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Glioblastoma (GBM) is the most frequent and lethal malignant brain tumor in adults. It emerges with an incidence of 3.2 per 100.000 in the US and 3.91 in Europe. Today, standard treatment after diagnosis consists of surgical removal of tumor tissue, followed by radiation therapy and adjuvant chemotherapy using temozolomide. Even after this rigorous therapy, patients show a median overall survival of only 15.6 months or 20.5 months when the tumor is additionally treated with so-called tumor treating fields. GBM is characterized by molecular heterogeneity within the same patient but also between different patients, which impedes development of novel therapeutics. During the last decades various immunotherapies including (multi-epitope) peptide vaccines, oncolytic viruses or immune checkpoint inhibitors against GBM were tested in small clinical studies, but failed to show a benefit in large studies. A novel kind of immunotherapies that showed great success in hematological tumors so far, is based on chimeric antigen receptors (CAR). These synthetic receptors can be introduced into immune cells to retarget their function towards tumor cells, independently of the major histocompatibility complex (MHC) that is often down regulated by tumors for immune evasion. A large hurdle for treatment of GBM using immunotherapies such as CAR-T cells, is antigen heterogeneity that limits the effect of therapies against single targets and renders the need for discovery of novel targets to enable treatment of a wide variety of patients with high success. Analyzing publicly available data and performing RT-qPCR experiments with RNA isolated from GBM tissue of a local cohort of patients, overexpression of two candidate GBM antigens, namely TMEM158 and PTPRZ1 were observed. Overexpression of both antigens in GBM in comparison to normal brain tissue and low-grade gliomas (only TMEM158) was revealed. In addition, a negative correlation between expression and patient survival was detected, as well as a correlation between TMEM158 and CD44 expression, the latter being a marker for GBM stem cells and the mesenchymal GBM subtype. Induction of chemoresistance by TMEM158 seems likely for GBM, since this was already discovered for several other tumor entities. Protein expression of TMEM158 was confirmed by Western blot analysis of different GBM cell lines. Since cell surface expression of a target protein is a prerequisite for targeting by a CAR-therapy, the expression of TMEM158 on cells from GBM cell lines was analyzed by flow cytometry. For this analysis a fluorescence-labeled peptide, based on sequence information of a known naturally occurring TMEM158 ligand (BINP) was designed. Binding to T98G and U-87 MG was observed, while only very low binding to the neuroblastoma cell line SH-SY5Y was seen in flow cytometry. Partial knockdown of TMEM158 was achieved using DsiRNAs, followed by Western blot (antibody staining) and flow cytometry (peptide staining), confirming the specificity of binding detectable by both methods. A recombinant fusion protein, consisting of the extracellular part of TMEM158 and a human Fc-antibody fragment was produced in 293T cells by transient transfection of an expression vector. The expected size of the protein produced was confirmed by Western blot. Furthermore, binding of the BINP-peptide to the recombinant protein was analyzed and compared to a scrambled BINP-peptide. In these experiments specific binding of the BINP-peptide was observed, also indicating the functionality of the recombinant protein. Next, CAR-constructs were designed using the original sequence information from BINP as binding domain and additional variants with amino acid exchanges at different positions. Significant cytotoxicity of all BINP-CAR-T cells was observed against T98G, which showed highest binding of BINP when analyzed by flow cytometry. A BINP-CAR version in which phenylalanine 11 was exchanged with alanine (BINP-F11A-CAR) showed significantly higher cytotoxicity against T98G than the BINP-CAR containing the original BINP sequence (BINP-WT-CAR). Against the U-87 MG cell line, only a version of the BINP-CAR containing an RGD- (arginine-glycine-aspartic acid) motif showed significant cytotoxicity. RGD-motifs are known to bind integrins like αVβ3, which was abundantly present on this cell line, as it was confirmed by flow cytometry within this work. Using this BINP-RGD-CAR version, targeting of both antigens at the same time seems possible. No significant cytotoxicity of the different CAR versions was observed against the TMEM158- and αVβ3-low cell line SH-SY5Y. In conclusion, overexpression of TMEM158 and PTPRZ1 and their negative influence on survival of patients, as found in recent literature, was confirmed for glioblastoma. Significantly higher expression of TMEM158 in GBM in comparison to low-grade gliomas as well as the correlation with CD44 hint at an association of TMEM158 with the aggressive phenotype of GBM. For all of these reasons, targeting of TMEM158 appears to be very feasible. Cytotoxicity of the produced BINP-CAR-T cells, which are the first CAR-T cells targeting TMEM158 so far, was demonstrated against GBM cells. Additional to cytotoxicity of the CAR-T cells, other in vitro assays and in vivo models should be utilized to determine more aspects of CAR-T cell function, in the future. For example, proliferation, cytokine release, invasion of tumor tissue, and inactivation of CAR-T cells by the tumor milieu should be quantified. To estimate how many patients could benefit from a therapy against it, percentage of patients and distribution within the tumors should be determined.

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

Yes / Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 106 cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis. / Replicel Life Sciences Inc, Vancouver, Canada.

Chronisches Nierenversagen kann zu einem endgültigen Funktionsverlust der Nieren führen. Nierenersatztherapien bleiben dabei die einzigen lebensrettenden Behandlungsmaßnahmen. Der globale Bedarf an Nierenorganen übersteigt jedoch bei weitem die Anzahl verfügbarer Spenderorgane. Humane induzierte pluripotente Stammzellen (hiPSC) stellen eine vielversprechende Quelle für die Generierung funktioneller Nierenzellen dar. Die hiPSC-abgeleiteten Nierenzellen könnten zukünftig für zelluläre Ersatztherapien verwendet werden. Innerhalb dieser Arbeit sollten die aus hiPSC-abgeleiteten Nierenzellen auf ihre Immunogenität überprüft werden. Dafür wurden hiPSC-Linien, reprogrammiert aus primären Urinzellen gesunder Spender, generiert. Die hiPSC wurden daraufhin in renale Vorläuferzellen (IM-Zellen) und proximale Tubuluszellen (PT-Zellen) differenziert. Die immunphänotypische Analyse ergab eine generell verminderte Expression immun-relevanter Gene verglichen zu adulten Urinzellen. Für die Immunigenitätsbestimmung wurden die Nierenzellen jeweils mit autologen oder allogenen Zellen des peripheren Blutes (PBMCs) kokultiviert. Die IM- sowie PT-Zell-Kokulturen mit PBMC gesunder Spender sowie von Patienten mit diabetischer Nephropathie ergaben keine Proliferation autoreaktiver sowie allogener T-Zellen, im Vergleich zu primäre Urinzellen mit gleichen HLA-Merkmalen. Des Weiteren wiesen IM- und PT-Zellen aktive immunsupprimierende Eigenschaften auf. Diese konnten in langzeit-kultivierten PT-Zellen nicht mehr beobachtet werden. Zusätzlich zeigten die hiPSC-abgeleiteten Nierenzellen eine Anfälligkeit gegenüber autologe sowie allogene natürliche Killerzellen (NK-Zellen). Diese Studie ergab erstmalig Einblicke in die Dynamiken der T- und NK-Zell-Antworten renal differenzierter hiPSC. Die hier gewonnenen Erkenntnisse können für die Entwicklung zukünftiger Therapien beitragen, um eine sichere Transplantation von hiPSC-abgeleiteten Nierenzellen, selbst im allogenen System, zu ermöglichen. / Kidneys are essential for numerous vital processes. Chronic damage can lead to end stage renal disease with the requirement of kidney replacement therapies. Kidneys are nowadays the most frequently transplanted human organs, however, the transplant demand is far exceeding the number of available donations. Human induced pluripotent stem cells (hiPSC) represent a promising alternative approach for the generation of functional renal differentiated cells suitable for autologous cell replacement therapies (CRT). Yet, the immunogenic potential of hiPSC and their progenies are among the major concerns. This study aimed to analyze immunogenic effects of hiPSC-derived renal cells in autologous and allogeneic settings in vitro. Therefor, primary urinary cells (pUC) from healthy donors were reprogrammed into hiPSC, which were differentiated into renal progenitors (IMC) and into proximal tubular cells (PTC). Immune-phenotypic characterization revealed overall reduced expression of immune-relevant genes in hiPSC and renal derivatives compared to pUC. Co-culture experiments of IMC or PTC with either autologous or allogeneic peripheral blood mononuclear cells of healthy donors or patients with diabetic nephropathy revealed no induction of T cell proliferation in comparison to pUC cells with the same HLA-types. IMC and PTC showed immunomodulatory effects on allogeneic T cell proliferation. These immunosuppressive capacities were lost in long-term cultivated PTC. hiPSC-derived renal cells showed to elicit autologous as well as for allogeneic natural killer cells (NK cells). This study provided insights about the immunogenic properties of hiPSC-derived renal cells. In vitro experiments revealed new hints about the dynamics of T- and NK cell responses dependent on the differentiation status of hiPSC-derived renal cells. These data show translational potential for the development of future CRT strategies aiming safe transplantation of hiPSC-derived renal cells across HLA barriers.