Cancer Immunotherapy. Oncolytic Viruses and CAR-T Cells

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1703

Cancer Immunotherapy

Oncolytic viruses and CAR-T cells

JING MA

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-1074-9 UPPSALA urn:nbn:se:uu:diva-425970 2020 Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, Friday, 22 January 2021 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Karl-Johan Malmberg (Department of Cancer immunology, Oslo University Hospital (OUH) ). Abstract Ma, J. 2020. Cancer Immunotherapy. Oncolytic viruses and CAR-T cells. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1703. 49 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1074-9. Various forms of cancer immunotherapy have developed rapidly with improved survival and quality of life for cancer patients. Cancer immunotherapy aims to educate the patient’s immune system to eliminate cancer cells, including immune checkpoint inhibitors (ICIs), adoptive cell transfer (mostly T cells), oncolytic viruses (OVs) and cancer vaccines. Especially ICIs have induced durable responses in patients with many different types of cancers. Chimeric antigen receptor (CAR)-T cell therapy has shown good efficacy in treating hematologic malignancies. However, there is still a significant number of patients that do not benefit from these treatments due to immune evasion. Strategies to modify cancer immunotherapies with immunomodulating agent needs to be investigated to maximize the effect of immunotherapy. Helicobacter pylori Neutrophil Activating Protein (HP-NAP) could be used as an immunomodulating agent to recruit, activate and mature immune cells, such as dendritic cells (DCs), monocytes and neutrophils, and also induce T helper type 1 (Th1)-polarized response. In this thesis, we examined to arm oncolytic virus or CAR-T cells with HP-NAP. Papers I and II investigate oncolytic viruses. In paper I, we investigated wild-type Adenovirus (Ad), Semliki forest virus (SFV) and Vaccinia virus (VV), for their ability to mediate lysis of tumor cells, which was found to be associated with the release of danger-associated molecular patterns (DAMPs) and subsequently triggered phagocytosis and maturation of DCs. However, only SFV-infected tumor cells triggered significant Th1-cytokine release by DCs and induced antigen-specific T cell activation, while VV induced immunosuppressive responses. In Paper II, we armed VV and SFV with the tumor-associated antigen GD2 and HP-NAP. We found that arming these OVs with HP-NAP resulted in distinct anti-tumor immune response and therapeutic benefit. VV-GD2m-NAP showed significantly increased therapeutic efficacy compared to VV-GD2m, associated with elevated antiGD2 antibody production. In contrast, there was no additive antitumor effect for SFV-GD2m-NAP compared with SFV-GD2m. Due to intrinsic properties of OVs, engineering OVs with immunomodulating agents needs careful consideration. Engineering SFV or similar viruses, which is very immunogenic, should focus on improving oncolysis, de-bulking tumor and release of tumor-associated antigens, while for VV or similar viruses, with immunosuppressive properties, the focus can be on arming the virus with immune modulators to improve anti-tumor immune response. Papers III and IV investigate CAR-T cells. In paper III, CAR-T cells were engineered to inducible secrete HP-NAP upon antigen recognition (CAR(NAP)-Ts). CAR(NAP)-Ts successfully reduced tumor growth and prolonged survival of mice in several solid tumor models with epitope spreading and initiated endogenous anti-tumor immune responses. Secreted HP-NAP created an immunologically hot tumor microenvironment with enhanced infiltration of immune cells (DCs, neutrophils, macrophages, and cytotoxic natural killer cells). In paper IV, we developed CAR T cells targeting CD20 (rituCD20CAR T cells). We found that rituCD20CAR T cells could efficiently kill CD20-positive lymphoma cell lines (U2932, Karpas422, DB, U698, Raji, Daudi) as well as primary mantle cell CD20-positive lymphoma (CD20+ MCL) cells accompanying with IFNγ secretion. Both rituCD20CAR and NAP-armed rituCD20CAR(NAP) T-cell treatment delayed tumor growth and prolonged mice survival in the murine lymphoma A20-hCD20 model. In summary, combing OVs and CAR-T cells with the immunomodulating agent HP-NAP is a promising way of maximizing the benefit of immunotherapy to combat cancers. Keywords: Cancer immunotherapy, Oncolytic viruses, chimeric antigen receptor (CAR)-T cells, Helicobacter pylori Neutrophil Activating Protein Jing Ma, Department of Immunology, Genetics and Pathology, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-513-1074-9 urn:nbn:se:uu:diva-425970 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-425970)

“Do not follow where the path may lead. Go instead where there is no path and leave a trail.”

~Muriel Strode

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Ma J†, Ramachandran M†, Jin C†, Rubio C, Martikainen M, Yu D* and Essand M*. (2020) Characterization of virus-mediated immunogenic cancer cell death and the consequences for oncolytic virus-based immunotherapy of cancer. Cell Death & Disease, 11:48 II Ma J, Čančer M, Jin C, Ramachandran M* and Yu D*. Concur- rent expression of HP-NAP enhanced antitumor efficacy of oncolytic vaccinia virus but not for Semliki Forest virus. Manu- script under revision III Jin C†, Ma J†, Ramachandran M, Yu D* and Essand M*. Expres- sion of bacterial virulence factor enhances the efficacy of CAR T cell therapy against solid tumors. Submitted manuscript IV Sarén T†, Ma J†, Kim H, Amini RM, Enblad G, Yu D* and Essand M*. CD20-targeting CAR T cell therapy based on rituxi- mab for the treatment of CD19-negative B cell malignancies. Manuscript

Reprints were made with permission from the respective publishers.

† Shared first author * Corresponding author

Contents

The Human Immune System ...... 13 The Innate and Adaptive Immunity ...... 13 Antigen presentation ...... 14 T cells activation and polarization ...... 15 The immune system in cancer ...... 18 Oncolytic virus (OV) therapy and Immunogenic cell death (ICD) ...... 21 Oncolytiv virus (OV) therapy...... 21 Immunogenic cell death (ICD) ...... 22 T cell-based immunotherapies ...... 25 Adoptive T-cells transfer ...... 25 Chimeric antigen receptor (CAR) T cell therapy ...... 26 CAR-T cell therapy for solid tumor ...... 28 Method ...... 30 Helicobacter pylori Neutrophil Activating Protein (HP-NAP) ...... 30 Present Investigations ...... 32 Paper I ...... 32 Paper II ...... 32 Paper III ...... 33 Paper IV ...... 34 Svensk sammanfattning ...... 35 Future Perspectives ...... 37 Immunogetic cell death indced by oncolytic viruses ...... 37 CAR(NAP)-T cell therapy in the clinic ...... 37 OVs/CAR-T cell combination therapy for solid tumor ...... 38 Acknowledgements ...... 39 References ...... 42

Abbreviations

ACT Adoptive Cell Therapy Ads Human Adenovirus ALL Acute Lymphoblastic Leukemia APC Antigen Presenting Cell ATP Adenosine Triphosphate BBB Blood-Brain Barrier BCR B Cell Receptor CAR Chimeric Antigen Receptor Caspase cysteine-aspartic proteases CCL Chemokine (C-C motif) Ligand CD Cluster of differentiation CEA Carcinoembryonic Antigen CI Checkpoint Inhibitor CLIP Class II-associated Ii Peptide CLL Chronic Lymphocytic Leukemia CRS Cytokine Release Syndrome CTLs Cytotoxic T-lymphocytes CNS Central Nervous System CR Complete Remission/Response CRT Calreticulin CXCL Chemokine (C-X-C motif) Ligand DAMPs Danger Associated Molecular Patterns DC Dendritic Cell DLBLC Diffuse Large B-cell Lymphoma DNA Deoxyribonucleic Acid EMA European Medicine Agency ER Endoplasmic reticulum FDA Food and Drug Administration GD2 A disialoganglioside expressed on tumors of neuroecto- dermal origin GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor GMB Glioblastoma Multiforme HDIs Histone Deacetylase Inhibitors HER2 Human Epidermal Growth Factor Receptor 2 HLA Human Leukocyte Antigen HMGB1 High Mobility Group Protein B1 HP-NAP Helicobacter pylori Neutrophil Activating Protein HSCs Hematopoietic Stem Cells HSV-TK Herpes Simplex Virus-Thymidine Kinase HSP Hot Shock Protein HVR Hyper Variable Region ICAM Intercellular Adhesion Molecule ICD Immunogenic Cell Death iCasp9 Inducible Caspase-9 IDO Indoleamine 2, 3-dioxygenase IFN Interferon Ii Invariant Chain IL Interleukin imDCs Immature Dendritic Cell ITAM Immunoreceptor Tyrosine-based Activator Motif Kbp Kilo base pair LAG-3 Lymphocyte-Activation Gene 3 LFA Lymphocyte Function-Associated Antigen MCL Mantle Cell Lymphoma mDCs Mature Dendritic Cells MDSC Myeloid-derived Suppressor cells MHC Major Histocompatibility Complex MIP Macrophage Inflammatory Protein MICA/B MHC class I chain-related protein A and B NK Natural Killer OV Oncolytic Virus PAMP Pathogen-Associated Molecular Pattern PBMC Peripheral Blood Mononuclear Cell PD-1 Programmed Cell Death-1 PDI Protein Disulfide Isomerase PRR Pattern Recognition Receptor PSCA Prostate Stem Cell Antigen RAE-1 Retinoic Acid Early Inducible Gene 1 ROS Reactive Oxygen Species ScFV Single Chain Variable Fragment SFV Semliki Forest Virus TAA Tumor-Associated Antigen TAP Transporter associated with Antigen Processing TCR T-cell Receptor TF Tissue Factor TfH Follicular T Helper Cells TGF-β Transforming Growth Factor-β Th cells Helper-T Lymphocytes TILs Tumor Infiltrating Lymphocytes TIM-3 T-cell Immunoglobulin and Mucin-domain Containing-3 TLR Toll-like Receptor TME Tumor Microenvironment TMZ Temozolomide TNF Tumor Necrosis Factor Treg Regulatory T cell T-VEC Talimogene laherparepvec V-CAM Vascular Cell Adhesion Molecule VGF Vaccinia Growth Factor VSV Vesicular Stomatitis Virus VV Vaccinia Virus

The Human Immune System

The immune system is a host defense system with a complex network of specialized cells, tissues, organs and effector molecules that can recognize, defense against and remember millions of different enemies, such as foreign mi- crobes (bacteria, fungi and parasites), virus, toxins as well as endogenous cancer cells. The immune system consists of lymphoid organs that control the production and maturation of immune cells. The key primary lymphoid organs are the thymus and the bone marrow, which provide an environment for production and early selection of lymphocytes. The secondary lymphoid organs are the places where the immune cells get “educated”. These organs mainly include the spleen, lymph nodes, tonsils and other specialized tissues in the mucous membranes of the bowel.

The Innate and Adaptive Immunity The immune system can be simply divided into two “lines of defense”: innate immunity and adaptive immunity. Innate immunity represents the first line to defense against invaders. It is an antigen-independent defense that can fight back immediately or within hours upon encounter with pathogens to protect the host. Innate immunity consists of many components, including physical barriers like the skin and mucous membranes, phagocytes such as macrophages, neutrophils, mast cells and natural killer (NK) cells, as well as inflammatory cytokines released by infected cells and immune cells [1]. The innate immune response can rapidly recognize a wide variety of pathogens sharing common structures which are named pathogen-associated molecular patterns (PAMPs) through their pattern recognition receptors (PRRs). Once an infection agent enters the host, innate immune cells can rapidly be recruited to the site of infection and release inflammatory cytokines and chemokines such as tumor necrosis factor (TNF), interleukin (IL)-1 (IL-1) and IL-6, to initiate a local inflammation to clear the pathogen [1]. Unlike the innate immune response, adaptive immunity is antigen-dependent and can distinguish “non-self” from “self” antigens in a refined man- ner. It takes more time to develop an adaptive immune response as specific cell clones must be educated and expanded. Another feature of adaptive immunity is that it can develop an immunologic memory which can trigger a

13 faster response if the same pathogen attacks again. There are two types of adaptive immune responses: the cell-mediated immune response, which is mainly mediated by T cells, and the humoral immune response, which is carried out by B cells. T cells are the main cells involved in destruction of cells infected by pathogens and elimination of tumor cells. T cells are activated and expanded through engagement between the T-cell receptors (TCRs) and antigen presenting cells (APCs) carrying specific foreign or mutated peptides presented on the major histocompatibility complex (MHC) molecules. Cytotoxic CD8+ T cells (CTLs) attack target cells by releasing substances, most importantly per- forin, granzymes and Fas-ligand (FasL), as well as secretion of chemokines and cytokines such as interferon-γ (INF-γ) and tumor necrosis factor while CD4+ T helper cells (Th) primarily mediate the immune response by releasing cytokines that can affect other immune cells, such as antigen present cells (APCs), B cells and CTLs [2]. After clearing an infectious pathogen, most T cells die and are cleared by phagocytes and only a few cells remain as memory cells that can be quickly re-expanded into large amounts of effector cells to eliminate the same pathogens at a later occasion. Unlike T cells, B cells can recognize antigens directly through B-cell antigen receptors (BCRs), which are membrane-bound immunoglobulin expressed on the surface of B cells. When B cells are specifically activated by a target antigen, they proliferate and differentiate into antibody-secreting plasma cells and/or memory B cells. The antibodies produced by plasma cells then enter the circulation and tissues and eliminate pathogens by specifically binding to antigenic epitopes on the surface of pathogens, leading to clearance through complement activation or antibody-dependent cellular cytotoxicity (ADCC) via Fc receptor-mediated engulfment by phagocytes [3]. Similar to T cells, most plasma cells undergo apoptosis after the pathogen has been cleared. However, pathogen-experi- enced memory B cells are retained and they can response rapidly to produce large amounts of antibody when the same infectious agents attack again.

Antigen presentation T cells get activated by recognizing proteolyzed antigens through their TCRs, which interact with antigenic peptides presented by MHC molecules on the surface of APCs. CD8+ T cells recognize peptides associated with MHC class I (MHC-I), while CD4+ T cells recognize peptides bound to MHC class II (MHC-II). CD8 and CD4 function as co-receptors to strengthen the TCR in- teractions by direct interaction with the MHC-I and MHC-II molecules, re- spectively. MHC-I molecules are expressed by all nucleated cells. Antigen peptides are generated from endogenously expressed self and non-self-proteins, which are degraded by cytosolic proteasomes and then transferred to endoplasmic reticulum (ER) via transporter associated with antigen presentation (TAP) and

14 loaded onto MHC-I. Chaperone proteins, such as calreticulin, ERp57, protein disulfide isomerase (PDI) load into MHC-I to stabilize them before a peptide binding. When a processed peptide (typically 8-9 amino acids) is loaded into the peptide-binding groove of MHC-I, the chaperons are released and the peptide-MHC-I complex is then translocated to the cell surface to present the peptide to CD8+ T cells [4, 5]. Unlike MHC-I, MHC-II molecules are primarily expressed on professional APCs including dendritic cells, macrophages and B cells, and present peptides derived from exogenous antigens, taken up by the APCs, to CD4+ T cells. In the ER, transmembrane α and β-chains of MHC-II and invariant chain (Ii) are assembled to form Ii-MHC class II complex (MIIC), which in turn is transported into a late endosome. In MIIC, Ii is digested into class II-associated Ii peptide (CLIP) that binds into the peptide-binding groove to form MHCII/CLIP complex [6]. Chaperone proteins HLA-DM facilitates loading of a specific peptide (typically 15-24 amino acids) to assemble peptide-MHC- II complex by releasing CLIP [7, 8]. The matured peptide-MHC-II complex are transported to the cell membrane and present the peptide to CD4+ T cells. The presentation of exogenous antigens on MHC-I is the third antigen presentation process termed cross-presentation. Dendritic cells (DCs) are the most efficient cross-presenting APCs to present the peptides derived from exogenous proteins to CD8+ T cells. Efficient cross-presentation has been shown crucial for initiation of cytotoxic CD8+ T cell responses against cancer [9, 10].

T cells activation and polarization The precursor T cells, termed thymocytes, develop from bone marrow-derived hematopoietic stem cells (HSCs). After entering the thymus, the thymocytes complete their maturation and undergo the positive and negative selections to become naïve T cells. During positive and negative selections, the receptors of thymocytes are tested for the interaction with self-peptides on MHC molecules to generate T cells able to recognize self MHC but not “self” antigens. Once naïve T cells leave the thymus, they travel through the blood to peripheral lymphoid tissues such as lymph nodes, where they encounter antigens and consequently get activated. The fate of naïve T cells is determined by three signals that are provided by pathogen-primed matured APCs (Figure 1). The antigen stimulatory signal 1 is from the ligation between TCRs and pathogen- derived peptides presented by MHC molecules on APCs, which determines the antigen-specificity of the response. The co-stimulation signal 2 is mainly the interaction between CD28 on T cells, and B7-1(CD80) and B7-2(CD86) on APCs [11, 12]. In the absence of the co-stimulatory signal, T cells become anergic, which might induce tolerance. TCR stimulation and co-stimulation signals are required for naïve T cells developing into protective effector/memory cells, normally accompanied by secretion of selective sets of cytokines that determines the fate of the T cells. CD8+ T cells differentiate into

15 CTLs while CD4+ T cells becomes helper T (Th) cells of various kind such as Th1, Th2, Th9, Th17 or regulatory T cells depending on the cytokine environment, which is regarded as signal 3.

Figure 1. T-cell stimulation and T helper (Th) polarization require three DC- derived signals. Signal 1 is the antigen-specific signal that is mediated through the T-cell receptor (TCR), triggered by peptide MHC complex. Signal 2 is the co-stimulatory signal, mainly mediated by triggering of CD28 binding with CD80 and/or CD86 on the DC. Signal 3 is the cytokine factors secreted by DCs and other cells that direct T cell polarization.

Dendritic cells (DCs) are one of the main APCs important for Th cell polarization. DCs usually reside in peripheral tissues at an immature statue. For example, classical myeloid CD11chi DCs always reside in the epithelia of skin and mucosal tissue [13]. When immature DCs encounter and recognize pathogens through PRRs, such as toll-like receptors (TLRs) [14, 15], they take up cell debris from infected cells, upregulate expression of costimulatory molecules, mature into immunostimulatory effector mature DCs and migrate to draining lymph nodes for education of T cells [16, 17]. After arrival in the lymph node, mature DCs provide signals to naïve Th cells: 1) Antigen-specific signal 1, which triggers TCRs by pathogen-derived peptides-MHC-II complex on CD4+ T cells; 2) costimulatory signal 2, which prevents the development of tolerance. The combination of signal 1 and signal 2 results in antigen-specific activation of naïve T cells and development into effector/memory cells. In addition, mature DCs release polarizing cytokines, called signal 3, which determine the phenotype of effector Th cells, including Th1, Th2, Th17 or regulatory T cells [18]. Interleukin-12 family members IL- 12, IL-23 and IL-27 [11, 19] and interferon γ (IFN-γ) are the critical cytokines for promotion of the development of Th-1 cells and cytotoxic T cells to defense against intracellular virus, bacteria and cancer cells [20-22]; IL-4 are critical for Th-2 immune response, which is important for the induction the humoral immune response to eradicate extracellular parasites and bacterial infection [23]; IL-10 [24] and transforming growth factor-β (TGF-β) induce

16 Tregs [25, 26] , while the combination of TGF-β and IL-6 induces Th17 response [27, 28]. Through cross-presentation the mature DC can also provide all signals needed for activation of CD8+ T cells.

17 The immune system in cancer

Cancer immunotherapy has established itself as an effective treatment over the last decade. Unlike more conventional cancer treatment, immunotherapy aims to cure patients by educating the host immune system to initiate antitumor responses and eliminate cancer cells. William Coley was the first person attempting to harness the immune system for cancer treatment in the late 19th century. Coley used a mixture of bacteria, known as “Coley’s toxin”, to treat cancer patients and achieved favorable responses in some types of malignancies [29]. Unfortunately, the early attempt of immunotherapy did not attract oncologists’ attention due to the lack of knowledge about the mechanism involved and the high risk of systemic bacterial infection [30]. With better understanding of the relationship between cancer cells and the immune system, the concept of cancer immunotherapy resurfaced during the last dec- ades and propelled the development of new immunotherapeutic approaches to fight against cancers [29]. Clinical studies, translating basic findings and un- derstandings, have shown the true potential of cancer immunotherapy. An important recognition of this is the 2018 Nobel Prize for Physiology or Medicine awarded to James P Allison and Tasuku Honjo for the discovery and use of immune checkpoint blockade [31]. Cancers are genetic disease caused by genetic mutations and epigenetic modulation, which lead changes of amino acid composition of cellular proteins and facilitate cells immortalization and outgrowth. T cells has the potential to recognize mutated “non-self” or “unnatural” proteins in cancer cells and protect the host [32]. When cancer cells die, they can release their antigenic load, which mostly consists of self-antigens but also mutated or over-expressed tumor-associated antigens (TAAs). These antigens can be captured by APCs, such as DCs. The DCs can get activated and matured, and present the captured antigens on MHC molecules. Then the matured DCs can migrate to draining lymph nodes and prime and activate of T cell recognizing the TAAs. The activated effector T cells then traffic into the circulation and can infiltrate into tumor to kill tumor cells expressing the TAA. During destruction of tumor cells, more TAAs will be released accompanying with inflammatory cytokines secretion, which will further recruit more immune cells into the tumor and activate more DCs, thus promoting the cycle to continue. This cyclic events are known as the cancer-immunity cycle [33] (Figure 2).

Figure 2. The Cancer-Immunity Cycle. The generation of immunity to cancer is a cyclic process that can be divided into seven major steps. Dead cancer cells release tumor antigens that can be taken up and cross-presented by APCs and prime effector T cells (Step 1-3). The antitumor specific T cells will circulate and migrate into tumor sites (step 4-5), recognize tumor cells and lysate them (Step 6-7). The lysed tumor cells will release new antigens and start another round of the cancer-immunity cycle.

Cancer cells have the capability to develop multiple mechanisms to escape immune recognition and promote cancer development and progression. This process is termed cancer immunoediting (Figure 3), which goes through three sequential phases known as elimination, equilibrium and escape. The elimination phase is the protective phase. During the elimination phase, the innate and adaptive immune system work together to detect tumor cells and destroy them before they get too aggressive. The tumor cells may express stress-induced molecules, such as MICA/B (human) or RAE-1 (mouse), damage-associated molecular pattern molecules (DAMPs) such as HMGB1 [34] and type I IFNs [35, 36], which are able to activate resident DCs. These DCs can take up tumor antigens and present them to T cells [37] and induce antitumor immune response, as the cancer immune cycle process mentioned above. The tumor cells may express NKG2D ligands which make them recognized by NK cells, thus inhibit tumor cell proliferation. Other immune cells, such as macrophages (M1), may also contribute in the elimination phase. These immune cells release immunomodulatory cytokines such as IL-12, TNF-α, IL-1 to create a pro-inflammatory microenvironment that facilitate the induction of tumor antigen-specific immune responses [38]. If tumor cells sur-

19 vive through antitumor immune attacks during the elimination phase, they enter the equilibrium phase. In this phase, the tumor cells remain in a dormancy state where they are unable to grow in numbers. The relative balance of immune suppressive cells and immune effector cells in the tumor microenvironment is associated with keeping the equilibrium phase [39]. The tumor cells could acquire immune evasive mutations and become low immunogenic during this phase [38, 40]. Once the tumor cells get the capability to escape immune recognition, these “immune-invisible” tumor cells will enter the escape phase and grow progressively. Tumor cell escape may occur through production of immunosuppressive cytokines such as IL10, TGF-β, upregulation of negative immunoregulatory molecules expression such as IDO and PD-L1, or recruitment of regulatory immune cells such as Tregs and myeloid-derived suppressor cells (MDSCs) to create an immunosuppressive microenvironment at the tumor lesion [41], which further promote tumor growth by inhibiting effector immune attacking.

Figure 3. Cancer immunoediting. Cancer immunoediting is a complex process consisting of three sequential phases: elimination, equilibrium and escape. In the elimination phase, immune response is triggered to destroy tumor cells. If this phase goes to completion, the tumor cells are cleared completely. If a few of transformed cells survive through the elimination phase they may enter into equilibrium phase, where immunity prevents tumor cell out growth and keeps them in functional dormancy. Immunogenic editing of tumor cells also happens during equilibrium phase. If tumor cells become low immunogenic under immune selection pressure, these tumor cells may enter the escape phase with growth beyond the control of the immune system.

20 Oncolytic virus (OV) therapy and Immunogenic cell death (ICD)

Oncolytiv virus (OV) therapy Oncolytic viruses are designed to selectively replicate in tumor cells with min- imal toxicity to healthy cells as malignant cells have features that favors OVs replication. These features involve an immune suppressive environment, dys- functional type I IFN signaling, uncontrolled proliferation and resistant to apoptosis [42]. OVs have shown potential advantages for cancer therapy. One reason is that OVs appear to favor specific replication in and lysis of tumor cells. A second reason is that OVs can elicit antitumor immunity by releasing tumor antigens into tumor microenvironment, which could be cross-presented to T cells by APCs [43]. Moreover, the viral genome could be modified by deleting or limited expression of virulence genes to reduce pathogenicity [44] and/or armed with genes encoding for example cytokines to enhance antitumor activity [45]. Furthermore, OVs are immunogenic cell death (ICD) inducers that could induce cancer cells to release danger signals (DAMPs and PAMPs), such as cell surface translocation of calreticulin (CRT) and release of ATP and HMGB1 into the extracellular compartment. These characteristics have increased the interests of using OVs for cancer treatment. T-VEC, one of the most promising oncolytic virus, was approved by Food and Drug Administration (FDA) in 2015 and European Medicines Agency (EMA) in 2016 for advanced melanoma treatment [46]. T-VEC is developed from a herpes simplex-1 virus (HSV-1) engineered by deleting the ICP34.5 gene for preferential replication in tumor cells and to eliminate neuropatho- genicity and the ICP47 gene to enhance antigen presentation to promote antiviral and anti-tumor immunity, and express granulocyte macrophage colony- stimulating factor (GM-CSF) which recruits APCs to the tumor microenvironment [47, 48]. Recent clinical data have demonstrated that T-VEC treatment can induce antitumor immunity and reverse immunosuppression [49]. How- ever, like other forms of immunotherapy, there are some challenges that need to be considered and addressed for OVs-based immunotherapy, including poor delivery and replication in tumor, and inefficient spreading to distant metastases. Pharmacological complementation might be an option to improve virus spread and distribution [50, 51]. Utility of Histone deacetylase inhibitors

21 (HDIs) has been shown to enhance spread and replication of vesicular stomatitis virus (VSV) in cancer cells and promote tumor-specific immunity in animal models [52]. Another hurdle is the immune response against the OV itself. The antiviral immunity may clear OVs and reduce their therapeutic efficacy [53, 54]. Therefore, the appropriate injection location and timing should be taken into consideration carefully. Local injection of OVs is one strategy to bypass this limitation [55]. Using immunosuppressive drugs such as type-I IFN inhibitors before OVs administration could dampen antiviral response, enhance viral infection and replication in tumor cells and subsequently initiate anti-tumor immune response [56, 57]. Many clinical researches have shown that OVs immunotherapy as a monotherapy has limited overall efficacy for cancer treatment. To broaden effective use of OVs for cancer therapy, combination therapy with other immunotherapies, especially with cell therapy and immune checkpoint inhibitors is a current trend for OV-based cancer immunotherapy [49, 55].

Immunogenic cell death (ICD) Immunogenic cell death (ICD) is a type of cell death that could induce an immune response against antigens derived from dead cells [58]. Tumor cell ICD is characterized by the release of danger signals (DAMPs) that can recruit immune cells to the tumor bed, activate and mature DCs along with engulfment of tumor antigens, and cross-priming of T cells to eliminate tumor cells [59]. There are three key DAMPs featured when characterizing ICD, cellular surface exposure of CRT, and release of ATP and HMGB1. Surface exposed calreticulin (ecto-CRT) act as an “eat me” signal that is important for recognition and phagocytosis by APCs [60]. Extracellular ATP, acts as a “find me” signal, and promote immune cells recruiting to the dying cells [61]. Extracel- lular ATP also could stimulate DC maturation via inflammasome activation [62]. HMGB1 promotes DC maturation and facilitates antigen presentation to T cells [63]. Conventional therapeutic ICD inducer include chemotherapeutic agents (such as Cyclophosphamide and Doxorubicin) and physical reagents (photodynamic therapy (PDT) and radiotherapy) [64]. OVs are also potent inducer of ICD that renders the infected cancer cells highly immunogenic [64]. OVs could lyse tumor cells accompanying with release of DAMPs as for conventional ICD inducers, but also releasing PAMPs. These DAMPs and PAMPs can recruit DCs to the tumor bed and activate and mature them and promote TAA phagocytosis by DCs. After migration to lymph nodes, matured DCs can present peptides from TAAs to T cells to prime a cellular anti-tumor immune response (Figure 4).

Figure 4. Properties of immunogenic cell death (ICD) induced by oncolytic viruses (OVs). OVs infect tumor cells and induce ICD, leading to release of pathogen- associated molecular patterns (PAMPs), such as double-stranded viral RNA and damage-associated molecular patterns (DAMPs), such as CRT, ATP and HMGB1, along with tumor-associated antigens (TAAs). This facilitates DCs recruitment into the tumor bed, uptake of TAA by DCs and cross-presenting TAAs to T cells, result- ing in the triggering of an antitumor immune response.

Oncolytic adenovirus (Ad) is one of the most well-studied OVs used in OV- based immune therapy and one Adenovirus-based OV named HB101 has been approved for head and neck cancer treatment in China [65]. The genome of Ads is well-understood and has a large cloning capacity. The adenoviral genome does not integrate into the targeted cell genome. Ad is a non-enveloped virus and compared with enveloped viruses, which release from intact cells through budding, Ad infects and replicates in targeted cells and eventually destructs cells, which is favorable for oncolysis [66, 67]. hTERT-Ad, an oncolytic adenovirus with replication controlled by the hTERT promoter, has been shown to induce immunogenic apoptosis of hepatocellular carcinoma cells, and trigger antitumor immunity [68]. The oncolytic Delta-24-RGD adenovirus has been shown to induce autophagic cell death of glioma cells [69] and initiate an tumor-specific immune response [70]. When combine oncolytic adenovirus Ad5/3-D24-GMCSF with temozolomide (TMZ), this regimen could also induce autophagy of tumor cells and induce ICD featured with increased ecto-CRT and releasing ATP and HMGB1 into the extracellular tumor microenvironment [71]. This combination therapy in patients with solid tumors could induce anticancer specific T cell response and improve overall survival [71].

23 Semliki Forest virus (SFV) is a promising oncolytic virus for central nervous system (CNS)-related tumor treatment due to its native neurotropism. However, SFV has been only used in experimental therapy in animal models so far [72, 73], and not yet in any clinical trial. The ICD inducing capacity of SFV has not yet been determined. Vaccinia virus (VV) is another agent used for oncolytic viral therapy because of several advantages. One is that VVs have a large genome and the potential of incorporating large transgene without disturbing virus replication. Another advantage is that VVs replicate in cytoplasm which reduce the chance of transgene integration into cell genome and thus reduce the risk of viral in- sertional oncogenesis [74]. VVdd, a vaccinia virus with TK and VGF double gene deletion, induces increased release of HMGB1 and ATP in infected human breast AT3 cells [75]. JX-GFP+/hGM-CSF, an oncolytic vaccinia virus, which is engineered to express GM-CSF, could lyse human melanoma cells accompanying with elevated CRT on cell surface and secretion extracellular HMGB1. It could also activate and mature DCs and CTLs when combined with 5-fluorocytosine (5-FC) [76]. Modified vaccinia virus Ankara (MVA) has been shown to induce autophagy of murine bone marrow-derived dendritic cells (BMDCs) and subsequently activate OVA-specific T cells, accompanying with increasing secretion of extracellular ATP [77].

24 T cell-based immunotherapies

Adoptive T-cells transfer T cell-based immunotherapies are the therapeutic strategies that utilize either tumor-infiltrated or genetically modified T cells into cancer patients to mediate anti-tumor immune responses [78]. Currently, T cell-based immunotherapies are classified into three major types, including adoptive transfer of tumor- infiltrating lymphocytes (TILs), T cells engineered with T cell receptor (TCR- Ts) and chimeric antigen receptors (CAR-Ts) [79]. Dr. S. Rosenberg was the first person that attempted to transfer TILs for cancer treatments in mouse models [79, 80] and human metastatic melanoma [81]. In the standard TIL treatment protocols, TILs are isolated from resected tumors and after expansion ex vivo, infused back into patients after lymphodepletion preconditioning by total body irradiation or chemotherapy such as cyclophosphamide and fludarabine [78]. The preconditioning regimen should create “physical space” by depletion of endogenous lymphocytes and decreasing competition for cytokines such as IL-7 and IL-15 [82]. In addition, it could remove immunosuppressive cells, such as Tregs, to disrupt a suppressive microenvironment and maintain TIL function [83]. TIL therapy has shown promising clinical results especially in melanoma patients [84, 85]. Difficulties of anti-tumor TIL isolation and expansion however limits it’s utility [86]. T cell isolation from the blood of cancer patients opens up for the production of genetically redirected T cells with anti-tumor specificity to bat- tle cancer. The process of TCR- and CAR T-cell therapies includes modification of T lymphocytes from cancer patients ex vivo with a non-viral or viral vector, expansion of the modified T cells and infusion back into patients. However, the mechanisms of tumor antigens recognition are different between these two regiments. TCR-T cells are engineered with a defined TCR to recognize a tumor antigen peptide presented by a specific MHC-I molecule [87], while CAR-T cells utilize antigen recognition fragments from a tumor antigen-specific antibody to bind to antigens on the surface of cancer cells. In terms of advantages of TCR-T cell therapy, firstly, TCR-T cells could recognize a wider range of antigens once including intracellular or cell surface or neo- antigens presented by MHC-I. Secondly, TCR-T cells retain the natural signal transduction pathway of TCRs, and thus could be easily fully activated to initiate cytotoxic activity when meeting tumor cells [88, 89]. A drawback is that

25 TCR-T cell therapy is restricted to a unique MHC-I molecule. Thus, the cancer patient must have an HLA haplotype that includes that very MHC-I to be eli- gible for treatment. Furthermore, tumor cells may also down-regulate surface expression of MHC-I as a mean to avoid T cell recognition.

Chimeric antigen receptor (CAR) T cell therapy CAR molecules are hybrid receptors and typically contain an extracellular antigen-recognition domain, a hinge, a transmembrane domain and one or more intracellular signaling domains [90] (Figure 5). The extracellular-recognition domain is typically a single chain variable fragment (scFv) derived from an antibody, which could recognize and bind to an antigen expressed on the surface of tumor cells. Unlike native or TCR- engineered T-cell, the specificity of CAR T-cell is based upon the targeting capability of scFv and thereby bypassed MHC restriction and avoids problems of MHC-I downregulation. The hinge region, also called as a spacer, is the extracellular domain that connects the extracellular-recognition domain with the transmembrane domain. Majority of CAR molecules are designed to use hinges of immunoglobulin (Ig)-like domain (IgG1, IgG2 or IgG4), CD28 or CD8α. The spacers provide the stability of CAR expression and also keep ad- equate distance to form proper immunological synapse to improve CARs’ activity. The transmembrane domain is the region that anchor the CAR molecule on the T-cell surface. Commonly used transmembrane domains are mainly derived from CD3, CD8α or CD28. Proper combination of intracellular domain with corresponding transmembrane region may improve CAR expression or stability. Incorporating the inducible T cell costimulatory (ICOS) intracellular domain with ICOS transmembrane, enhanced anti-tumor function and persistence of CAR T cells [91]. The intracellular domain is re- sponsible for signal delivery into the CAR T cells. It is classically derived from the zeta chain of CD3 together with signaling transduction domain of costimulatory signal molecules, such as CD28 family (CD28 and ICOS) and tumor necrosis factor receptor (TNFR) family (41BB and OX40). Based on the number of stimulatory signaling molecules, the CAR T cells can be cate- gorized into three generations. The first generation CARs only have CD3 zeta chain (CD3ζ), which can only deliver activation through signal 1 and induce transient T cell activation [92]. One or more costimulatory signal molecules are incorporated in the second or third generation CARs to provide costimulatory signal 2 that are important for enhancing CAR T cells antitumor function, prolonging CAR T cells persistence and promoting proliferation [93]. Furthermore, a fourth generation CARs have developed to co-express inflammatory cytokines, such as IL-12, IL-18, IL-7 or CCL19, or costimulatory ligands, such as CD40-L or 41BB-L, leading to enhanced anti-tumor immunity. Such CAR T-cells have been shown to remodel tumor microenvironment for

26 T cell activation and persistence, as well as recruit and activate other endogenous immune cells to generate an immune response against CAR antigen-negative tumor cells [94-98].

Figure 5. The structure of four generations of CAR. The conventional CAR consists of a binding domain, the light (VL) and heavy (VH) variable fragments of a TAA-specific monoclonal antibody followed by a flexible hinge and a transmembrane domain. The intracellular parts are different between the generations. The first generation CAR only has the CD3-zeta chain (CD3ζ) signal domain. The second and third generation CAR also incorporate one or more co-stimulatory domains (CD28, ICOS, 4-1BB, OX40). The fourth generation CARs are typically based on the second generation CARs but include inducible secretion of an inflammatory cytokine or costimulatory ligands.

In November 2020, 350 clinical trials involved CAR-T cells were ongoing (www.clinicaltrials.gov), mostly in the USA and China. Most of these clinical trials are evaluating CD19-specific CAR T cells in hematological malignancies including chronic lymphocytic leukemia (CLL), B cell lymphoma, and acute lymphoblastic leukemia (ALL) [99]. High rates of complete remission have been demonstrated using CD19 CAR-T cell treatment in the clinics. The success has led to approval of three CD19 CAR-T cell products, Kymriah™ (tisagenlecleucel) and Yescarta™ (axicabtagene ciloleucel) which are approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of ALL and diffuse large B-cell lymphoma (DLBCL) [100-102] and TecartusTM (brexucabtagene autoleucel) which is approved by the FDA for the treatment of relapsed or refractory mantle cell lymphoma (MCL) [103]. Clinical experience has indicated that CD19 CAR-T cell therapy could induce B-cell aplasia, an “on-target-off-tumor” side effect. This can be managed with immunoglobulin infusion treatments [104], especially for children, who have weaker humoral immunity [105]. The main adverse side effects of CAR- T cell therapy are cytokine release syndrome (CRS) and neurotoxicity. CRS is driven by massive release of cytokines from infused CAR T cells upon antigen recognition, like interferon-γ, IL-1 and IL-6 [106, 107]. Toxicity of CRS

27 involves symptoms including fevers, cardiac dysfunction and kidney failure. CRS can be managed by using steroids and/or an IL-6 antagonist (Tociliumab) [108, 109]. Neurotoxicity has also been observed in CD19 CAR-T cell therapy, with symptoms like tremor, seizures and cerebral edema [110]. It is not known exactly how neurotoxicity occurs. Based on observations from patients with neurotoxicity syndrome, there is a hypothesis that neurotoxicity might be mediated by an increased inflammatory cytokine, which lead activation and disruption of vascular endothelium, and an increased blood-brain barrier (BBB) permeability [111, 112]. In most cases where neurotoxicity occurs, the condition usually reversed with only supportive care but in some cases it can be fatal.

CAR-T cell therapy for solid tumor Due to the success of CAR-T cell therapy for hematological disease, research- ers also put a lot of efforts on investigating the possibility of CAR-T cell therapy for solid cancer, such as CAR-T cells targeting epidermal growth factor receptor (EGFR) for glioblastoma, or targeting human epidermal growth factor receptor 2 (HER2) for breast and ovarian cancers, osteosarcomas and glioblastoma multiforme (GBM), or targeting carcinoembryonic antigen (CEA) for advanced liver malignancies [30]. However, the translation of CAR-T cells therapy to solid tumor treatment has not become successful. There are multiple factors affecting this translation and the main problems are 1) identifying a proper target antigen that is uniquely and homogenously expressed on tumor cells, 2) T cell trafficking and infiltration into the tumor bed, and 3) the immunosuppressive tumor microenvironment. It is difficult to select an ideal target for CAR-T cell design. Due to that solid tumor cells are derived from healthy cells, most antigens expressed on tumor cells are also likely to be expressed on normal tissue cells [113]. GD2 for example, is over expressed on neuroblastoma cells, but is also expressed on normal nerve cells in the brain. GD2 CAR T cells can efficiently kill tumor cells but can result in fatal encephalitis [114]. One solution to address this obstacle is to make multi-targeted CAR-T cells, in which co-targeting of two different antigens in needed to activate CAR-T cells to reduce the risk of CAR- T cells killing of normal cells. Combination targeting of HER2 and IL13Rα2 by bispecific CAR-T cells has been shown to be potent in eliminating glioblastoma tumor cells [115]. Different from hematological tumors, solid tumors are more complex with high tumor heterogeneity, therefore it is difficult to find a single target antigen that is expressed by all tumor cells within a tumor. Targeting tumor stem cells might be one way to address tumor heterogeneity [116]. In order to control unwanted toxicity induced by CAR T cells, co-expression of a suicide gene, such as inducible caspase 9 (iC9), truncated EGFR or

28 HSV-TK, has been used to improve the safety issue [117]. The CAR T cells can then be eliminated by addition of the prodrug that is activated by the suicide gene. Even if a good target can be identified, the CAR-T cells need traffic into the tumor site to kill tumor cells. Physical and chemical barriers like stromal cells, low pH, and abnormal vasculature can prevent CAR-T cells from entering the tumor. One strategy to address the “getting into” problem is to express a chemokine receptor to attract CAR-T cells into the tumor. Local secretion of IL-7 and CCL19 by CAR-T cells has showed to improve CAR-T cell infiltration and persistence into tumors [97]. An alternative approach is local delivery of CAR-T cells directly into tumor, which could also limit systemic toxicity. There is a paucity of clinical trials evaluating the local delivery of CAR-T cells into tumors, such as EGFR variant III (EGFRvIII)-targeting CAR T cells and IL13Rα2-targeting CAR T cells for glioblastoma and carcinoembryonic antigen (CEA)-targeting CAR T cells for colorectal adenocarci- noma [118-121]. The immunosuppressive tumor microenvironment is another important issue that needs to be considered. Armored CAR T cells with secretion of cytokines, such as IL-12 and IL-15, have been shown to reshape tumor microenvironment and initiate endogenous immune response to destroy CAR antigen negative tumor cells [122, 123]. Combination with checkpoint inhibitors is another option to enhance CAR-T cell cytotoxicity, prolong their persistence and induce expansion [124-126].

29 Method

Helicobacter pylori Neutrophil Activating Protein (HP- NAP) The thesis focus on investigating whether Helicobacter pylori Neutrophil Ac- tivating Protein (HP-NAP) can be used as an immunostimulatory agent to improve responses to cancer immunotherapy. HP-NAP is a 150kDa dodecameric protein that acts as a major virulence factor of H. pylori infection. It was termed NAP because of its capacity to stimulate neutrophils to produce reactive oxygen radicals [127]. HP-NAP forms a ball-shaped dodecameric multi-protein and each unit contains four helix-bundled subunits (17kDa) [128, 129]. Similar to DNA-binding protein and ferritins, HP-NAP has originally been identified as an iron-binding protein [128]. Every dodecamer of HP-NAP could bind up to 500 atoms of iron and this seems important to keep the dodecamer structure stable [130]. Iron-loaded HP-NAP plays an important role in bacterial protection by binding to bacterial DNA by reducing the oxidative stress [130, 131]. HP-NAP is considered to be a pro-inflammatory molecule, which can induce inflammation in infected tissue and support H. pylori growth by nutrients released from the inflamed tissue [132]. HP-NAP exerts a crucial property in recruitment of polymorphonuclear leucocytes into H. pylori-infected tissue. HP-NAP can upregulate β2 integrins expression on the surface of rolling neutrophils and monocytes in blood ves- sel, which induce adhesion of leukocytes to endothelium and extravasation to infected area [133, 134]. Furthermore, HP-NAP can stimulate PMNs to release cytokines and chemokines, including TNF-α, which can upregulate adhesion molecules V-CAM and I-CAM [135] on endothelial cells, and secretion of IL-8 (also named CXCL8), macrophage inflammatory protein 1 alpha (MIP-1α, also named CCL3) and MIP-1β (also named CCL4) [134]. These cytokines and chemokines can recruit more neutrophils, monocytes and lymphocytes to infected tissue [134]. HP-NAP can induce NF-κB activation through Toll-like receptor-2 (TLR- 2) [136]. As an TLR-2 agonist, HP-NAP stimulates monocytes and DCs to secrete IL-12 and IL-23, which creates an Th1-rich environment [136]. HP- NAP can also induce monocyte and DC activation and maturation by upregu- lating expression of CD80, CD86 and HLA-DR [136]. Co-culturing T cells with HP-NAP has been shown to induce a remarkable increase in the number

30 of IFN-γ secreting T cells and decrease IL-4 producing T cells, which is in line with a significant Th1-type immune response [136]. T helper responses to allergens usually result in Th2 polarization. For example, mite allergen- specific Th cells mainly consist of Th2 cells (89%), while Th1 and Th0 cells only accounted for 1% and 10%. However, when adding HP-NAP, a significant shift from Th2 to Th1 response was observed, inducing increased number of Th1 cells (38%) and Th0 cells (33%) and decreased number of Th2 cells (29%). Also, these HP-NAP stimulated allergen-specific Th cells showed strong cytolytic activity [136]. Due to the immune properties and multi-dimensional immune stimulatory capacity of HP-NAP, it has been investigated for clinical use, such as for diagnosis of H. polyri infection, vaccine development to prevent H. polyri infection and drug development to protect against H. polyri infection or other diseases. To diagnose H. polyri infection, an ELISA assay based on recombinant HP-NAP has been developed and evaluated to detect HP-NAP specific antibodies in H. polyri infected patients [133, 137]. HP-NAP can be also used as a biomarker for prediction of the outcome of H. polyri infected patients [138, 139]. For vaccine development, it is an attractive vaccine target candidate for prevention of H. polyri infection. Prophylactic vaccination based on recombinant HP-NAP can induce high levels of HP-NAP-specific antibodies and T cells and protect mice from H. polyri infection [133, 140, 141]. Addi- tionally, HP-NAP can serve as an immunoadjuvant to enhance the immunogenicity of some poor immunogens when expressed concurrently [142]. Due to that HP-NAP has been shown to induce Th1 response and inhibit Th2 response, this protein is an attractive candidate to boost cancer immunotherapy. Co-expression of HP-NAP by oncolytic viruses (adenovirus and measles) has been documented to induced Th1 response and exhibited antitumor activity [143, 144].

31 Present Investigations

Paper I Aims: Immunogenic cell death (ICD) is the form of cell death that can induce an immune response through activation of dendritic cells (DCs) and adaptive immune cells. Inducers of ICD are characterized by their ability to stimulate the release of damage-associated molecular patterns (DAMPs) from dying cells, such as extracellular ATP (“find-me” signal), cell surface exposure of calreticulin (CRT) (“eat-me” signal), and release of high mobility group box 1 protein (HMGB1) (activation signal for immune cells). Oncolytic viruses are ICD inducer that can trigger antitumor immune response. We aimed to investigate the cell death properties of wild type Adenovirus (Ad), Semliki Forest virus (SFV) and Vaccinia virus (VV) and characterize their ability to induce ICD. Major findings: We found that Ad primarily induce autophagy of tumor cells, SFV activate immunogenic apoptosis while VV mainly activate necrop- tosis. All three virus could induce the release of DAMPs from dying tumor cells including CRT, ATP, HMGB1 and HSP90, and trigger phagocytosis and maturation of DCs. However, only SFV-infected tumor cells were able to induce DCs to secrete Th1 cytokines and initiated antigen-specific T cell activation in vitro.

Paper II Aims: Oncolytic viruses (OVs) are promising therapeutic agents for cancer treatment by tumor selective lysis and induction of anti-tumor immunity. En- gineering OVs with immune-modulating agents are used to provoke stronger antitumor immunity. The neutrophil-activating protein (NAP) from Helico- bacter pylori is a toll-like receptor (TLR)-2 agonist that can act as chemoat- tractant and activator of immune cells, such as neutrophils, monocytes, and dendritic cells (DCs). NAP has the potential to drive Th1 polarization by creating an IL-12 and IL-23 enriched milieu. We aimed to analyze whether arming oncolytic GD2-expressing Vaccinia virus (VV) and Semliki Forest virus (SFV) with the immune stimulatory NAP can further enhance their anti-tumor immunity.

32 Major findings: We found in the mouse NXS2 neuroblastoma tumor model that arming oncolytic SFV-GD2m with NAP (SFV-GD2m-NAP) did not improve therapeutic efficacy, compared to unarmed SFV-GD2m. How- ever, NAP boosted an anti-SFV antibody response. On the other hand, arming oncolytic VV-GD2m with NAP (VV-GD2m-NAP) could significantly reduce tumor growth and prolonger mice survival in comparison to unarmed VV- GD2m. This induced a higher anti-GD2 antibody response. These data suggest that arming OVs with immunomodulating factor needs careful and critical evaluation.

Paper III Aims: Chimeric antigen receptor (CAR)-T cell therapy has received remarkable successes in treatment of hematological malignancies. However, treatment of solid tumors with CAR-T cells has met multiple obstacles. It is mainly due to the immunosuppressive microenvironment in tumor beds and the lack of ideal tumor-associated antigen that is homogenously expressed on the surface of tumor cells. Many strategies and approached have been tried to overcome these obstacles, including arming CAR-T cells with cytokines/chemokines and combination of CAR-T cells therapy with other treatments. Our pre- vious findings showed that NAP, a major virulence factor for Helicobacter pylori, can activate and mature DCs and induce Th1-polarization, which co- operate to induce antitumor T cell immunity. We therefore armed CAR-T cells with inducible secretion of NAP and evaluated whether NAP could enhance the antitumor capacity of CAR-T cells for solid tumors. Major findings: We observed that NAP secreted from CAR(NAP)-T cells upon target antigen recognition promoted DC maturation and Th-1 polarization, which in return, enhanced the cytolytic capacity of the CAR(NAP)-T cells. CAR(NAP)-T cells could successfully reduce/delay tumor growth, and promote survival of mice compare with conventional CAR-T cells in various murine tumor models. In NXS2-mCD19 tumor model, secreted NAP created an immunologically hot microenvironment, enhancing infiltration of immune cells (DCs, neutrophils, macrophages, and cytotoxic NK cells), and higher levels of Th1-related cytokines and chemokines. Treatment with CAR(NAP)- T cells induced epitope spreading and initiated an endogenous T cell response that recognized and kill also “CAR-antigen-negative” tumor cells. In ovalbu- min-modified (a non-targeted bystander antigen) tumor models (NXS2- mCD19-OVA and Panc02-mCD19-OVA), CAR(NAP)-T cells yielded significantly more OVA-specific CD8+ T cells. And these T cells exhibited activated (CD69+CD107a+), high proliferative (Ki67+) and memory-like phenotype (CD62L+CD44+CD127+). In a heterogeneous tumor model (a mixture of CAR antigen negative and positive tumor cells), CAR(NAP)-T cells significantly reduced tumor growth and prolonged mouse survival.

33 Paper IV Aims: Chimeric antigen receptor (CAR)-T cell therapy targeting CD19 has received remarkable success in the treatment of hematological malignancies. However, numerous clinical trials have shown that some patients relapse after CD19 CAR-T-cell treatment, and that the recurrent cancers can display downregulation or complete absence of CD19 (the CAR-target antigen). We aimed to develop CAR-T cell targeting CD20 (rituCD20 CAR-T cells) and pre-clin- ically evaluate their cytotoxic capacity on tumor cell lines and patient-derived mantle cell lymphoma (MCL) cells. If effective rituCD20 CAR-T cells may be used for patients relapsing with CD19-negative tumors after CD19 CAR-T cell therapy. Regarding of a risk of antigen (CD20)-loss after rituCD20 CAR- T cells treatment, we also aim to arm rituCD20 CAR T cells with NAP to induce bystander immune response against other tumor-associated antigens. Major findings: For CD19 and CD20 double-positive lymphoma cell lines (U2932, Karpas422, DB, U698, Raji, Daudi), we found rituCD20 CAR-T cells showed similar cytotoxic capacity and interferon gamma (IFNγ) secretion as CD19 CAR-T cells. rituCD20 CAR-T cells could efficiently kill primary mantle cell lymphoma cells (CD20+ MCL) accompanied by IFNγ secretion. In murine lymphoma A20-hCD20 model, which stably expressed human CD20, both rituCD20 CAR-T cell and NAP-armed rituCD20 CAR-T cell treatment delayed tumor growth and prolonger mice survival compared to Mock-T cell- treated mice.

34 Svensk sammanfattning

Immunterapi har utvecklats snabbt och etablerat sig som en ny behandlings- form av cancer som i många fall leder till komplett bot även där andra behand- lingsformer inte längre fungerar. Syftet med immunterapi är att aktivera krop- pens försvarssystem (immunförsvaret) att känna igen och döda tumörer. Så kallade ”checkpointhämmare”, vilka ofta beskrivs som blockerare av T cellers broms har visat sig ytterst framgångsrika vid behandling av vissa cancerfor- mer, likaså har behandling med genetisk modifierade T celler, så kallade CAR-T celler nått stor framgång vid behandling av hematologisk cancer. Onkolytiska virus och cancervacciner är också under stark utveckling. Den största andelen cancerpatienter drar dock tyvärr ännu inte nytta av utvecklade immunterapier och strategier för att ytterligare förbättra behandlingarna efter- strävas.

En virulensfaktor från Helicobacter Pylori som benämns NAP är starkt im- munaktiverande och skulle därför kunna användas för att ytterligare öka immunsvaret mot cancer. NAP har visat sig kunna aktivera dendritiska celler och få dem att inducera ett starkt så kallat T-hjälpar typ 1 (Th1) svar, vilket är den typ av immunsvar man vill uppnå vid immunterapi. I min doktorsavhandling har jag undersökt om onkolytiska virus och CAR-T celler kan förstärkas ge- nom att bära med sig genen som kodar för NAP.

Delarbete I och II avhandlar onkolytiska virus. I delarbete I analyserar jag i detalj den tumörcellsdöd och det immunsvar som orsakas av Adenovirus (Ad), Semliki Forest virus (SFV) och Vaccinia virus (VV). Samtliga virus visade sig ha förmåga att aktivera immunsystemet men det är enbart SFV som indu- cerar ett Th1 svar med frisättande av cytokiner och aktivering av dendritiska celler och genererande av ett antigenspecifik T-cellssvar. I Paper II modifierar jag VV och SFV att uttrycka ett tumörantigen tillsammans med NAP och un- dersöker deras terapeutiska effekt i in vivo. NAP genererade ett distinkt immunsvar och terapeutiska fördelar för VV som ledde det till en signifikant förhöjd produktion av antikroppar mot tumörantigenet. Slutsatsen är att vid modifiering av SFV, sett virus som är mycket immunogent, bör man fokusera på att förbättra den onkolytiska effekten som leder till tumörcellsdöd och fri- släppande av tumörantigen från tumörcellerna medan för VV, ett virus med

35 immunhämmande egenskaper, bör man fokus på att beväpna viruset med im- munmodulering, exempelvis i form av NAP, för att förbättra immunsvaret mot tumören.

Delarbete III och IV avhandlar CAR-T celler. I delarbete III genererades CAR-T-celler med inducerbar utsöndring av NAP. Dessa CAR (NAP)-T celler var betydligt bättre än konventionella CAR-T celler att bekämpa tumörtill- växt och förlänga överlevnad hos möss i flertalet solida tumörmodeller. Ut- söndrandet av NAP skapade en immunologiskt ”het” tumörmikromiljö med förbättrad infiltration av immunceller vilket ledde till en aktivering av kropps- egna T celler som kunde attackera tumören, inklusive tumörceller som sak- nade det antigen som CAR-T cellerna riktade sig mot. I delarbete IV utvecklade vi CAR-T celler riktade mot CD20, vilket är ett antigen som uttrycks av lymfom. Vi fann att CD20-riktade CAR T-celler effektivt kunde döda CD20- positiva lymfomcellinjer samt primära tumörceller från lymfompatienter. Både CD20-riktade CAR-T-celler och CD20-riktade CAR(NAP)-T celler kunde bromsa tumörtillväxt och förlängde överlevnad hos möss med lymfom.

Min avhandling visar att onkolytiska virus och CAR-T-celler utrustade att bära med den starkt immunmodulerande molekylen NAP är lovande för att maximera effekten av dessa former av immunterapi för att bekämpa cancer.

36 Future Perspectives

Immunogetic cell death indced by oncolytic viruses Immunogenic cell death (ICD) is a cell death modality that can induce antitumor immune response. Oncolytic viruses (OVs) can, besides infecting and lysing tumor cells and releasing TAAs, induce ICD accompanied with release of DAMPs that further enhance anti-tumor immunity. The golden standard to define ICD is based on its ability to induce protective immunity in a prophylactic setting in vivo. Only if mice get protected from wild-type tumor cells challenging after vaccination, the cell death modality is defined as bona fide ICD [64, 145]. We have shown that Ad5, SFV, and VV can induce the release of DAMPs including CRT, ATP, HMGB1 and HSP90 from dying cells but that only SFV infected tumor cells induced release of Th1-related cytokines from DCs and initiated antigen-specific T cell activation in vitro. We are now evaluating SFV-mediated ICD in different murine models, and investigate the pathways involved in immunological consequence induced by SFV-mediated ICD. Through dissecting the mechanism of viral oncolysis mediated ICD, we aim to provide a better rationale for future OV engineering.

CAR(NAP)-T cell therapy in the clinic Our data from mouse models show that CAR(NAP)-T cells are promising for cancer treatment. Our results indicate that CAR(NAP)-T cells can induce endogenous T cells that recognize and inhibit “CAR-antigen-negative” tumor cell growth in vivo. We next would like to exam the CAR(NAP)-T cells in human patients to evaluate its toxicity and efficacy profile. We have engineered CD20CAR (NAP) T cells and evaluate these cells in immunocompetent murine model. We are developing protocols for human CD20 CAR(NAP)-T cells to be used for treatment patients with lymphoma, and we are currently evaluating of every step involved in engineering of human CAR- T cells, including comparison of different viral delivery systems and different ways to activate and expand CAR-T cells.

37 OVs/CAR-T cell combination therapy for solid tumor CAR-T cell therapy has received remarkable success in hematological malig- nacies. However, for solid tumor CAR-T cell infiltration, an immunosuppressive tumor microenvironment, and antigen heterogeneity are challenges that must be overcome. Thus, combination therapies that can address these limita- tions are needed to improve performance of CAR-T cells in solid tumor. OVs could be excellent partners to synergize with CAR-T cell therapy. As de- scribed above, OVs can kill tumor cells and through ICD modulate tumor microenvironment from immunologically ‘cold’ to immunologically ‘hot’, which favor infiltration and activity of CAR-T cells. We have seen that SFV infection is able to generate a Th1 immune microenvironment. We are inter- ested to evaluate the safety and efficacy of combination of CAR(NAP)-T cells with SFV infection for solid tumor treatment.

38 Acknowledgements

The studies presented in this thesis have been carried out at the Department of Immunology, Genetics and Pathology at Rudbeck Laboratory, Uppsala Uni- versity. This work had financial support from the Swedish Cancer Foundation, the Swedish Research Council, the Swedish Children Cancer Foundation and the Chinese Scholarship Council.

I would like to take this opportunity to express my sincere gratitude and ap- preciation to all the people who have helped me during my PhD studies.

I would like to first thank my supervisor, Di Yu. You always come out with so many amazing ideas and are able to point out the main direction and potential problem for every project. Every time when I met problems, your door is always open and you are always there. You are really like a big brother to me and I have learned a lot of knowledge and experience from you both in science and life. You have been a real amazing supervisor.

I also would like to thank my co-supervisor Magnus Essand for giving me the opportunity to start my PhD study in your amazing group. Also, many thanks for your patient guidance and great ideas for every project.

Thanks to my other co-supervisors Mohanraj Ramachandran and Mia Phil- lipson. Mohanraj Ramachandran, you are always there to help me when I meet any problem and import a lot of ideas in our projects. You are so good at digging so many cool fantastic techniques. Mia Phillipson, you are always there when I need you.

Many big thanks to Chuan Jin, we have spent so much time together to design and perform experiments and analyze data. You are like my sister and I can share everything in my life with you. I have got so much support and trust from you. Grammatiki Fotaki, Kiki, thank you for all the nice discussion and all your support. You are such an inspiring friend and I miss you and the amazing office time with you so much. Also thank you to take care of my Meatball. My dear Tina Sarén, you are an awesome friend. Thank you for creating such nice atmosphere in our office. You are always there and let me know who I can rely on. I am looking forward to our future collaboration in our new amazing CAR-T project. Also, thanks for talking

39 with CiCi whenever she needs you. Good luck with your PhD. Tiarne van de Walle, thanks to share information about Rhapsody and also thanks for your help on my big sample isolation day. You saved me so that I could pick up Cici on time.

Miika Martikainen, you are so knowledgeable, especially in the virus field. Thank you for all the nice talks and collaboration, especially in our amazing ICD projects. You give me so much support in these projects. Minttu-Maria Martikainen, thanks for all your help in taking care of my mouse experiment, especially during my vacation. I would never manage so many mice for i.c injections without you. You are a good collaborator and I really enjoy the time we are together as well as our gosspy in the animal facility. Bertin Paranjothi Mary, welcome to our group. Trust me, you will enjoy the time in this group and love it.

Thanks also to current and former master students: Arwa Ali, Alexandros Iskantar, Giulia Saronio, Aishwarya Sivaramakrishnan, Menghan Gao and Xu Zhu. I would like to extend my gratitude to all current and former GIGers: Angelica Loskog, Sara Mangsbo, Berith Nilsson, Emma Eriks- son, Tanja Lövgren, Jessica Wenthe, Ann-Charlotte Hellström, Alireza Labani Motlagh, Mohamed Eltahir, Iliana Kyriaki Kerzeli, Sedigheh Naseri, and anyone else I am missing here. Thanks for creating so nice work- ing atmosphere and also for interesting data clubs. Special thanks to Anna- Karin Olsson, Maria Ulvmar and Anna Dimberg for discussions during our TME seminar and to all group members of Anna’s group: Roberta Lu- gano, Hua Huang, Kalyani Vemuri, Alessandra Vaccaro, Maria Geor- ganaki, Luuk Van Hooren, it is so nice to have our common TME meeting wih all of you. I have gained a lot of knowleadge from you as well as great ideas and comments for my work. Special thanks to Alessandra, thank you so much for giving me nice introduction to cell dissociator and vibratom. Also thank you so much for your help on that big day.

I also would like to thank my chinese friends in Uppsala, Yuan Xie, Yinghua Zha, Su-chen Li, Hua Huang, Ren Sun, Shengnan Dong, Yan Zhang, Lang Cheng, Anqi Xiong, Kun Wei, Rui Miao, Lei Chen, Lei Zhang, Xi Chen, Le Fu, Bo Tian and anyone else I am missing here, you have supported me so much. Thanks for making my life in Sweden so fun and memorable. Yuan and Kun, you are really good neighbor and thanks for your help during my last pregnant stage. 源儿，好想你哦！怀念源的有求必应。得空去西安看你。琨儿，感谢你分享各种经验给我，还有时不时的投喂。 Ying- huan 花儿, thank you for being so kind to me all the time and our wonderful trip to Norway with Yuan. Take care of yourself and your family in Stockholm. 怀念在 Flogsta 的时光，十分感谢你当时的陪伴和容忍我的小脾气。十分感谢你的信任。 My sister Su chen, you always shared home-made food

40 with me and cici, feed us fatter and fatter 多谢姐姐，总是关心我们“孤儿寡母”，总是分享漂亮的衣服给豆子，她非常非常的喜欢。Yan and Cheng, thanks to help me take care of cici when I need to work during week- end. I really enjoy the time of taking care of our girls together. Trust me, Joe is the sweetest girl in the world. 非常开心能认识你们俩口还有可爱的乔乔，你们陪我度过的艰难的独自带娃的日子。希望两个小宝贝能做最好的 “小闺蜜”，开心健康地长大。Hua, thanks for your patience to teaching me perfusing mice and using vibratome. 华，我们可以继续买买买哦！！！

还有多年陪伴我的国内的朋友们，虽然我们不能时长见面，但是你们总是不顾时差的陪伴，让我在异国他乡的生活没有那么孤单。尤其是珊儿，璐儿和小晶晶，一切尽在不言中，总之，有你们真好！

Lastly, I would give my biggest thank to my whole family. My sweet sister, Xue, thanks to help me take care of my parents, you are more like their daugh- ter girl than me. 老姐，多谢你的有求必应。多谢你帮我照顾我爸妈，帮我解决最大的后顾之忧。Thanks to my dear mom and dad, I know you so not expect me to study abroad, however, you allow me to do what I want to do. Thanks for you understand and support my study aboard, living so far away from you. I need give big thanks to my mom for taking care of cici. 多谢爸爸妈妈多年来的理解和支持，给我无限的自由去做各种决定。你们是我最最坚强的后盾。Thanks for my sweet baby girl, Cecilia, you are the most successful “project” during my PhD life and you make my life more wonderful and sunnier. Peng, my dear husband, thanks a lot for your unlimited support and understanding for all my life.

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49 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1703 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-425970 2020