following articles

1. Ramachandran M, Yu D, Wanders A, Essand M#, Eriksson F#. An infection-enhanced oncolytic adenovirus secreting Helicobacter Pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors. Mol. Ther., 21(11): 2008-2018, 2013.

2. Ramachandran M, Jin C, Yu D, Eriksson F, Essand M. Vector-encoded Helicobacter pylori neutrophil activating protein promotes maturation of dendritic cells with Th-1 polarization and improved migration. J Immu-nol., 193(5): 2287-2296, 2014.

3. Jin C*, Ma J*, Ramachandran M, Yu D, Essand M. CD19 CAR T-

cells with induced secretion of Helicobacter Pylori Neutrophil-Activating Protein (HP-NAP) yields improved anti-tumor activity and reduced immunosuppression. Manuscript

4. Ramachandran M*, Yu D*, Dyczynski M, Baskaran B, Zhang L, Lulla A, Lulla V, Saul S, Nelander S, Dimberg A, Merits A, Leja Jarblad J#, Essand M#. Safe and effective treatment of experimental neuroblastoma and glioblastoma using systemically delivered triple microRNA-detargeted oncolytic Semliki Forest virus. Clin. Cancer Res., 2016, Sept 16 [Epub ahead of print]

*Equal Contributions- First Author.

# Equal Contributions- Last Author

Reprints were made with permissions from the respective publishers

1. Open access under the terms of the Creative Commons Attribution-Non Commercial-No Derivs 3.0 Unported License (CC BY-NC-ND)

2. Copyright© 2014. The American Association of Immunologists, Inc.

4. Copyright© 2016. American Association for Cancer Research

Other related publications and work

1. Yu, D, Jin, C, Ramachandran, M, Xu, J, Nilsson, B, Korsgren, O, et al. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of pri-mary cell cultures. PLoS One, 8(1):e54952, 2013.

2. Hillerdal. V, Ramachandran. M, Leja. J, Essand. M. Systemic treat-ment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice. BMC Cancer, 18;14:30, 2014.

3. Jin. C, Fotaki. G, Ramachandran. M, Nilsson. B., Essand. M, Yu. D. Safe engineering of CAR T cells for adoptive cell therapy of cancer us-ing long-term episomal gene transfer. EMBO Mol. Med., 8(5):e201505869, 2016.

4. Anwar M., Arendt M., Ramachandran M., Essand M., Akusjärvi. G, Öberg. D. Ixovex, a novel oncolytic E1B-mutated adenovirus. Submit-ted: PLoS One, 2016

Main supervisor: Prof. Magnus Essand, IGP, Uppsala University, Sweden Co-supervisor: Dr. Di Yu, IGP, Uppsala University, Sweden Dr. Justyna Leja-Jarblad, IGP, Uppsala University, Sweden Faculty opponent: Prof. Richard G. Vile, Mayo Clinic, Rochester, Minnesota, USA Examination committee: Asc prof. Göte Svedberg, IMBIM, Uppsala University, Sweden Prof. Lars Hellman, ICM, Uppsala University, Sweden Asc prof. Gerald McInerney, MTC, Karolinska Institute, Sweden

Table of Contents

CANCER IMMUNOTHERAPY: ................................................................. 13 Introduction .............................................................................................. 13 Cancer Immunoediting ............................................................................. 15 Cancer Immunity Cycle ........................................................................... 16 

DENDRITIC CELLS .................................................................................... 20 

ONCOLYTIC VIRUS THERAPY FOR CANCER ..................................... 24 Oncolytic Virotherapy to Viro-Immunotherapy: the new perspective. .... 25 Adenovirus ............................................................................................... 27 

Adenovirus life cycle ........................................................................... 28 Adenovirus as oncolytic agents ........................................................... 31 

Semliki Forest virus ................................................................................. 34 SFV genome and life cycle .................................................................. 34 SFV neuro-pathogenesis ...................................................................... 35 SFV as oncolytic agents ....................................................................... 38 

ADOPTIVE CELL IMMUNOTHERAPY ................................................... 40 Chimeric Antigen Receptor engineered T-cells ....................................... 41 Lessons from clinical application of CAR T-cells ................................... 42 

METHODS ................................................................................................... 44 1. Arming Oncolytic Viruses and CAR T-cells with therapeutic transgene................................................................................................... 44 

Helicobacter pylori Neutrophil Activating Protein (HP-NAP) ........... 45 2. MicroRNA Detargeting ........................................................................ 49 

AIMS ............................................................................................................ 51 

RESULTS AND DISCUSSION ................................................................... 52 

FUTURE PRESPECTIVES .......................................................................... 56 

GLOSSARY ................................................................................................. 58 

ACKNOWLEDGEMENTS .......................................................................... 62 

REFERENCES ............................................................................................. 66 

Abbreviations

ACT Adoptive Cell Therapy

ADP Adenovirus Death protein

Ads Human Adenovirus

APC Antigen Presenting cell

ATP Adenosine triphosphate

CAR Chimeric Antigen Receptor

CCL Chemokine (C-C motif) ligand

CD Cluster of Differentiation

CNS Central Nervous System

CPV Cytopathic vacuoles

CR Complete Remission/Response

CRAds Conditionally Replication Adenoviruses

CRS Cytokine release syndrome

CRT Calreticulin

CTLA Cytotoxic T-lymphocyte-associated protein

CTLs Cytotoxic T-lymphocytes

CXCL Chemokine (C-X-C motif) ligand

DAMPs Danger Associated Molecular Patterns

DC Dendritic cell

DNA Deoxyribonucleic acid

GBM Glioblastoma Multiforme

GD2 A disialoganglioside expressed on tumors of neuroectodermal origin

GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor

HMGB High Mobility Group protein B

HP-NAP Helicobacter pylori Neutrophil Activating Protein

HSCT Hematopoietic Stem cell Transplantation

HSP Heat Shock Proteins

HVR Hyper Variable Region

ICAM Intercellular Adhesion Molecule

ICD Immunogenic Cell Death

IDA Iron Deficiency Anemia

IFN Interferon

IL Interleukin

Kbp Kilo base pairs

LAG-3 Lymphocyte-Activation Gene 3

LFA Lymphocyte Function-associated Antigen

MDSC Myeloid Derived Suppressor cell

MHC Major Histocompatibility Complex

MIP Macrophage Inflammatory Protein

miRISC miRNA-induced silencing complex

miRNA Micro Ribonucleic acid (microRNA)

mRNA Messenger Ribonucleic acid

NK Natural Killer

nsP Non-structural Protein

OR Objective Response

OV Oncolytic virus

PAI-2 Plasminogen Activator Inhibitor-2

PAMPs Pattern Associated Molecular Patterns

PD Programmed cell Death protein

pRb Retinoblastoma protein

RNA Ribonucleic acid

RNP Ribonucleo Protein

ROS Reactive Oxygen Species

scFv Single-Chain variable Fragment

SFV Semliki Forest virus

siRNA Small interfering Ribonucleic acid

TAA Tumor-Associated Antigen

TCR T-cell Receptor

TF Tissue Factor

TGF Transforming Growth Factor

Th T Helper cell

TILs Tumor-infiltrating Lymphocytes

TIM-3 T-cell Immunoglobulin and Mucin-domain containing-3

TLR Toll-like Receptor

TME Tumor Microenvironment

TNF Tumor Necrosis Factor

Treg Regulatory T-Lymphocyte

UTR Untranslated Region

VCAM Vascular cell adhesion protein

VEGF Vascular Endothelial Growth Factor

13

CANCER IMMUNOTHERAPY:

Introduction The immune system is the guardian of an individual and its principle role is to protect and fight against invading pathogens and infections. It can also identify abnormal cell in the body like neoplastic lesions and cancer cells. As we learn more about tumor biology and its interaction with the immune sys-tem it has become clear that the tumor immune microenvironment is an im-portant factor determining cancer progression. For example, cancer patients undergoing hematopoietic stem cell transplantation for the purpose of replac-ing their bone marrow after chemotherapy have a significantly lower risk of relapse indicating a graft vs. tumor response1, 2 and patients undergoing organ transplants and as a consequence are under constant immune suppres-sion are more susceptible and have higher risk of developing de novo can-cers3. This knowledge has led to re-definition of the hallmarks of cancer by Hanahan and Weinberg, where they define “avoiding immune destruction” and “tumor-promoting inflammation” as emerging hallmarks and enabling characteristics for cancer progression respectively4. However, the role of immune system in cancer can be diverse ranging from pro-tumorigenic: e.g. immunosuppression in the tumor microenvironment a situation when the cancer cells manipulate and deceive the immune system to protect itself from immune destruction, to anti-tumorigenic: e.g. certain immune cell (especially cytotoxic T-cell) infiltration to the tumor microenvironment, leading to in-creased overall survival and better prognosis5.

Immunotherapy for cancer is a treatment by which one tries to re-educate the immune cells of their own abilities and boost an effective immune response to eradicate cancer. The first attempt to stimulate immune response in the purpose of cancer treatment was tried more than a century, ago in the 1890s, when William Coley used a mixture of killed bacteria to treat cancer6. Col-ey’s attempts led to some successes but were never carefully controlled. In the 1970s a cancer vaccine based on these bacterial toxins (Coley toxins)7 and administration of Bacillus Calmette-Guerin8 , a vaccine primarily used against tuberculosis, were shown to be beneficial for some types of bladder cancer. These early promises of cancer immunotherapy unfortunately did not live up to the expectation and it is not until recently that cancer immunother-apy have made major breakthroughs. Several immunotherapeutic drugs are

14

now approved for cancer treatment9, 10. This success can be attributed to studies in understanding how the cancer cells interact with immune cells and eventually evade immune recognition. To celebrate the recent success and acknowledge the potential that cancer immunotherapy holds to cure like cancer, Science Magazine in 2013 announced it as the “Breakthrough of the year” 11.

General overview of the immune system The immune system can be broadly divided in to two parts depending on the speed and specificity of the immune reaction as innate and adaptive respons-es respectively. The first response to an invading pathogen is mediated by the innate arm (non-specific response), which comprises of anatomical and biochemical barriers (skin and mucous membranes). It also comprises un-specific cellular (neutrophils, monocytes, macrophages dendritic cells and, natural killer (NK) cells) responses and soluble factors (serum proteins, cy-tokines, chemokines and complement system). The innate immune response is characterized by quick recognition and elimination of pathogens. The ana-tomical barriers prevent entry and proliferation of pathogens, and if the bar-riers are breached the cellular and soluble factor part of the innate immune response are summoned12, 13. These cells are usually equipped with pattern recognizing receptors that recognize pathogen associated molecular patterns (PAMPs) and initiate a generalized attack that either phagocytose pathogens or kill infected cells 14.

The adaptive arm can be categorized as humoral and cellular immunity mediated by B-lymphocytes (B-cells) and T-lymphocytes (T-cells) respec-tively. The adaptive arm is characterized as precise and long-lasting immune response, which involves use of antigen-specific receptors and antigenic memory. An antigen is any substance or molecular pattern that can give rise to a specific immune response, e.g. a viral protein, bacterial protein, or mu-tated molecule expressed in tumors. In the first step of adaptive immune response, the antigen is presented to lymphocytes by antigen presenting cells (APC) in the lymphoid tissues. This primes and activates the lymphocytes to initiate an effector response to the antigenic stimuli. In case of B-cells, plas-ma cells are formed and antibodies are secreted that neutralize extracellular toxins and microbes. In case of T-cells, they leave the lymphoid tissue to the site of disease where they kill infected and abnormal cells, e.g. a virus-infected cell or a cancer cell12, 13.

The innate immune arm also co-ordinates with the adaptive immune arm to initiate long-lasting and specific responses. Cytokines and chemokines se-creted by the innate immune cells are vital signals for stimulation of effec-

15

tive B-cell and T-cell responses. APCs such as dendritic cells (DCs) and macrophages play a key role in bridging the two arms of the immune sy-stem12. Through this well-structured and inter-connected organization the immune system is able to keep our bodies in check from invaders and dis-eases. DCs will be discussed in more details later in this thesis.

Cancer Immunoediting The interaction between the immune system and cancer is a dynamic process and the immune system can actively both aid and counteract cancer progres-sion. Understanding this process is essential to develop new immune thera-peutic strategies to fight cancer. This complex dynamic process of interplay between cancer and immune system is defined as “cancer immunoediting” and has 3 phases: elimination, equilibrium, and escape15.

The Elimination phase is the cancer immuno-surveillance hypothesis rede-fined. Arising neoplastic cancer cells are actively identified by both the in-nate and adaptive immune system and destroyed before they are diagnosed (immune protection). How the cancer cells are recognized is not fully under-stood. However, some classical hypotheses like “danger signals” e.g., type I interferon (IFN) secretion by developing tumors can serve as stimuli for DCs and other innate immune cells16. Also, expression of stress ligands such as MICA/B (human) and RAE-1 (mouse) by tumors and release of damage-associated molecular pattern molecules (DAMPs) like high mobility group box 1 (HMGB1) by dying tumor cells are thought to play an important role in triggering an anti-tumor innate immune response leading to the production of IFN-γ (Table 1)15, 17. The initial effects of an innate immune response are amplified, leading to production of chemokines that recruit more immune cells like NK cells, macrophages and DC that can kill tumor cells and liber-ate tumor antigens. This drives the development of a tumor-antigen-specific adaptive immune response that actively eliminates cancer cells15. This pro-cess is also known as the cancer-immunity cycle and is in detail discussed in the next section. Effectiveness of the elimination process is entirely depend-ent on the characteristics of the cancer cell such as its rate of growth, origin and anatomical location.

In the Equilibrium phase the immune system and the cancer cells that have escaped surveillance enter into a dynamic equilibrium. This is thought to be the longest phase of the immunoediting process where the adaptive immune system maintains the tumor cells in a dormant state and also determines the immunogenicity of the tumor cells15, 17. The adaptive immune compartment, specifically T-cells (both CD4 and CD8) and the vital cytokines interleukin (IL)-12 and IFN-γ are responsible for controlling tumor cell growth18. This

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distinguishes the equilibrium from elimination phase where both innate and adaptive immune cells actively take part in tumor eradication. The end result of the equilibrium process is the arising of tumor clones that have acquired immune evasive mutations.

In the Escape phase the clones that have evaded immune recognition grow, leading to the development of diagnosable and progressive disease. Tumor escape and progression can occur in several mechanisms, by genetic aberra-tions in the tumor cells and by establishment of an immunosuppressive mi-croenvironment. Possible genetic changes in the tumor cells are: (a) clones that become resistant to cytotoxic immune cells by either activation of anti-apoptotic effector molecules like BCl-2 or continuous activation of pro-oncogenic factors like STAT-3; (b) clones that down-regulate tumor-associated antigens (TAA), or down-regulate major histocompatibility com-plex (MHC) class I (MHC-I) and thereby down-regulate antigen presentation or loss of antigen processing capacity within the tumor cell and ultimately becoming invisible to immune surveillance15, 19. Tumor cells can induce an immunosuppressive microenvironment and make it unfavorable for active tumor elimination. This is mediated by secretion of immunosuppressive cytokines by the tumors themselves, such as transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), or galectin to name a few. Actively progressing tumors also recruit regulatory immune cells like regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) that inhibit anti-tumor immune responses. Both the regulatory immune cells and tumor cells express molecules that dampens the T-cell immune response, such as cytotoxic T-lymphocyte-associated protein (CTLA)-4, programmed cell death protein (PD)-1 on T-cells and a ligand for PD-1 (PD-L1) on tumor cells (Table 1)17.

Cancer Immunity Cycle A sequence of organized, step-wise, iterative events need to be initiated to mount effective anti-cancer immune response. The seven steps involved in this process are referred to as the cancer immunity cycle by Chen D.S. et. al. (Figure 1, Table 1)20. A well-known characteristic of cancer cell is accumu-lation of genetic aberrations, which leads to expression of neoantigens and global loss of DNA methylation, which lead to reactivation of embryonal antigens referred to as cancer testis antigens. In the first step of cancer im-munity cycle, these antigens are released from dying cancer cells and phago-cytosed by DCs. In order to yield an effective anti-tumor T-cell response, strong immunogenic signals must be accompanied by immunogenic cancer cell death to activate DCs. These signals can be cytokines, danger signals released by dying tumor cells, etc. (Table 1). In the second step activated

17

DCs carrying the tumor-associated antigens (TAA), present them to T-cells via both MHC-I (cross-presentation) and MHC-II. The naïve T-cells are primed and activated to become effector T-cells in the lymphoid organs (step 3). This is an important step because it determines if the cancer antigen is viewed as foreign or an antigen which has escaped the central tolerance. Also, the nature of the immune response mounted and the final outcome of an effective anti-tumor response is determined by this step, which depends on the ratio of Tregs versus antigen-specific effector T-cells. Activated effec-tor T-cells leave the lymphoid organs (step 4), and infiltrate the tumor mass (step 5). In the final few steps the effector T-cells recognize antigens pre-sented on MHC-I by cancer cells in a specific manner via the T-cell receptor (TCR) (step 6) and kill them by releasing granzymes and perforins (step 7). This leads to release of more TAA and the immunity cycle returns to step 1. However, in cancer patients not all these processes are optimally functional, leading to poor anti-tumor immunity (Table 1)20. Immunotherapeutic ap-proaches aim to trigger or release the breaks in any, or preferably multiple, of the above steps to initiate an effective self-sustaining anti-tumor cycle.

Cancer immunotherapy comprises of a broad range of treatment approaches that target different steps in the immunity cycle. A few examples of success-ful immunotherapies are mentioned here. The most successful immunothera-peutic approach available today is immune checkpoint inhibiting antibodies (anti-PD-1, anti-PD-L1 and anti-CTLA-4) to release the breaks of antitumor T-cells. The US Food and Drug Administration (FDA) has approved the CTLA-4 inhibitor Ipilimumab for the treatment of metastatic melanoma21, the PD-1 inhibitor Nivolumab for treatment of for melanoma, lung cancer, kidney cancer and Hodgkin’s lymphoma and the PD-1 inhibitor Pembroli-zumab for the treatment of metastatic for melanoma, non-small cell lung cancer, and head-and-neck cancer (sarcoma)22. European and Asian regula-tors are not far behind. Adoptive transfer of chimeric antigen receptor (CAR) T-cells engineered to recognize the pan-B-cell marker CD19 is also very successful in the clinic for the treatment of B-cell malignancies23 and may reach FDA approval within 1-2 years. . Talimogene laherparepvec (T-vec) an oncolytic herpes simplex virus (HSV)-1 secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), was approved by the FDA in 201524 and in 2016 approved in Europe25 for treatment of un-operable melanomas.

18

s

Figure 1 The Cancer-Immunity cycle: The steps involved in mounting an effective, self-propagating anti-tumor immune response. Steps 1-3: initiating and propagat-ing anti-tumor immune response. Steps 4-5: immune cell emigration from the lymph node and accessing the tumor. Steps 6-7: tumor cell recognition in an antigen-specific manner and execution of cytolytic activity. This is a cyclic process; dying tumor cells release more tumor cell antigens that initiate a new round of the immun-ity cycle

 

Table 1 Positive and negative regulators of the Cancer-Immunity cycle and current-ly researched immunotherapeutic approaches (Table adopted and modified from20).

Step Positive regula-tors

Negative regula-tors

Immunotherapeutic approaches

1. Release of cancer antigens

Necrotic cell death

Immunogenic cell death

Programmed or tolerogenic cell death

Chemotherapy

Radiotherapy

Targeted therapies (oncolytic viruses)

2. Cancer antigen capture and presenta-

Adjuvants re-leased from dy-ing tumor cells (ATP, HMGB1, CRT)

IL-10, IL-13, IL-4 Vaccines

GM-CSF

19

Step Positive regula-tors

Negative regula-tors

Immunotherapeutic approaches

tion Pro inflammato-ry cytokines (TNF-α. IL1, IFN-α)

Immune stimula-tors (CD40/CD40L, GMCSF)

Microbial prod-ucts (TLR lig-ands)

Anti-CD40 agonist

TLR agonist

3. Priming and activa-tion

CD28:B7.1, OX40:OX40L, CD27:CD70, CD137 (4-1BB)/CD137L, GITR, IL-2, IL-12

CTLA-4, PD-L1, prostaglandins

Anti-CTLA4

Anti-OX40 agonist

IL-2

IL-12

4. Homing to tumor

CX3CL1, CXCL9, CXCL10, CCL5

Chemokine receptor expression in CAR T-cells

5. Vascular extravasa-tion

LFA1:ICAM1,

Selectins

endothelin B re-ceptor, VEGF

Anti-VEGF

Sunitinib

6. Tumor recognition

TCR: MHC-I Reduced peptide-MHC-I expression on cancer cells

CAR T-cells

7. Killing of cancer cells

Granzyme B,

Perforins,

IFN-γ

TIM3, LAG3, Arginase

PD-L1:PD1, PD-L1:B7.1, IDO, TGF-β

Anti PD-1

Anti-PDL-1

IDO inhibitors

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DENDRITIC CELLS

DCs belong to the innate immune system and were identified by Steinman and Chon in 197326, for which Steinman post-mortem received the Nobel prize in 2011. DCs are specialized APCs hence they mediate the link be-tween innate and adaptive immune system (B and T cells). They are also known as ‘professional APCs’, as they are very efficient in stimulating even low number of T-cells to respond to the antigens they carry and present. DCs can be of different origins, i.e. myeloid or lymphoid and can also be mono-cyte-derived in vitro under the right environmental conditions27, 28. They are usually in their immature state (immDC) and reside in the peripheral tissues, epithelia and blood. immDC have high capacity to capture, internalize and process antigens29. They mature when in contact with activation signals like toll-like receptor (TLR) ligands, CD40L, danger signals and chemokines29. Mature DCs (mDCs) have upregulated surface expression of MHC class I and class II, through which antigenic peptides are presented to T-cells. Non-peptide antigens like lipids are presented to T-cells through molecule of the CD1 family by the DCs30. The maturation process is also accompanied by the upregulation of co-stimulatory molecules (CD80, CD86, and CD40), maturation markers CD83, cytokine secretion and migration to the draining lymph nodes, which have important roles in T-cell activation (Figure 2). They present captured antigens to naïve T-cells in the lymphoid organs to initiate a specific immune response.

Antigen presentation Antigen presentation by DCs is mediated by MHC-I and MHC-II complexes, and DCs are one of the few cells in the human body that have expression of both the complexes. MHC-I belongs to the immunoglobulin superfamily, having an alpha chain with three domains and a beta chain (β2 microglobu-lin) for stability. They usually present small endogenous peptides (8-9 amino acids) to CD8+ T cells. This type of presentation is common in viral infec-tions, intracellular bacterial infections and in cancers where mutated/ over expressed self-proteins are presented31. MHC-I complex is expressed by all nucleated cells of the body. MHC-II complex on the other hand is only ex-pressed on APCs like macrophages, DCs and B cells. They are heterodimers made of an alpha and a beta chain and usually present peptides from exoge-nous proteins (microbes, pathogens and TAAs) that are phagocytized and

21

processed in the endosomes. MHC-II presentation is very important for priming of CD4+ T-cells (also known as T helper cells)32.

DCs can mediate a third mode of antigen presentation called cross-presentation. The source of antigens for DCs is rarely endogenous, because DCs mostly uptake exogenous antigens by phagocytosis. An important char-acteristic of DCs called “cross priming” has been described, where endoge-nous antigens (viral proteins and mutated/over-expressed tumor proteins) were phagocytosed from dying/dead cells and presented on MHC-1 complex by DCs to CD8+ T-cells33, 34. This is thought to be very important to activate CD8+ T cell response against cancer, because DCs usually lack an internal source of TAAs.

Figure 2 DC maturation and their activation of T-cells: Immature DCs mature upon stimulation by pathogens or inflammation. This process is accompanied by changes in morphology, upregulation of co-stimulatory markers migration patterns and secretion of cytokines that help T-cell activation.

T helper cell polarization (Th1 vs Th2) Apart from activation of T-cells by stimulation of the TCR by MHC-antigen complex recognition on DCs (signal 1), and co-stimulation by CD28 binding to CD80, CD86 on DCs (signal 2), DCs can modulate the type of T cell re-sponses by a third signal (Figure 2) mediated by the type of cytokines se-creted by DCs. The signals 2 and 3 can differ depending on the type of stim-uli the DCs come across35. Depending on the DC cytokine profile and mi-croenvironment, T cell responses can be polarized towards T helper cell (Th)-1, Th2, Th9, Th17 or regulatory T cell (Treg) responses. Th1 polarizing cytokines like IL-12, IL-2 and Tumor necrosis factor (TNF)-α help in initiat-ing a pro-inflammatory response, which is important for activating cytotoxic T-lymphocytes (CTLs) and clearance of infected cells and tumor cells36. Whereas, PGE2 and nitric oxide secretion, surface expression of OX40L by DCs are Th2 polarizing and produce an anti-inflammatory response. This usually occurs to promote humoral responses (B cell response) and prevent

22

extensive tissue injury due to inflammation36, 37. So, Th1 polarized response is usually beneficial in the context of cancer therapy.

Dendritic cells and tumor microenvironment Most human and mice tumors have DC infiltration, but the activation status of DCs depends on the tumor microenvironment (TME). The microenviron-ment is very complex and a few important findings related to DC are dis-cussed here. Tumors talk with resident phagocytes and DCs through differ-ent signals which include “find me”, “eat me” and “do not eat me”38. The “do not eat me” is the most common signal in tumors and it negatively regu-lates DC function. Through this signal cancer cells can disguise the immune system to promote immune escape. Interaction of the “do not eat me” signal-ing molecules CD47 or lactoferrin with signal-regulatory protein-α (SIRPα) on phagocytes could prevent phagocytosis and antigen uptake. A blocking antibody against CD47 in combination with rituximab enhances phagocyto-sis of dead lymphoma cells and improved therapeutic effect in mice39.

Tumors can alter DC function by inhibiting or altering their antigen presen-tation capacity. For example, tumor-associated proteins like mucin 1 are endocytosed by DCs and end up in the endosome and inhibit antigen pro-cessing and presentation by DCs to T-cells40. Several tumor-derived factors like cytokines and membrane-bound ligands can also alter the DC matura-tion process. IL-10 and TGFβ are inhibitory cytokines secreted by tumor, which can lead to antigen specific anergy in the TME41. DCs in the TME also express OX40L, which polarizes a Th2 type immune response. As dis-cussed above, a Th2 type-skewed response is not favorable for tumor im-mune therapy. IL-4 and IL-13 are characteristic Th2 cytokines and they promote tumor growth by inhibiting apoptosis, and stimulating tumor-associated macrophages to secret epidermal growth factor (EGF)42. Addi-tionally, DCs can also directly promote tumor growth, for example, by pro-moting angiogenesis in ovarian cancer43.

DC in vivo targeting & reprogramming inflammation for cancer therapy Ex vivo differentiated, TAA-loaded and matured DCs have been used for cancer therapy since the 1990s for the treatment of melanoma, renal cell carcinoma lymphoma etc. It has been proven to be safe to use, however the clinical benefit has been a mere 10-15% 44. One of the reasons for this mini-mal response rate could be attributed to poor migratory capability of the ex vivo generated DC to migrate to the lymph nodes to prime an effective adap-tive immune response45. Alternatively, DCs can be reprogrammed and load-ed with antigen in vivo to improve their functionality. DCs express several surface molecules that can be targeted for reprograming and improving their function in the TME. Antibodies are used to stimulate surface molecules of

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DCs. For example, using an agonistic CD40 antibody to activate DC mimics the CD40-CD40L interaction46. Another common target for immune therapy is to stimulate receptors on DCs that recognize pathogens like TLRs, C-type lectins and intracellular helicases. This strategy depends on non-specific activation of anti-tumor immunity invoked by pathogen associated molecular patterns (PAMPs)47, 48. The use of Bacillus Calmette Guerin (BCG) vaccine for local therapy of bladder cancer is a classic example of this strategy48. Also, several TLR ligands, CpG oligodeoxynucleotides (TLR9)49, Imiquimod (TLR7 and TLR8)48 and polyinosinic-polycytidylic acid (pol-yI:C) (TLR3 or helicases)50 have been used for therapy in different settings.

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ONCOLYTIC VIRUS THERAPY FOR CANCER

Oncolytic viruses (OVs) exhibit anticancer properties by selectively replicat-ing in, and killing cancer tissues. Spontaneous cancer regression in patients after certain viral infections was observed almost a century ago51, which marked the onset of oncolytic virotherapy. The first clinical study using on-colytic virus for therapy was in 1949, when hepatitis virus was used for treatment of Hodgkin’s disease52. Since then many unmodified viruses (Egypt 101 virus53, Adenovirus54, Mumps virus55) were used for cancer treatment. The advancement of genetic engineering and recombinant DNA technologies in the past few decades has provided great platforms for under-standing of virus biology. A number of viruses have been genetically modi-fied for the purpose of cancer therapy, with focus on increasing potency, selectivity and safety. Oncolytic viruses belonging to different families (Ad-enovirus, Herpes simplex virus, Measles Virus, Vaccinia, and Reovirus etc.) have been engineered and successfully tested in clinical trials56-58.

Viruses have a natural preference for replication in cancer cells. This can be attributed to the typical hallmarks of cancer cells described by Weinberg and Hanahan like resistance to apoptosis, uncontrolled growth, evading growth suppressors and immune suppression, which favor virus replication4, 56. In addition, certain features of OVs give them an edge over conventional can-cer therapies (chemo and radiotherapy): (a) as OVs use multiple mechanisms for tumor cell killing (discussed below), there is less probability to genera-tion of resistance, (b) they can be engineered to replicate in a tumor selective fashion, thus reducing systemic toxicity and pathogenicity of the virus, (c) they are self-amplifying drugs, meaning virus dose in the local tumor micro-environment increases due to in situ virus replication, and (d) they can be genetically engineered to carry therapeutic transgenes and safety features can be built in to reduce toxicity59. As mentioned above, there are several mech-anisms by which OVs can mediate tumor destruction, briefly (a) direct lysis of tumor cells, (b) cytotoxicity of viral proteins, (c) induce anti-tumor im-mune responses and (d) induce sensitization to chemo and radiation therapy60. These multiple of facets make oncolytic viruses potent agents for cancer therapy. However, there are some notable drawbacks of OV therapy that need be carefully assessed and addressed. The major drawback is the

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immune response against the virus itself, which can be a double edged-sword (both promoting anti-virus immune response and anti-tumor immune response). There can be preexisting antibodies in case of human viruses, making it virtually impossible to use systemic virus administration. Also, anti-viral immune response mounted at the first round of treatment can lower the effect for multiple dose treatments59. However, it is now believed that the direct oncolysis of tumors by viruses may not be the only factor determining the therapeutic efficacy, rather the synergistic anti-tumor immune response that is generated upon viral oncolysis is more important61. With this note, OV are now thought to be a new class of immunotherapeutic cancer drugs.

Oncolytic Virotherapy to Viro-Immunotherapy: the new perspective. The primary objective of OV therapy is to specifically replicate in and lyse cancer cells as mentioned above. However, with more preclinical and clini-cal investigations it is becoming evident that infection of tumors by OV cre-ates an inflammatory storm capable of activating both adaptive and innate immune responses against the tumor62, 63. Viral oncolysis is immunogenic, capable of altering the cytokine milieu in the TME and directly influencing the anti-tumor immune response. One of the first observations of this phe-nomenon was when advance melanoma patients were vaccinated with an oncolytic vaccinia virus encoding GM-CSF. The clinical responses correlat-ed with anti-tumor immune response with enormous amounts of immune cell infiltration to injected tumors64.

Following infection and replication of an OV in tumor cells, they can direct-ly kill and eliminate viable tumor cells. During this process they induce sys-temic immune response by release of danger signals, TAA and the efficacy of immune response elicited also depends on the type of cell death induced by the OV (pyroptosis or necrosis are more immunogenic forms of cell death than autophagy or apoptosis). The cell death and types of molecules released is dependent on the OV used and the nature of the tumor cells. However to summarize, the immune activation signals released during OV cell lysis may include virus associated PAMPs, host cell associated danger associated mo-lecular pattern signals (DAMPs) (e.g. HSPs, CRT, HMGB, and ATP), all of which can prime and activate locally residing APCs. Activated APCs capture TAA released upon viral oncolysis, migrate and activate CD4+ and CD8+ T-cell responses establishing an anti-tumor immunity65, 66. This is a key process in effective tumor eradication because viral oncolysis can release tumor as-sociated neo–antigens (antigens previously hidden to the immune system due to lack of presentation or a newly developed antigen due to mutation in the

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tumor cell). T-cells directed against these antigens are in particular very effi-cient in tumor eradication67 (Figure 3). However, the T-cell response is also directed against viral antigens that lead to clearance of the virus68, 69. The kinetics of anti-viral immune response and its influence in mounting an anti-tumor immune response is still unclear and influenced by many factors in the TME.

Figure 3 Schematic representation of anti-tumor immunity induced by OVs: Tu-mor cells initiate an antiviral response upon OV infection. This consists of release of reactive oxygen species (ROS) and pro-inflammatory cytokines from the infected tumor cells, which subsequently stimulates immune cells (antigen presenting cells, CD8+ T-cells, and natural killer (NK) cells). The OV replication in tumor cells leads to oncolysis, release of viral progeny, DAMPs, PAMPs, transgenes (payloads engineered to be expressed by OVs) and TAAs (which includes neo-antigens). The released progeny virus infects neighboring tumor cells and initiates a cascade of the above mentioned processes. The PAMPs and DAMPs trigger immune cell activation (DCs, NK cells etc.) via Toll-like receptors (TLRs), cytokine and chemokine recep-tors during which antigen presenting cells (APCs) also uptake the released TAAs (including neo-antigens). Antigen carrying and activated APCs prime immune re-sponses against the initially virus infected tumor cells and as well as de novo im-mune responses against TAAs/neo-antigens displayed by uninfected tumor cells.

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OV infection induces a strong proinflammatory cytokine milieu that influ-ences the type of immune cell infiltrate into the tumor and is capable of re-versing the rather immune suppressive TME. Virus infection in human can-cer induces secretion of proinflammatory cytokines IL-6, IL-8, type-I IFNs, TNF-α, RANTES, IL-12 and macrophage inflammatory protein (MIP)-1α/β and reduces levels of the immunosuppressive cytokine IL-1065, 70. Most of the cytokines mentioned above are known to have potent anti-tumor activity by themselves or through recruitment of other immune mediators. Apart from being able to induce a proinflammatory milieu themselves, OVs are also engineered to express immune stimulating genes locally in the TME. These immune modulating proteins help in altering the immune suppressive microenvironment and boost an anti-tumor immune response63. More about this is discussed in the section arming OVs with therapeutic transgene.

Adenovirus Human adenoviruses (Ads) are icosahedral shaped, non-enveloped viruses that belong to the Adenoviridae family. They have a linear, double-stranded DNA genome of about 26-45 kilo base pairs (kbp) in size, which codes for about 30-40 genes (Figure 4)71. Ads were first isolated from human adenoid tissue cultures in 195372, and since then about 57 serotypes of Ads have been identified and characterized73. Ads are further classified based on their ge-nome sequence and immunological distinctiveness into 7 species namely A-G73. Adenoviruses infect a wide range of vertebrate hosts; however their replication in the host is species specific. Human Ads do not replicate effi-ciently in other hosts, with a few exceptions and in replication permissive hosts they have a high lytic activity and can be used as an anti-tumor agent74. Ads primarily infect epithelial cells of the respiratory tract causing mild symptoms like nasal congestion, cough, runny nose, and conjunctivitis. Hex-on, penton base and fiber proteins form the capsid of an Ad. The icosahedron of Ads is formed by 240 homotrimeric hexon proteins and 12 penton bases with extending trimerized fibers occupy their vertices72, 75. Depending on the surface location of the hexon proteins, they are further divided into four groups (H1-H4)76. Up to 9 flexible hyper variable regions (HVR) have been identified based on amino acid diversity on the surface of hexon. This diver-sity contributes to the difference in serotypes of Ads77, and are good targets for introducing genetic modifications.

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Adenovirus life cycle Ads infect a wide variety of dividing and non-dividing cells and tissues. Many Ad serotypes bind to the coxsackie-adenovirus receptor using their high affinity fiber knob to initiate binding to host cells78. Then, the arginine-glycine-aspartic acid (RGD) motif on the penton base of the virus interacts with αvβ integrins on the cell surface, which leads to endocytosis of the virus via clathrin-coated pits79, 80. After successful entry into the host cell, Ads reside inside endosomes. As early endosomes mature to late endosomes the pH is lowered, which promotes endosome escape for the Ads. During this process the fiber knobs and the penton base dissociate from the capsid lead-ing to endosome disruption and virus escape81. Cellular microtubules and dynein assist translocation of partially dissociated virus particles to the nu-clear pore, after which the viral DNA is deposited in the nucleus80. Viral DNA binds cellular histones and subsequently viral DNA replication is initi-ated, which depends on the host cell replication machinery (Figure 5).

The Ad life cycle can be divided into two phases, depending on its gene expression pattern (a) early phase gene expression (E1-E4, non-structural proteins), occurring before virus DNA replication and (b) late phase gene expression (L1-L5, structural proteins), which occurs after DNA replication. The transcription takes place from both DNA strands and have scattered open reading frames that are under the control of multiple promoters (Figure 4)72. Detailed description of the adenoviral genes is summarized in Table 2.

Figure 4 Transcriptome of the adenovirus genome: Early (E) phase genes (in red), late (L) phase genes (in green). The promoter for each gene is indicated by brackets (pink for early genes, light green for late genes). LITR is the left inverted terminal repeat while RITR is the right inverted terminal repeat. Ψ is the packaging se-quence. VA I/II are viral-associated non-coding RNA transcribed intermediate early. Picture credits: Dr. Di Yu, Department of Immunology, Genetics and Pathology.

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Figure 5 Life cycle of an adenovirus armed with secreted HP-NAP protein: Host cell entry in initiated by binding of fiber to cell surface receptors (e.g. coxsakie adenovirus receptor (CAR) for Ad5.). Virus enters the host by endocytosis, partially disassembles and is translocated to the nucleus where the viral DNA is released. Early gene expression (E1A and transgene, HP-NAP) is initiated, after which viral DNA replication is initiated by early proteins. In the later stages structural proteins are synthesized, in to which the viral DNA is packaged and progeny virus particle is assembled. Finally the host cell is lysed and progeny virus particles are released.

The E1A gene (also referred to as “immediate early” gene) is the first to be expressed and plays a vital role in initiating and creating a favorable envi-ronment for Ad replication. E1A proteins bind to retinoblastoma protein (pRb) and subsequently releases the transcription factor E2F and thus forc-ing the host cell cycle into S-phase. E1A protein also trans-activates the ex-pression of other Ad genes82. In the intermediate phase proteins IVa2 and IX regulate the expression of late genes by controlling transcription through the major late promoter (MLP). Two viral associated non-coding RNAs, VA I and VA II are also encoded by the virus genome in the intermediate phase. They regulate translation of both early and late genes and act as RNA inter-ference (siRNA and miRNA)83. After translation of the structural proteins they translocate to the nucleus, where packaging for viral DNA and assem-bly of progeny virus occurs (Figure 5, Figure 6). Some groups of Ads (group C) accumulate adenovirus death protein (ADP) at the later stages, and this protein is thought to play an important role in cell lysis84. Other groups of Ads (group B) lack expression of ADP, and in this instance the mecha-nism of cell lysis is not clearly elucidated. The progeny virus particles lyse the host cells and are ready for another round of infection.

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Table 2 Proteins encoded by the adenovirus genome and their functions

Gene Proteins and their function

Early Phase

E1A a. Binds pRb to release transcription factor E2F to push cell cycle to S-phase

b. Trans-activates other viral promoters E1B a. 55kD subunit binds p53, and transport of late viral mRNA

b. 19kD subunit is Bcl-2 homologue, anti-apoptotic

E2A a. Preterminal protein (pTP) and DNA polymerase, DNA replication

E2B a. Single-strand DNA binding protein, DNA replication

E3 a. gp19kD, inhibit surface expression of MHC I b. 10.4kD/14.5kD RID complex, internalize TNF receptor and de-

grade Fas ligand, inhibit TNF mediated apoptosis c. 14.7kD, inhibit TNF mediated apoptosis, stabilized NFκB d. 11.6kD ADP, induce cell lysis

E4 a. orf 1-6/7 modulate viral mRNA metabolism, block host protein synthesis, promote virus DNA replication

b. orf 3 forms network in infected cell and sequestrate tumor sup-pressor

Intermediate Phase

IVa2, IX Initiate major late promoter which regulate late gene expression

VA I, VA II Non-coding RNA, stimulates translation of viral proteins, blocks double stranded RNA protein kinase R during IFN response

Late Phase

L1-L5 Structural proteins

a. L1 - IIIa b. L2 - penton base, V, VII c. L3 - hexon, VI, virus protease d. L4 - VIII e. L5 – fiber

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Figure 6 Replicating adenovirus particles: Transmission electron micrograph of ade-novirus particles in the nucleus of Human bone osteosarcoma cells (HOS), 48 hours post virus infection. Adenoviral particles can be identified by the icosahedral shape. Magnifications 9300X, Inset image 68000X. Picture credits: Dr. Di Yu.

Adenovirus as oncolytic agents The human Ad serotype 5 (Ad5) is most commonly used in the field of on-colytic viro-immunotherapy because of several advantages. First, it is capa-ble of infecting a wide range of dividing and non-dividing cell types. This is important for treatment of cancer stem cells, which usually maintain a low proliferation rate57, 85. Ads can be selectively engineered to kill cancer cells and are self-amplifying drugs i.e. a single round of replication in a cancer cell would lead to release of many viral progeny particles. At least in theory Ad infection can persist until the tumor is completely eradicated. Ads can be advantageous for treatment of therapy-resistant tumors because they have complex killing mechanisms and do not solely rely on apoptotic and necrotic cell death (Table 3)56, 60, 86. A second major advantage of Ads is that they remain and replicate in the host cells in an unintegrated episomal state, which reduces the risk if oncogenesis by insertional mutagenesis87. The ade-novirus genome is well studied, and it can be easily manipulated to insert and code for therapeutic genes.

However, wild-type Ads have several downsides as oncolytic agents. They infect all tissues including normal healthy tissues, lacking tumor specificity at the infection stage. Ads are highly immunogenic, which can be a double-edged sword, aiding in inducing anti-tumor immunity, but also inducing anti-viral immunity, which can lead to rapid elimination of the oncolytic virus. Lack of option to administer Ads systemically, especially to pre-exposed recipients’ calls for new approaches to improve virus delivery to tumor cells.

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Conditionally replicating adenoviruses (CRAds) Targeting Ad replication to tumor cells can be achieved at different levels

I. One of the first strategies devised to restrict Ad replication to tumor cells was achieved by mutating or deleting essential viral genes. The loss of these genes is usually complemented by mutations in tumor cells that indi-rectly favor Ad replication. One of the first examples is the E1B-55kD delet-ed ONYX-015 virus88, which was also the first virus to enter clinical trials. The Δ24 Ad is another example where the E1A gene bears a 24bp deletion (dl922-947). E1AΔ24 lacks the ability to bind to the pRb tumor suppressor, which regulates the G1-S-phase check-point in the cell cycle. Thus this virus replicates only in pRb mutant cells and in cells with deregulated pRb path-ways89 (Figure 7). Also to note, pRb mutation or dysfunctional pRb path-ways is one of the most commonly found mutations in cancer.

Table 3 Mechanisms by which adenovirus proteins can influence tumor killing

Mechanism Examples of adenoviral

genes involved 

Direct cytotoxicity E3 11.6kD90

E4ORF491

Induction of anti-tumoral immunity

a. CTL infiltration

b. tumor cell death, antigen release

c. immunostimulatory cytokine induc-

tion

d. enhanced sensitivity to cytokines

E3 gp19kD*

E3 11.6kD

E3 10.4/14.5, 14.7kD*

E1A92

Sensitization to chemo and radiation therapy E1A93

Expression of therapeutic genes GM-CSF, CD40L, HP-NAP

(exogenous proteins)

*Viral protein inhibits anti-tumoral mechanism (Table adopted from86).

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II. Transcriptional and post-transcriptional targeting is achieved by con-trolling expression of E1A gene with a tissue or tumor-specific promoter (transcriptional) or tagging the E1A gene with a microRNA (miRNA) target sequence (post-transcriptional). E1A is the first gene to be expressed and it is the most important gene for initiation of the Ad life cycle. So, controlling E1A expression with a promoter active only in tumor cells or certain tissues restricts Ad replication to such tumor cells or tissues. Many promoters have been reported, a few examples, a recombinant prostate-specific promoter (PPT) restricts Ad replication to normal and neoplastic prostate cells94, and the chromogranin A (CgA) promoter restricts Ad replication to normal and neoplastic neuroendocrine and endocrine cells95. miRNA targeting is a rela-tively new strategy, where the E1A 3’UTR is tagged with a sequence com-plementary to a tissue-specific miRNA. This makes the E1A transcript a target for miRNA recognition in target tissues and hence degraded. This strategy has been used to prevent off target toxicity. For example in the liver, modification of the E1A gene with liver-specific miR122 target sequences suppresses Ad replication in normal hepatocytes, where miR122 molecules are highly expressed96 (Figure 12). Detailed mechanism is explained in the methods section.

Figure 7 Tumor-selectivity of Ad5(E1AΔ24): Illustration of replication of an ade-novirus with 24bp (8 amino acids) deletion in the E1A region (E1AΔ24) in tumor cells with mutant pRb protein or deregulated pRb pathways while replication is blocked in normal cells with functioning pRb

III. Transductional targeting can be achieved by modifying the surface of the virus, either to unspecifically enhance virus transduction or to selectively enhance transduction of tumor cells alone. To unspecifically enhance trans-duction researchers have switched fibers or fiber knobs from different ade-novirus serotypes (e.g. Ad5 fiber replaced with Ad35 fiber97). Also cell-penetrating peptides have been introduced to the fiber or hexon of Ad5 to enhance transduction of primary cells (e.g. Tat from HIV-198). For restricting virus entry to tumor cells, the cyclic FWKT motif from somatostatin was

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inserted in the HI-loop of the fiber to facilitate binding to somatostatin re-ceptor99. In another instance, the RGD motif has been inserted into HI-loop of the fiber to selectively infecting tumor cells expressing high levels of in-tegrin100.

Semliki Forest virus Semliki Forest virus (SFV) is an alphavirus and belongs to the Togoviridae family. It was first isolated from the Semliki Forest in Uganda from mosqui-toes in 1942 and that is where it got its name from101. Their vertebrate host is still not known but viral infections have been found in monkeys, horses and humans. SFV infection is spread primarily by mosquito bites. It is not trans-mitted though aerosols or gastrointestinal exposure in mammals expect in rodents where they can be transmitted through intranasal inhalation102. It causes severe encephalitis in rodents, but only a few outbreaks in humans have been reported with mild symptoms like headaches, fever and fatigue103. SFV is considered non-pathogenic in humans hence it is classified as a bi-osafety level-2 virus for research purposes. However, to note there has been one reported death of an immune-compromised laboratory personnel who was exposed to larger amounts of the virulent Osterrieth strain of SFV104.

SFV genome and life cycle SFVs are enveloped viruses with a positive (+) single-stranded RNA genome of approximately 11-12 kilo bases in length. The RNA genome is both capped at the 5’ end and polyadenylated at the 3’ end. The SFV genome is very simple and encode for nine virus proteins that includes four non-structural proteins (nsP1-4), four structural proteins (capsid protein C, and envelope proteins E1-3) and a small 6 kDa protein that is not incorporated into the virions. Protein C forms the icosahedral capsid of the virus, which is also enveloped by a lipid bilayer (Figure 8). E1 and E2 heterodimers form the spike proteins which cover the entire outer surface of the virion. Alpha-viruses use their spike proteins to bind the target receptor on the host cells and are taken up by the cells by receptor-mediated endocytosis. However, the route of entry of SFV is much debated because the primary receptor for SFV binding is unknown and in conditions inhibiting endocytosis, alpha-virus RNA can enter the cells via pores generated by virus and host proteins105. Low pH in the endosome initiates the dissociation of E1-E2 het-erodimer and trimerization of the E1 fusion peptide with the endosomal membrane. This creates a pore in the endosome facilitating release of the viral capsid and eventually the virus genome into the cytoplasm106 (Figure 9). The non-structural proteins are translated from the 42S RNA viral ge-nome and the structural proteins are encoded from the subgenomic 26S

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RNA107 (Figure 8). The initial transcription product from the 42S RNA is nsP1234 fusion protein, which is further processed by nsP2 protease activity to individual nsPs. Very early after infection, nsP4 is cleaved and forms a replicase complex along with nsP123.The replicase complex duplicates the 42S RNA to its negative strand in cytopathic vacuoles (CPVs), which are smooth bag-like structures on the plasma membrane derived from lysosomal and endosomal membranes108 (Figure 9, Figure 10). Later in the infection cycle, the nsP123 polyprotein is cleaved in trans to individual nsPs that form the late replicase complex that synthesize the plus strand RNA genome.

The plus strand can be either synthesized from the subgenomic or the ge-nomic promoter and transcription from the former usually leads to more than threefold excess subgenomic 26S RNA. The detailed roles of the individual nsPs are listed in Table 4106, 107, 109. The subgenomic 26S RNA codes for the viral structural proteins in the form of a single polyprotein (C-pE2-6K-E1). The capsid protein is first cleaved from this polyprotein by auto-proteolysis, leaving the N-terminus of pE2 containing signal sequence for ER transloca-tion exposed. The capsid protein bind the newly synthesized positive strand viral RNA and from the nucleocapsid. The E1 and pE2 are processed and post-translationally modified in the ER, form heterodimers and are trans-ported to the cell surface via Golgi complex. Before reaching the cell mem-brane the pE2 is cleaved to E3 and E2 proteins by furin in the Golgi. Un-cleaved pE2 also can be packed into a new viral particle, but this processing of pE2 is necessary to form an infectious viral particle. The cytoplasmic domain of E2 binds the nucleocapsid at the plasma membrane. The budding progeny viral particle is enveloped by E1-E2 heterodimer containing the host plasma membrane106, 107, 109. The schematic diagram of SFV life cycle is de-picted in Figure 9.

SFV neuro-pathogenesis Different strains of SFV have been isolated and exhibit varying neuro-pathogenicity in adult mice. The first SFV isolate was the L10 (as known as SFV4) from Aedes abnormalis mosquitoes in Uganda in 1942101. SFV4 pre-dominantly infects and replicates in neurons, is lethal to adult mice causing severe encephalitis. Later a second strain, A7 was isolated from mosquitoes in Mozambique110. This strain is avirulent in adult mice and is not lethal. These is also another strain derived from the A7, called the A7(74) which is also avirulent in adult mice but lethal to newborn mice111.

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Figure 8 Genome of Semliki Forest virus: The SFV genome is 11-12kb (+) ssRNA which is capped and polyadenylated. The genome has two open reading frames coding for the non-structural (nsP1-4) and structural (C-E3-E2-6K-E1) polypro-teins. The subgenomic mRNA contains downstream hairpin loop (DLP) that pre-vents translation shutoff mediated by protein kinase R activation by the host. Picture modified and adapted from ViralZone (http://viralzone.expasy.org).

Table 4 Nonstructural proteins encoded by the SFV genome and their functions

Gene Proteins and their function

nsP1 Methyl and guanylyl transferase activities, involved in capping the viral RNA

Initiation of negative-strand RNA synthesis and mobilization of early replicase complex to plasma membrane

nsP2 Helicase activity, RNA duplex unwinding

Regulates 26S subgenomic RNA synthesis

Cessation of negative-strand synthesis

Contains a papain-like proteinase domain, processing the non-structural polyprotein

nsP3 It is a phosphoprotein, regulates of RNA synthesis

Exact function unknown, major determinant of neurovirulence

nsP4 RNA-dependent RNA polymerase

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Figure 9 Semliki Forest virus replication cycle: (1) SFV E glycoprotein binds to host cells receptors and enters via clathrin-mediated endocytosis. (2) Viral capsid dissociates in the endosome and viral RNA is released. (3) + ssRNA are translated in to non-structural polyprotein (nsp1-4). (4) nSP polyprotein forms the replication complex spherules in the plasma membrane (5) and are subsequently internalized to form cytoplasmic viral factories called cytopathic vacuoles (CPV-I) where dsRNA is synthesized from the ssRNA viral genome. (6) The subgenomic RNA is translated into structural proteins, (7) the enveloped proteins are processed in the ER and Golgi, (8) and eventually transported to the plasma membrane. (9) dsRNA is tran-scribed to provide new + ssRNA genomes which is packed in to the viral proteins. (10) At the plasma membrane the newly formed viral capsids associate with enve-lope proteins and bud out new virions.

Figure 10 Cytopathic vacuole formation dur-ing SFV replication: Transmission electron micrograph of SFV replicase complexes in cytopathic vacuoles (CPV) in HOS cells 48 hours post infection. CPVs are formed by fu-sion of several small spherules containing replicase complexes with endosomes. Magnifi-cations 9300X, Inset image 68000X. Picture credits: Dr. Di Yu.

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Upon intraperitoneal (i.p) administration in adult mice, high levels of plasma viremia is observed 24 hours post infection that usually drops after 48 hours and after 4 days to undetectable levels for both strains. During this period SFV replication is observed in muscles cells, mainly the smooth, skeletal and cardiac muscles112, 113. However, central nervous system (CNS) infection and ability to cause encephalitis is strain specific. Both the SFV4 and A7 are able to enter the brain and a perivascular foci of infection are observed at the initial stages112. When administered intranasally, CNS entry occurs through infection of the olfactory nerves114. After entry in to the CNS both strains predominantly infect neurons and oligodendrocytes, but they are rarely ob-served to infect astrocytes, meningeal, ependymal or choroid plexus cells115,

116. The infection pattern and tropism are identical for both strains, but the neurovirulent SFV4 spreads rapidly in the brain from the original perivascu-lar foci. In neonatal mice this infection destroys the neurons and leads to mice death, however in adult mice at the time of death the neurons are in-fected and have a normal morphology112. Spread of SFV4 is associated with increase in pro-inflammatory cytokine profile in the brain117. In a study on Sindbis virus, a close relative to SFV, CD8 T-cell and Th1/Th17 helper cell infiltration was observed. So it is thought that this inflammation is cause of Sindbis virus related encephalitis rather than virus mediated cell death118.

The avirulent A7 strain infection in brain and pathogenicity varies with mice age. In neonatal and young mice less than 12 days old, A7 spread in the brain is rapid and similar to SFV4. However, in mice older than 14 days there is a sharp decrease in A7 spread in the brain restricted to the perivascu-lar foci and mice survive virus infection112, 119. The reason for age depend neurovirulence of the A7 strain is not well known but may dependent on the maturity of the CNS cells. The A7 strain is able to infect and replicate effi-ciently in maturing neurons and as neurons mature replication of A7 redu-ces114, 119. Another possible mechanism for age related neurovirulence in-creased antiviral immune response in mature neurons. Also, it is believed that neurovirulence of alphaviruses is associated with their ability to coun-teract type-I IFN-mediated antiviral responses120. A chimeric virus created by substituting nsP3 from A7 with that from SFV4 was completely neuro-virulent117, 121. nsP3 is the major determinant of SFV4 neurovirulence and is also thought to be involved in counteracting type-I IFN responses122.

SFV as oncolytic agents The studies using SFV as oncolytic agents have been restricted to preclinical settings. The A7/74 strain was traditionally preferred as an oncolytic agent due to its reduced neurovirulence in adult mice. A7/74 strain was successful-ly used in several xenograft models as oncolytic agent successfully. To name a few: human melanoma123, osteosarcoma124, lung cancer125 and human glio-

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blastoma126. However, due to the natural neuro-tropism of SFV its use as an oncolytic agent has largely focused on CNS-related tumors and in particular glioblastoma. Early results with the A7/74 strain in U87 glioma xenograft mice model and BT4C syngeneic rat model were very promising126, 127. But, the attenuated A7/74 strain failed to cure mice bearing gliomas in syngeneic immune competent mouse models and surprisingly even mouse glioma cells pre-infected with virus in culture were able to develop tumors when implant-ed i.c. in mice. This is thought to be because of IFN-β sensitivity of the A7/74 strain and inability to overcome type-I antiviral defense128, 129.

One of the main determinants of SFV neurovirulence is the ability to coun-teract type-I IFN response. Hence, the neurovirulent SFV4 strain is a natural choice, since it is partially able to resist type-I IFN responses. However, SFV4 is lethal to mice and studies involving SFV4 as an oncolytic agent has been limited. As mentioned earlier, SFV is considered nonpathogenic to humans; it is still worth developing strategies to attenuate its neurovirulence to avoid potential toxicities. SFV4 has been tested as oncolytic agent at least in a couple of instances. One study was with mice bearing colon adenocarci-noma and murine sarcoma virus-transformed fibroblasts that were treated with SFV4. In this study the mice were first immunized with SFV4 virus like particles (viral particles capable of only one round of replication, lack struc-tural protein coding genes in their genome) prior to administration of onco-lytic SFV4 to prevent virus related toxicities. SFV4 replicated in the tumors however no long-term response was obtained130. In the next study miR124-tagged SFV4 showed improved safety in adult mice and had improved ther-apeutic efficiency in the syngeneic CT-2A glioma mouse model122. In this thesis, more miRNA targets were inserted in the SFV4 genome to improve the safety in mice for the purpose of oncolytic virus therapy. miRNA target engineering and principle is discussed in detail in the methods section.

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ADOPTIVE CELL IMMUNOTHERAPY

Adoptive cell therapy (ACT) involves isolation and reinfusion of immune cells (in particular T-cells) to patients to induce antitumor effect. T-cells have the natural ability to specifically and effectively eliminate diseased cells that are widespread in the body. T-cells also have the ability to infiltrate inflamed tissue and studies show a favorable prognosis in cancer patients who have high CD8+ T-cell infiltration131. A fraction of patients with ad-vanced metastatic melanoma and renal cell carcinoma have long-term com-plete tumor regression when T-cell responses were manipulated by IL-2 administration132. This has provided much encouragement in developing ACT therapies for cancer. However, tumors evolve and develop ways to shield themselves from T-cell attack and current research aims for ACT therefore focus on developing ways for the T-cells to overcome this barrier. The objective of ACT is similar to that of a vaccination, i.e., priming and expansion of antigen-specific T-cells that are able to attack and kill diseased cells, but it is carried out ex vivo.

The first hint of successful ACT was observed in the allogeneic hematopoi-etic stem cell transplant (HSCT) setting when a subset of donor T-cells rec-ognized and killed tumor cells (graft-versus-tumor response)133. Following this early observation different forms of allogeneic T-cell transfers were developed like donor leukocyte infusion134, and virus specific T-cell infus-ion135. Since HSCT infusion includes the risk of graft-versus-host (GVH) disease, autologous T-cell transfer strategies have been developed. Admin-istration of T-cells isolated and activated from resected metastatic tumors, so called tumor-infiltrating lymphocytes (TILs), has resulted in very good, du-rable and reproducible response rates in melanoma patients (20% CR and 50% OR)136. Optimization of ex vivo culture conditions and lymphoid deplet-ing pre-conditioning of patients with chemotherapy and total body irradia-tion before TIL administration extended the objective tumor regression to 50-70% in melanoma patients (with durable CR beyond 10 years in some patients)136. Besides melanoma, TILs have been isolated from ovarian can-cer, breast cancer, and renal cell carcinoma. However, inaccessible tumor sites, poor TIL recovery and poor antigenicity of some tumors are some of the roadblocks in expanding TIL therapy as a global cancer therapy opt-ion131. Also, transfer of ex vivo expanded bulk T-cells has been tried in pa-tients. This approach relies on activated cells with lowered triggering thresh-

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olds for clinical benefit, but does not enrich for TAA specific T-cells137. A promising strategy for developing TAA specific T-cells is to engineer them with artificial TAA-specific receptors e.g. a new TCR or a chimeric antigen receptor (CAR).

Chimeric Antigen Receptor engineered T-cells As the name suggests, a CAR is a chimera of domains from different pro-teins assembled together to create a functional receptor. These novel recep-tors initiate a functional downstream effector T-cell signaling pathway when they encounter target antigen, usually the TAA on a cancer cell. This gives the opportunity to engineer a large variety of TAA-specific receptors target-ing a broad range of cancer types. CARs typically contain four domains (a) extracellular antigen binding domain: It confers the antigen-specificity to the engineered T-cell. A majority of the engineered CARs for cancer therapy have antibody-derived antigen binding domains called single-chain variable fragment (scFv). CARs containing a scFv extracellular domain retain the specificity of an antibody. A major advantage of having scFv extracellular domain is that it bypasses the need for antigen presentation by MHC-I on tumor cells, as antibodies directly bind to cell surface antigens. (b) Spacer or hinge region: It gives flexibility and length to allow proper dimerization of scFv, thus improving its stability. The most commonly used spacer re-gions are derived from IgG Fc CH2-CH3 domains, CD28 hinge domain and CD8α spacer domain (c) Transmembrane domain: It determines the stabil-ity of CAR expression on cell surface. The most commonly used transmem-brane regions are derived from CD3ζ CD4, CD8 and CD28 molecules138 and (d) Cytoplasmic signaling domain(s): This region has the domains that provide the necessary downstream signaling for T-cell effector functions. CARs are classified into different generations based on the number of cyto-plasmic signaling domains namely first, second and third generation CARs. First generation CARs have only one cytoplasmic domain, usually T-cell activation signaling domain (CD3ζ chain). In addition to the T-cell activa-tion domain second generation CARs have one extra co-stimulatory signal-ing domain, e.g., CD28, 4-1BB, ICOS or OX40 and third generation CARs have two extra co-stimulatory domains, e.g. CD28/OX40 or CD28/4-1BB138-

140 (Figure 11).

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Figure 11 Structure of a Chimeric Antigen Receptor (CAR): A CAR typically has an extracellular single chain variable fragment (scFv) antigen-binding domain derived from an antibody, linked to the cytoplasmic signaling region by a hinge and transmembrane regions. The cytoplasmic region includes CD3ζ signaling domain from the T-cell receptor (1st generation), or in combination with one co-stimulatory domain usually CD28 (2nd generation) or in combination with two co-stimulatory domains, CD28 and 4-1BB/OX40 (3rd generation). The newer next generation CARs is derived from 2nd generation CAR that is modified to express transgenic payloads upon T-cell activation. Picture modified from Dr. Chuan Jin, Department of Immu-nology, Genetics and Pathology.

Lessons from clinical application of CAR T-cells CAR T-cells have been very successful in treating patients with CD19+ he-matological malignancies (chronic lymphocytic leukemia, acute lympho-blastic leukemia and Non-Hodgkin’s lymphoma), with many patients having long term complete remissions23. Second generation CAR against CD19 with either CD28/CD3ζ or 41BB/CD3ζ have been mostly used in the clinic. Tu-mor regression correlates well with CAR T-cell proliferation in vivo and release of cytokines. Lympho-depleting preconditioning helps proliferation and persistence of CAR T-cells in some patients, which may be associated with elimination of immune suppressive cells like Tregs

141 and increase in levels of cytokines IL-15 and IL-7 that enhanced expansion of infused T-cells and persistence of T-cells with a central memory phenotype142, 143. The main adverse effects of CAR T-cell therapy is Cytokine Release Syndrome (CRS), associated with excessive release of pro-inflammatory cytokines like IL-6, IFN-γ and TNF-α by activated CAR T-cells. CRS is reversible by monotherapy with anti-IL-6 receptor antibody (tocilizumab) or TNF-α anti-body (infliximab), sometimes in combination with steroids while milder CRS appears to be self-limiting144. However, immunosuppressive interven-tions may also affect CAR T-cell therapeutic efficacy23, 145. Tumor lysis syn-

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drome (metabolic changes due to sudden, massive tumor cell lysis) has also been observed in patients treated with CD19 CAR T-cells. These patients are at risk of potential renal injury and for this reason patients are usually under i.v. hydration144. Neurological toxicities have also been observed in patients treated with CD19 CAR T-cells with symptoms like confusion, seizures, delirium aphasia etc146. The cause of such after effects is still unknown but they were reversible.

The application of CAR T-cells to solid tumors is however limited and less successful. Some examples of CARs used for solid tumor in clinical trials are HER2 CAR T-cells for patients with osteosarcoma, and glioblastoma multiforme (GBM), GD2 CAR T-cells for treatment of neuroblastoma, and ErB2 CAR T-cells for ErB2+ glioblastoma, sarcoma, head and neck squa-mous cell cancer. The most successful ones are GD2 CAR T-cells for treat-ment of neuroblastoma (3/11 CR)147 and HER2 CAR T-cells for sarcoma (4/17 CR)148. Problems associated with developing CAR T-cell therapy for solid tumors are multifactorial. The major hindrances are choice of target antigen, T-cell trafficking, infiltration and immune suppression149, 150. Solid tumors especially have high TAA heterogeneity and promote antigen escape. Therefore it is very difficult to find an ideal target for CAR T-cell develop-ment of solid tumors. Targets like carbonic anhydrase IX, which is highly expressed in tumor cells like renal cell carcinoma but expressed in low levels on healthy cells have been evaluated, but some patients developed cholangi-tis and damage to the epithelial cells of the bile duct due to expression of carbonic anhydrase IX151, hence choice of antigen must be made carefully. To circumvent TAA heterogeneity, bispecific CARs have been used to target two antigens like HER2 and IL13Rα2 to treat experimental glioblastoma152 or target PSMA and PSCA to treat prostate cancer153. Fundamental prerequi-site for efficient CAR T-cell therapy is that they need to efficiently traffic to and infiltrate tumor mass. Tumor stroma surrounding solid tumors have a huge impact on T-cell trafficking and infiltration. Therefore strategies that aims at antigen independent destruction of stroma and immune cell infiltra-tion needs to be developed. The anti-angiogenic drug Sunitinib is known to normalize tumor vasculature and promote immune cell infiltration, in partic-ular T-cells154. Therefore, such combination with CAR T-cells is an option to promote T-cell infiltration to the tumor.

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METHODS

Detailed methods can be found in the respective articles. The two important components used to evolve viruses and CAR T-cells are described here

1. Arming Oncolytic Viruses and CAR T-cells with therapeutic transgene OVs are being engineered to express immune modulating therapeutic genes as payloads in an attempt to increase their therapeutic index. This provides an opportunity to modulate the immune suppressive TME and mount an effective anti-tumor immune response, providing an additional way for kill-ing tumor cells besides oncolysis63. There have been a number of viruses armed with different transgenes, some of the most successful and relevant transgenes are mentioned here. The most successfully used immune stimu-lating agent is GM-CSF, a protein which recruits DCs and induces anti-tumor cytolytic T-cell response155. T-Vec, the first FDA approved oncolytic virus drug is a Herpes simplex virus-1 armed with GM-CSF24. Another suc-cessfully used immune stimulating protein for arming Ads is CD40 ligand (CD40L), which binds to CD40 receptor on DCs to activate them, and sub-sequently induce anti-tumor immune response156.

As discussed above CAR T-cells have multiple roadblocks when it comes to treatment of solid cancers. Lymphodepleting conditioning is thought to en-hance efficacy of CAR T-cells through activation of innate immune cells. Innate immune cells in particular DCs are very important in providing co-stimulatory signals to aid efficient function T-cells. One of the reasons why CAR T-cells efficiently cures B-cell malignancies is likely that the B-cells are able to provide CAR T-cells co-stimulatory signals138. However, solid tumors have a very immune suppressive microenvironment that does not support efficient T-cell activity. Administration of cytokines like IL-12157, IL-15158, IL-7157 have been evaluated in preclinical setting and have shown to boost cytolytic activity of T-cells in the tumor microenvironment. CAR T-cells engineered with inducible IL-12 expression have significantly better cytolytic activity, reverse the immune suppressive TME and destroy the

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tumor stroma159, 160. Thus promoting nonspecific innate immune cell activa-tion seems to enhance CAR T-cell activity.

This thesis discusses about oncolytic adenovirus and CAR T-cells armed with Helicobacter pylori Neutrophil Activating Protein (HP-NAP) as the immune modulating transgene.

Helicobacter pylori Neutrophil Activating Protein (HP-NAP) HP-NAP is a major pathogenic protein isolated from the cell-free water ex-tracts of H. pylori161. HP-NAP got its name because it was first identified for its ability to activate neutrophils to produce reactive oxygen species (ROS)161. The gene encoding HP-NAP is called napA and it is usually found accumulating in the bacterial cytosol162. The napA gene is highly conserved within the different species of H. pylori. It is a spherical 150kDa dodecamer-ic protein made of 12 monomeric subunits161. Each monomer is about 17kDa and is made of four α-helices163. The monomers interact and bind to each other with at least 20 hydrogen bonds per subunit, making the dodecameric structure of HP-NAP very stable164. Sequence analysis reveals high similari-ties between HP-NAP and the family of DNA-protecting proteins164, with structural similarities to ferritin proteins. Therefore, it was originally thought to be an iron-binding protein, which latter evolved to a pro-inflammatory molecule165. The surface of HP-NAP is highly positively charged, which might account for its ability to interact with and activate human leukocy-tes163.

During H. pylori infection, HP-NAP is exposed to the surface of the bacte-rium and promotes adhesion of bacterium to gastric mucosa by binding to mucin166. The role of HP-NAP to facilitate sialic acid binding-mediated ad-hesion of sialylated antigen on host cell surface167 and protect bacterium from oxidative stress168 has been studied in napA knock-out experiments. HP-NAP is a DNA binding protein, and thus protect bacterial DNA from damage due to free radicals168. Another mode by which HP-NAP protects bacterial DNA damage is by its ability to sequester free iron molecules and reduce oxidative stress produced by ferrous ions169. HP-NAP is also a pro-tective antigen, aiding better survival of H. pylori during infection168.

H. pylori infection is usually associated with severe gastric conditions like peptic ulcer, gastric adenocarcinoma, MALT lymphoma170 and possibly also extra-gastroduodenal diseases including iron deficiency anemia (IDA)171. Until now there is only one reported case of H. pylori associated gastric can-cer patient with high levels of HP-NAP specific antibodies in sera172 and no reports show direct association of HP-NAP with H. pylori-induced gastric conditions. However, a positive association of HP-NAP with IDA has been

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reported which can be attributed to the iron binding property of HP-NAP173. HP-NAP is also thought to be associated with pathology of aquaporin-4 an-tibody-related neural damage in Multiple Sclerosis patients174. These patients had a positive correlation between anti-HP-NAP antibodies and anti-aquaporin-4 antibodies174. Hence, more convincing proofs are needed to associate HP-NAP with disease pathology.

Effects of HP-NAP on neutrophils

HP-NAP plays a critical role in promoting trans-endothelial migration of neutrophils to inflamed mucosal tissue during H. pylori infection161, 175, 176. HP-NAP upregulates surface expression of β2 integrins, on rolling neutro-phils and monocytes177, which promotes strong binding to endothelium and extravasation. HP-NAP induces conformational change of β2 integrins, which results in a stronger binding capacity to the endothelium176. HP-NAP also induces secretion of TNF-α by macrophages and mast cells178, 179, which in turn can activate expression of adhesion molecules like VCAM and ICAM180 on endothelial cells. TNF-α can also directly activate expression of integrins on neutrophils181. In addition, HP-NAP induces secretion of several cytokines and chemokines like IL-8 (also called CXCL8), macrophage in-flammatory protein 1 alpha (MIP-1α, also called CCL3) and MIP-1β (also called CCL4) by neutrophils176. These chemokines can further attract chemo-taxis of neutrophils to the site of inflammation.

HP-NAP is a TLR-2 agonist182 and binds to the receptor on neutrophils via its C-terminal region183. The inflammatory response in the gastric mucosa can be attributed to the activation of neutrophils by HP-NAP to produce ROS and myeloperoxidases184. The activation of ROS depends on the as-sembly of NADPH oxidases, which occurs through activation of a G-protein coupled receptor pathway leading to phosphorylation of PI3-K, Src family kinases and influx of calcium to the cytosol165, 177.

Effects of HP-NAP on monocytes

Monocytes produce tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2) upon HP-NAP stimulation179. This contributes to the progression of inflammation and gastritis during H. pylori infection because tissue healing could be interrupted due to antifibrinotic and procoagulant properties of TF and PAI-2179. Monocytes and DCs secrete remarkable amounts of IL-12p35 and IL-12p40 (subunits of the active form IL-12) and IL-23p19 (complexes with IL-12p40 to its active form IL-23) upon HP-NAP stimulation178. IL-12 and IL-23 are important T helper type 1 (Th1) polarizing cytokines. H. pylo-ri also induce secretion of other inflammatory cytokines like IL-6, IL-8 and TNF-α by monocytes. However HP-NAP null H. pylori bacterium, even at

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the highest dose (5x105 CFUs/ml) resulted in poor secretion of IL-12 by monocytes178. This suggests HP-NAP plays an important role in induction of IL-12 secretion. TLR-2 binding of HP-NAP is necessary for NF-κB activa-tion and subsequent release of cytokines by monocytes178. A TLR-2 blocking study elucidates the importance of TLR-2 in HP-NAP stimulated secretion of IL-6 in splenocytes185. HP-NAP induces progressive differentiation of monocytes to DCs by inducing expression of HLA-DR (MHC-II molecule), CD80 and CD86 (co-stimulatory molecules)178, 186.

Effects of HP-NAP on lymphocytes: Th1 polarization

HP-NAP can induce neutrophils, monocytes and DCs to secrete IL-12, a strong Th1 polarizing cytokine177, 178. In addition, HP-NAP also interacts with lymphocytes. Human T-cells when cultured with HP-NAP secreted high levels of IFN-γ and low levels of IL-4, suggesting a Th1 polarized T-cell response. This is also consistent with reports indicating a strong Th1 polarizing response associated with gastric pathology in H. pylori infected humans187. T helper (Th) cell responses to allergens are generally Th2 polar-ized. However, when a mite allergen-specific Th cells were cultured with HP-NAP, a predominant Th1 polarized response was observed (38% Th1, 33% Th0 and 29% Th2). Whereas, Th cells cultured without HP-NAP had strong Th2 response (89%) with high secretion of IL-4 and IFN-γ37, 178. Also, Th cells cultured with HP-NAP displays strong cytolytic activity. HP-NAP also induces a bystander effect, by inducing Th1 polarization on non-antigen specific T-cells188. There are several in vivo studies in mice models with allergic asthma189 and mice infected with Trichinella spiralis (Ts)185 that emphasize the anti-Th2 property of HP-NAP.

Clinical applications of HP-NAP

HP-NAP has a wide range of applications in the clinic like diagnostics, vac-cine and drug development for H. pylori infection and other diseases like cancer. This can be attributed to its immune modulating properties and its multiple modes of action on many immune cells.

I. Diagnostics: Non-invasive tests like serological test, C-Urea breath test and stool antigen tests are being used for detection of H. pylori infection and are easily accepted by the patients compared to the invasive tests190. However, these tests are not very reliable and outcome depends on the choice and nature of the antigen. HP-NAP being a highly immunogenic antigen and being highly conserved among the 12 different species is thought to be a good infection marker for diagnosis of H. pylori infection. But, whether HP-NAP could be used or not still needs to be validated and investigated. The possibility of using recombinant HP-NAP as a target for detecting serum anti-HP-NAP antibodies during H. pylori infection using

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ELISA has been evaluated. H. pylori infected patients were 60%177 and 89.4%191 positive for anti-HP-NAP antibodies in two different studies. HP-NAP is also associated with certain clinical outcomes of H. pylori infection. For example, H. pylori-associated gastric cancer patients have higher anti-HP-NAP antibodies than chronic gastritis patients172. In another study pa-tients with gastric cancer have higher HP-NAP protein levels than patients with duodenal ulcer192. Thus, HP-NAP can be used as a prognostic marker for prediction of H. pylori-related gastric cancer.

II. Vaccine development: HP-NAP being one of the major viru-

lence factors of H. pylori, is a good target for vaccine development. Both prophylactic and therapeutic vaccination strategies against H. pylori using HP-NAP have been evaluated with success in animal models. About 80% of mice are protected from H. pylori infection after oral prophylactic vaccina-tion177. HP-NAP has also been combined with other virulence factors of H. pylori like CagA and VacA, and it reduces H. pylori colonization in dogs193 and this vaccine is also safe and immunogenic in humans194. However, ther-apeutic validation of such multicomponent prophylactic vaccine is needed. Also, HP-NAP is being delivered with live vectors like attenuated S. typhi-murium195, and viral vectors like attenuated measles virus196 for vaccination purpose. Positive anti-HP-NAP antibody and cell-mediated responses were observed196. HP-NAP is being used as an adjuvant to stimulate immune re-sponse against weak immunogens, for example HP-NAP-tagged human im-munoglobulin λ-chain induces a strong immune response against λ-chain in mice197. So, HP-NAP has multiple applications in vaccine development. It can be used as a protein vaccine, DNA vaccine or as an adjuvant.

III. Drug development: HP-NAP is a candidate drug in cancer

immunotherapy and for treatment of allergic diseases. Recombinant HP-NAP (bladder cancer)198, as well as measles virus-encoded HP-NAP (breast cancer)199 exhibit anti-cancer properties by inducing a cytotoxic Th1 type immune response. HP-NAP is being used as a target for treatment of H. py-lori infection in certain cases. New drugs that target HP-NAP are being iden-tified and validated like arabinogalactan proteins from Chinos mastic gum, which itself is an inhibitor of HP-NAP (blocks neutrophil activation and adhesion to endothelial cells)169, 200

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2. MicroRNA Detargeting Viruses usually have a broad range of cell types they can infect and tissue-specific targeting of viral vector is one of the most important requirements for improving their safety. MiRNA dependent restriction of viral replication and transgene expression in certain tissues has been one of the emerging and successful tools in gene therapy201, 202. They are small non-coding, 21bp long RNA molecules involved in regulating processes like cellular differentiation, proliferation, development and cell death203. They mediate these processes by post-transcriptional silencing of the messenger RNA (mRNA) molecules and it is the case in about 60% of the protein-encoding genes in humans. MiRNA expression is complex and diverse; more than 2500 sequences have been identified (http://www.mirbase.org/)204. The expression profiles of miRNA in cell types are unique; sometimes are abnormally expressed in cancer205 and other diseases206. This gives the opportunity to control virus replication in a particular cell type, or tissue.

Pri-miRNAs (approx. 70bp), the precursors of mature miRNAs are encoded by the genome and form hairpin loop structures. The pre-miRNAs are pro-cessed by the ribonuclease III enzymes Drosha and Dicer in two steps to form the miRNA duplex (approx. 21bp, with 2-nucleotide 3’ overhangs). The 5’end with less thermodynamically stable base paring of the duplex is chosen as mature miRNA and the other strand is degraded. Mature miRNAs assemble together with ribonucleoprotein (RNP) to form miRNA-induced silencing complex (miRISC), after which they bind the target sequences in mRNAs (Figure 12). However, a single mature miRNA can bind hundreds of mRNAs due to imperfect base pair complementarity needed for binding. miRISC silences protein translation by several different mechanisms. Brief-ly, (a) when miRISC binds to 3’end of the target mRNA it induces dead-enylation and subsequent degradation of the target mRNA, (b) miRISC can repress cap recognition or block 60S joining and subsequently repress trans-lation initiation, (c) repression of protein synthesis can occur even after initi-ation of translation by slowing elongation or forced ‘drop off’ of ribosome induced by miRISC and (d) miRISC along with an unidentified protease ‘x’ can induce proteolysis of nascent peptide207 (Figure 12).

To restrict virus replication or transgene expression by viral vectors in cer-tain tissues, miRNA target sequences are inserted, typically into the 3’UTR of the viral genome or transgene cassette. This will lead to recognition of the target sequences by the miRNAs and degradation of viral mRNA or attenua-tion of viral RNA translation in specific tissues201, 202 (Figure 12). In the case of OV therapy, to attenuate virus replication in normal/healthy cells miRNA target sequences (complementary to miRNAs expressed in normal cells) are introduced into the viral genome. Coxsackie A21 OV was tagged with target

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sequences for miR206 and miR133a to attenuate off target replication in skeletal muscles and muscle respectively, was the first proof of concept208. Since then many RNA122 and DNA96 viruses have been engineered with miRNA target sequences to prevent off target toxicities and improve tumor specificity.

Figure 12 Tissue-specific restriction of OV replication by microRNA target (miRT) tagging: Primary miRNAs (Pri-miRNA) are transcribed from the genome and processed by Drosha and DGCR8 to precursor miRNA (Pre-miRNA) in the nucleus. After export of the pre-miRNA to the cytoplasm, it is processed to from mature miRNA by Dicer. MiRNA-induced silencing complex (miRISC) is formed with the mature miRNA that binds to the target sequences to execute the downstream processes. Illustration of selective replication of an oncolytic virus in tumor cells by tagging the viral genome with a tissue/normal cell-specific miRNA target sequence (in red). In cells where the miRNA complementary to the target sequence is ex-pressed to will lead to degradation of the viral mRNA and or block translation of viral protein and thus virus replication. In tumor cells where the miRNA expression is lost or low, viral replication is not inhibited.

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AIMS

The overall aims of this thesis are to use gene therapy as a tool to improve the immunostimulatory potential of oncolytic virus and CAR T-cells and improve safety profiles of oncolytic viruses for cancer therapy.

Specific Aims:

Article 1: To arm an infection-enhanced, surface-modified, oncolytic ade-novirus with secreted HP-NAP, and evaluate if this virus has enhanced ther-apeutic efficacy in a neuroendocrine cancer xenograft model.

Article 2: To investigate the effect of adenovirus-encoded HP-NAP on DCs maturation, migration and Th cytokine profile.

Article 3: To engineer CAR T-cells to express HP-NAP as an inducible protein that is expressed upon T-cell activation, and evaluate if HP-NAP can enhance anti-tumor activity of CAR T-cells.

Article 4: To evaluate the oncolytic potential of a triple miRNA-detargeted SFV4 in experimental neuroblastoma and glioma models.

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RESULTS AND DISCUSSION

Article 1: Recruitment and activation of innate immune cells to the tumor is critical for achieving successful cancer immunotherapy because these cells can mediate direct anti-tumor cytotoxicity and also induce a subsequent antigen-specific immune response209. Of the innate cells, neutrophil infiltration in tumors correlates with tumor regression upon OV therapy210. HP-NAP binds and activates immune cells via toll-like receptor-2, is a potent chemoattractant for several innate immune cells like neutrophils, monocytes and mast cells177. The aim of this study was to arm an infection-enhanced CRAd with secreted HP-NAP, to boost an anti-tumor immune response, in addition to viral oncolysis.

Arming oncolytic adenovirus with HP-NAP in the E1A region was well tolerated by the virus (Ad5PTDf35-[Δ24-sNAP]) and displayed equally good oncolytic efficacy in vitro when compared to the un-armed virus (Ad5PTDf35-[Δ24]). Ad5PTDf35-[Δ24-sNAP] treatment prolonged survival of mice bearing xenografts of human neuroendocrine tumors. Treatment with Ad5PTDf35-[Δ24-sNAP] also increased neutrophil infiltration to tu-mors leading to increased necrosis, indicating better tumor cell killing. Treatment with Ad5PTDf35-[Δ24-sNAP] also induced increased secretion of pro-inflammatory cytokines like TNF-α, MIP2-α and IL-12/23-p40, indi-cating activation of immune system by HP-NAP. In summary, this study describes that HP-NAP can serve as a potent immunomodulator in promot-ing antitumor immune response in the tumor microenvironment and enhance the therapeutic effect of oncolytic adenovirus (Figure 13). However, since human adenovirus do not replicate in mouse cells this study was performed with human tumors in immune-compromised mice that lack B-cells and T-cells, i.e., lack an adaptive immune system. Therefore, we were not able to study the role HP-NAP in the presence of an intact immune system. This was addressed in later studies.

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Article 2: HP-NAP, one of the major virulence factors of the bacteria, has gained much interest as a therapeutic agent. It is used as adjuvants to boost antibody re-sponse against therapeutic peptides197, and as an immunotherapeutic anti-cancer agent198. We (in article 1) and others have used it as an oncolytic vi-rus-encoded protein for the purpose cancer therapy with success in immune deficient mice199. It is known that HP-NAP mediates anti-tumor activity by inducing a Th1 polarized immune response198, however the mechanism be-hind this is partly unknown. The aim here was to study the role of adenovi-rus encoded HP-NAP in activating DCs, a key player in priming the adaptive immune system.

DCs treated with tumor cell supernatant containing HP-NAP displayed a mature and activated phenotype expressing T-cell co-stimulatory surface markers like CD80, CD83, CD86 and CD40. HP-NAP-matured DCs also secrete Th1 polarizing cytokines IL-12, TNF-α and IFN-γ. IL-12 is known for having potent anti-tumor effects by improving effector functions of CD4+ T-cells, CD8+ T-cells and NK cells160, 211. HP-NAP can reverse a rather Th2 polarized immune response to a Th1 polarized response, which is beneficial for cancer therapy178. HP-NAP mature DCs also expressed the lymph node homing receptor CCR7 in vitro and HP-NAP induced migration of activated DCs to draining lymph nodes in vivo. This is important because from recent finding it is evident that ex vivo generated DC based vaccines perform poor-ly after re-infusion45. This stresses the need for efficient in vivo priming and activation of DCs. HP-NAP-matured DCs efficiently induced antigen specif-ic T-cell proliferation in vitro that were functional when co-cultured with target cells expressing the target antigen. In summary, HP-NAP activated DCs displayed all the key phenotypes of functionally active DCs to prime the adaptive immune system in a Th1 direction (Figure 13). This further strengthens the notion that HP-NAP is an important therapeutic protein, and that it can be used as an adjuvant for DC-based cancer vaccines and cancer immunotherapy.

Article 3: CAR T-cells, targeting the pan B-cell marker CD19, have made remarkable progress in clinical studies for both acute and chronic B-cell leukemia. Suc-cess has also been observed for B-cell lymphoma although somewhat less so than for leukemia. The reason may be that that lymphomas exhibit a semi-solid structure leading to penetration obstacles and an immunosuppressive microenvironment within these tumors. HP-NAP promotes DCs maturation and induces a very strong Th1 polarization (Article 2)178. Activated DCs also provide T-cells with key co-stimulatory signals which is rather absent in the

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immune suppressive TME138. The aim of this study was to insert sequence encoding NAP into CD19-CAR T-cell, which is secreted upon T-cell activa-tion by target cell recognition.

We found that in engineered T-cells, NAP was secreted when the CAR mol-ecule recognized to CD19 on tumor cells. CAR T-cells appears to tolerate inducible NAP and the secreted NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved killing abilities in vitro (Figure 13). Secreted NAP also induced migration of innate immune cells in vitro as observed by us and others176, 178. NAP CD19-CAR T-cells also dis-play enhanced cytotoxicity in vivo promoting tumor regression and pro-longed survival of mice bearing syngeneic A20 lymphoma. The data indicate that inducible secretion of NAP from CAR T-cells reverses the immune sup-pressive TME by inducing maturation of DCs, attracting innate cells and increasing secretion of cytokines and chemokines, thus reducing tumor growth.

Figure 13 Proposed anti-tumor mechanism of an oncolytic adenovirus and CAR T-cell encoding secreted HP-NAP as a transgene

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Article 4: Glioblastoma multiforme (GBM) and high-risk neuroblastoma are cancers with no cure available. Immunotherapy in the form of neurotropic oncolytic SFV is a promising therapeutic strategy122; however type-I IFN response against RNA viruses like SFV remains a hurdle128. The partly IFNβ-insensitive SFV4 strain could be used but it is virulent and lethal to adult mice. Others have shown that miRNA124-tagged SFV4 has largely attenuat-ed replication in mouse CNS, with a few mice displaying viral replication in the oligodendrocytes of the corpus callosum212. To completely attenuate SFV4 replication in brain we constructed SFV4miRT, which is attenuated in normal CNS cells through insertion of miRNA target sequences for miR124, miR125 and miR134. SFV4miRT RNA genome in theory is degraded in cells where any of the above mentioned miRNAs are expressed.

We validated that healthy mouse brain, spine and in vitro differentiated neu-rons, astrocytes and oligodendrocytes expressed either one of the three miRNAs and that expression of these miRNAs is absent or low in most hu-man and mouse tumor cells. The introduction of miRNA target sequences for miR124, miR125 and miR134 into the virus genome did not affect virus replication in tumor cells but significantly reduced replication of virus in brain cells (neurons, astrocytes and oligodendrocytes) of adult mice without causing any virus related encephalitis when administered systemically. A single intravenous injection of SFV4miRT prolonged survival and cured 50% of mice with subcutaneous syngeneic neuroblastoma (NXS2), 27% mice with orthotopic syngeneic GBM (CT-2A) but did not improve progres-sion free survival of mice bearing GL261, another orthotopic syngeneic GBM model. The therapeutic response observed in the CT-2A model is in accordance with a previous report122. Although SFV4 is less sensitive to IFN-β than other SFV strains129, higher doses of type-I IFN inhibited SFV4 replication. Therapeutic efficacy in different tumor models inversely corre-lated to secretion of IFN-β by respective cells upon SFV infection in vitro. Killing efficacy of SFV4miRT on human glioblastoma cell culture (HGCC) lines inversely correlated to IFN-β response and interferon-α/β receptor (IF-NAR)-1 expression.

SFV4miRT has reduced neurotoxicity, while retaining its oncolytic poten-tial. SFV4miRT is an excellent candidate for treatment of GBMs and neuro-blastomas with low IFN-β secretion. A strategy to counteract the type-I anti-viral immune response should be addressed in order to improve the therapeu-tic efficacy.

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FUTURE PRESPECTIVES

I. HP-NAP has found several therapeutic applications (e.g. vac-cination, anti-cancer agent, adjuvant etc.) in the pre-clinical settings recently. Our future plans are to validate the anti-tumor immune modulatory effects of HP-NAP combined with viral oncolysis in an immune competent animal model. Adenoviruses lack or poorly replicate in murine cells213, so we intend to explore the possibility of using Semliki Forest Virus (SFV) or Vaccinia virus for this purpose. Developing successful cancer vaccines have been difficult because most TAAs are self-antigens, and the cancer usually devel-ops immune evasive mechanisms. Previously, it has been reported that fu-sion of a foreign immunogen (non-self) with a TAA helped evade this self-tolerance and successfully generate TAA-specific antibodies in mice 214, 215. HP-NAP is also a good adjuvant, known to boost immunity against weak immunogens197 and being a foreign immunogen it can be fused with TAA for cancer vaccination. So, we would like to take advantage of this property by tagging tumor antigens with HP-NAP to further boost specific anti-tumor response. Currently, our group is focusing on neuroblastoma immune thera-py. GD2 (disialoganglioside) is over-expressed on tumors of neuroectoder-mal origin216. GD2 is a weak immunogen, so we would like to use an onco-lytic virus armed with HP-NAP fused to a mimotope of GD2 (Mimotope D) to boost immunity against GD2. However, HP-NAP also promotes immunity against the viral vector (un-published data), which can lead to clearance of the virus and reduce effect of oncolysis. To overcome this, different viral vectors will be used in a prime boost strategy to avoid anti-viral immune response and increase therapeutic efficiency.

II. Our emerging data from CD19CAR T-cells secreting induci-ble HP-NAP seems promising for the treatment of lymphomas. The prelimi-nary results indicate that innate immune cells like DCs and neutrophils play an important role in the increased efficacy of CAR T-cells and suppressing tumor burden. So, we would like to investigate further if these cells do play an active role in eradicating tumor cells. Preliminary results also indicate that HP-NAP induces a proinflammatory TME rather than an immunosup-pressive TME. Detailed characterization of the tumor immune microenvi-ronment after treatment with NAP CD19 CAR T-cells needs to be performed to confirm this. Another of our aim is to expand NAP CAR T-cell treatment

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to solid tumors, where the conventional CARs have largely failed. We are developing inducible NAP CARs against GD2 and its efficacy will be tested in syngeneic neuroblastoma mice models.

III. SFV is a promising oncolytic agent for treatment of CNS re-lated tumors. However, there are still some important issues that need to be addressed. The insertion of multiple miRNA targets improved safety of the lethal SFV4 strain when administered systemically in adult mice. These three miRNA targets (miRNA124, miRNA125 and miRNA134) were screened for in healthy mouse CNS tissues and it is yet known if they are suitable miRNA targets in humans (expressed in healthy human brain). Suit-able miRNA targets for SFV4 attenuation in humans need to be screened and validated. SFV is sensitive to type-I IFNs and has low therapeutic efficacy in tumors that are responsive to type-I IFNs. We have ongoing efforts to make point mutation in the SFV genome (particularly in the nsP regions) to make them more IFN resistant and thus improve therapeutic efficiency.

IV. Virus-induced cell death is considered immunogenic and sev-eral reports suggesting the same217. Drug-induced immunogenic cell death (ICD) of cancer cells is advantageous for cancer therapy because dying cells can effectively induce DC activation and subsequent anti-tumor immunity218,

219. ICD induced by viruses haven’t been characterized in detail and it is one of our goals to characterize ICD induced by Ads, SFV and Vaccinia virus.

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GLOSSARY

Antigen Is a molecule (protein, toxin, carbohydrate etc., usu-ally foreign substance) against which a specific im-mune response can be mounted.

Antigen presenting cell

APC are cells that is capable of capturing proteins and present them as processed antigens on the MHC molecule to T-cells

Cancer A disease arising due to uncontrolled growth of cells with the ability to spread to other parts of the body.

Chemokines They are a class of cytokines that have the ability to induce chemotaxis (migration) of cells

Clinical Benefit Is a term used to define any condition upon treat-ment other than progressive disease.

Complete Response No detectable tumor after a particular treatment. However, it does not mean that a patient is cured upon receiving the treatment

Cytokine Small proteins secreted mainly by immune cells that play an important role in cell communication and signaling.

Cytotoxic T-Lymphocyte

Is a type of T-lymphocytes characterized by expres-sion of CD3 and CD8. As the name suggest they kill pathogen-infected (mainly virus) cells, damaged cells and cancer cells

Glioblastoma Multi-forme

GBM is a cancer that arises from the glial cells in the brain. It’s the most frequently arising primary cancer of the brain.

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Helper T-Lymphocyte

Is a type of T-lymphocytes characterized by expres-sion of CD3 and CD4. They mainly help other im-mune cells by secreting cytokines

Immunogenic Cell Death

Is a type of cell death induced by some drugs and viruses that induces an effective immune response against the antigens released by the dying cells.

Leukemia

Cancers arising as a result of abnormal growth of white blood cells, usually initiate in the bone marrow. Here we focus on leukemia in B-lymphocytes.

Lymphocyte They are white blood cells and part of the immune system, include B-lymphocytes, T-lymphocytes and NK cells

Lymphoma They are cancers that arise from the cells in the lym-phatic system (semi-solid tumors formed by lym-phocytes)

Major Histocom-patibility Complex

MHCs are molecules expressed on the surface of cells that present short peptides (antigens) to T-cells for subsequent activation of an immune response against the antigen.

microRNA They are approximately 22 nucleotide long non-coding RNA molecules that regulate gene expres-sion post-transcriptionally

Neoantigen It is an antigen that is newly formed due to muta-tions in malignant cells and not recognized by the immune system previously.

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Neuroblastomas They are extracranial cancers arising from the neural crest cells in the neck, adrenal gland, spinal cord or chest.

Neuroendocrine cancer

They are cancers arising from the uncontrolled growth of neuroendocrine cells, the cell type that connects the nervous system to the endocrine (hor-mone-producing) system. Some neuroendocrine cancers are referred to as carcinoids, which can arise from cells in the intestine or lungs.

Oncolytic Virus

It’s a virus that is engineered to preferentially repli-cate in and subsequently kill cancer cells. An onco-lytic virus can also be a naturally selected virus with preferential activity in tumor cells.

Overall Survival The time period for which a patient is alive from the time of diagnosis or start of a particular treatment. The death can be due to disease or any other cause.

Partial Response Decrease in tumor volume of up to 50% upon re-ceiving a treatment, but with tumor remaining.

Progression Free Survival

The time period for which a patient lives with the disease after receiving treatment. The death is due to disease progression.

Progressive Disease Continuous growth of the tumor or arising of new tumor lesions from the time of diagnosis.

Regulatory T-Lymphocyte

Tregs are a type of T-cells with immune suppressive activity, mainly to prevent autoimmunity by main-taining tolerance against self-antigens.

Solid tumor A mass of tissue arising due to uncontrolled prolif-eration of cells and usually does not contain any liquid areas and cysts.

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Stable Disease Refers to small amount of growth (less than 20% of original size) or small amount of shrinkage (typical-ly less than PR) of the tumor.

Tumor Infiltrating Lymphocyte

They are lymphocytes that are found in tumor and tumor stroma. Mainly composed of T-cells, but also B-cells and. NK cells.

Tumor microenvi-ronment

The complex cellular and extracellular environment in which a tumor cell propagates is referred to as the TME. It usually comprises of immune cells, blood vessels, extracellular matrix, fibroblasts, cytokines etc.

Tumor-associated antigen

TAA is an antigen that is produced or derived from the tumor cells. It is usually specific to or over ex-pressed in these cells.

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ACKNOWLEDGEMENTS

The studies presented in this thesis were carried out in the Department of Immunology, Genetics and Pathology at Rudbeck Laboratory, Uppsala Uni-versity. The work had financial support from Cancerfonden, Barn Cancer-fonden and Vetenskapsrådet.

I take this opportunity to express my sincere gratitude and appreciation to all the people who have helped me during my time in Uppsala during my PhD studies.

I would like to first thank Professor Magnus Essand, my supervisor for giving me this opportunity to do a PhD thesis. You have been a great super-visor and mentor. I have been inspired by you not only in science but also in music. One of my aims during these years was to learn guitar and play with you during my party, but unfortunately science has not given me enough time to do so. Thanks to my co supervisors Justyna-Leja Jarblad and Di Yu, especially spending a lot of time with me to design, perform and analyze the results for my experiments. The time all my supervisors have spent with me is invaluable and I have gained a lot of knowledge and experience from you. A special thanks to Di, you have been like a brother to me and I have learned a lot from you both in science and life.

Fredrik Eriksson, thank you for supervising me during my master’s thesis, which is one of the reasons I got this PhD position and also for spending his valuable time in providing me valuable feedbacks throughout the course of this thesis. Berith Nilsson, without you I would not had most of my viruses. Thank you for helping with many cloning’s that I could not manage, produc-ing the viruses and endless support to keep the lab running smoothly. I wish you a happy retirement.

Dr. Chuan Jin you have been sharing this journey with me from when we did our masters until PhD and continuing (hopefully ). You have been a great friend and colleague, thank you for all the contribution to this thesis, the lovely Chinese hotpot nights and dinner hangouts. Grammatiki Fotaki a.k.a. Kiki; you are such an inspiring colleague and an awesome friend; I will have never manage the long FACS hours that you are pulling off. Thank you for hosting the most amazing house warming party, I enjoyed it “a lot, a

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lot, a lot”, and will never forget it!! Good luck with your PhD, by the end you will realize that all the students go through the same feelings during the process of publishing a manuscript (so take it easy, Chuan will agree with me ). Jing Ma you are the best in cloning and thank you for willing to help me whenever needed. I wish you good luck with your PhD. Miika Mar-tikainen, you are my guru for SFV. You had arrived at the right time in our lab to provide me with important tips on my manuscript. Thank you for the time you spent with me discussing the results. Thank you, Dr. Alex Karls-son-Parra and Prof. Thomas Tötterman for joining us during the lab meet-ings, sharing with us you immense knowledge in immunology and about the clinical trials.

I would also like to thank the present and past members of the GIG group: Sara Mangsbo (for always keeping your door open for discussions and giv-ing me important comments on my second article), Victoria Hillerdal (for fun times at conferences and fruitful collaborations), Emma Svensson (thank you for unofficially taking over Berith’s duties to keep the lab in or-der), Hannah Karlsson (kindling my interest in dancing, I know now who I should bug if I want to learn), Matko Čančer (thank you for accepting to be the toast master at my party), Angelica Loskog, Erika, Jessica, Frida, Ga-briella, Wictor, Ioanna, Lina, Linda, Lisa, Dr. Hyeongsu Kim, Vanessa and Gunilla for your timely tips, advice and help with various techniques and instruments in the lab. I had a lot fun making the GIG movies with you guys.

I would express my gratitude to Anna Dimberg and the members of her lab, Luuk van Hooren, Maria Georganaki, Hua Huang, Lei Zhang and Rob-erta Lugano for their collaboration and interesting data clubs. Without your timely help I would not have managed to complete many of my experiments. Anna thank you for scrutinizing my manuscript like a reviewer, I think it was one of the main reasons that it could get through in CCR. Lei thank you for teaching me how to implant brain tumors and being a wonderful co-author. Luuk, a special thanks to you for hosting me in Maastricht after the BD course and showing me around your lovely city. It’s also been fun talk-ing science, Indian traditions and astrology with you. However, I failed to convince you that astrology is true (but, Maria could help me out with that). Maria it was interesting to know we share the same thoughts on this pseudo-science. I would also like to thank Anna-Karin Olsson and the members of her group for giving valuable inputs during the data clubs.

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All the students who I have supervised for their master courses, Chris Tebid, Jinar Rostami and Tina Sarén (you have been great, good luck with your future) thank you for your contributions. You have helped me a lot during my thesis.

I would like to express my special thanks to Ann-Charlotte Hellström (for performing some tail vein injections and teaching me how to perform them myself) and Minttu-Maria, for helping me with animal experiments.

Thank you, Andres Merits, for inviting me to Tartu and hosting me in your lab for a month. I learned a lot from you and your team. Sirle Saul and Age Utt thank you for not only teaching me how to produce SFV virus, but mak-ing feel at home in Tartu and the fun times playing beach volleyball.

I would like to thank all the co-authors (Sven Nelander, Valeria, Aleksei, Matheus and Alkwin) and the others who have helped me successfully completing my articles. Alkwin Wanders for his professional help with pathology. Patrik Johansson, thank you for tips, advice with statistics and R programming and also the wonderful Christmas dinner. Sara, Matyas, Pacho, and Jeremy (BioVis), thanks for teaching me and help with micros-copy, flow sorting and flow sight. Rodrigo for willing to collaborate and designing the flow chamber for us. Mia Philipson and Evelina for allowing me to use their IVIS imaging system at crucial times. Thank you Peerta Magnusson for helpings us with the blood loop models and accepting to be the chair person at my defense.

Thank you to all the fellow PhD students and members of the IGP PhD stu-dent council: Viktor, Emma, Sofia, Veronica, Lenor, Svea, Johanna, Sara, Chiara, Linnea and Dijana for the fun times during meetings, social evenings and conferences in Romme and Visby.

The Staff at IGP administration have always been available for me and very helpful. Christina Magnusson and Helene Norlin thank you for taking care of all the PhD students, fixing documents that I needed and calling Migra-tionsverket endlessly to get my resident permit processed.

Kailash singh, thank you for helping me get a house in Uppsala when I ar-rived to Sweden as a master student and providing me with positive feed-back on my publications. All my other friends at Rudbeck: Yuan, Anqxi, Soumi, Anja, Snehangshu, Maria K, and Iliana; thank you all for your support, making the lab life more interesting and fun. Argyris (did you think I forgot you?), I had great time hanging out with you and a big thanks for hosting us in Mykonos. It was great fun.

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Thanks to all my friends I have met in Uppsala through this journey, I have had fun with you guys and created some unforgettable memories: Olof Ols-son, Svenja Reinders, Erik Lundin, Madhu Elango, Lisa Rebello, Aditya Harisankar, Suhasini Udaykumar, Avinash Bayya, Vinoth Kumar, Shakthi, and Sunitha,

My family away from home: Vivek Anand, Satishkumar, Krishnapriya, Aravindh, Karthik G.M, Roshan Vaid, Ram Kumar, Jaya Kumar, Sha-rath, Monica, Ganesh and Javed. You guys have been more than just friends to me. You have made my stay in Sweden memorable, fun and less painful. Vivek, thank you for being tolerant with me and being my flat-mate right from our first day in Sweden and the only one surviving. Aravindh we just need one acceptance, don’t worry about the rejections (I know you un-derstand what I mean). Satish and Priya, thank you for the many movie nights, cooking, baking and badminton sessions, they have been real stress breakers. Priya, thank you very much in taking the pain to help me design the cover for my thesis even in the most difficult times. Karthik, good luck with your fatherhood. Aravindh, Karthik, Vivek and Satish it’s been fun travelling with you guys to many places and hope we continue to do so in the future.

Last but most importantly I wish to express my gratitude and to my amma and appa who have shaped me as a person, being an inspiration and standing by me all the time. I would also like to thank them for providing me with this opportunity and confidence to go abroad and study. I know how much you miss me (the same back).

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