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The adoptive transfer of cancer-specific T-cells has demonstrated success as a novel treatment strategy in some hematological malignancies but this approach has not yet achieved widespread curative potential in the majority of tumors. To circumvent many of the limitations currently facing adoptive cell therapies, our lab has recently developed a combination therapy involving the in vivo boosting of adoptively transferred tumor-specific memory T-cells with an oncolytic viral vaccine. While this represents a demonstrably powerful approach in preclinical models of cancer it is limited by its targeting of a single antigen. Therapeutic resistance is a common concern when targeting a single antigen or pathway and an ideal therapy would include built-in mechanisms to address the heterogeneity and mutability that is inherent to cancer. Thus the focus of this research involved the development of a strategy to target therapeutic resistance in the context of the adoptive cell transfer with oncolytic viral boost regimen. In order to address the single antigen limitations, the engineering of tumor-specific T-cells with a targeting capacity for a second antigen is described. In addition to their endogenous tumor target it is shown that these cells have specificity for and can kill cells expressing ligands for the natural killer group 2 member D receptor which are commonly upregulated on both cancer cells and components of the tumor microenvironment. Indeed it is demonstrated in an in vivo model of relapse that T-cells capable of targeting both antigens produce more consistent and prolonged remissions than those with only their endogenous targeting capacity. Furthermore pharmacological strategies for the enhancement of engineered T cell survival and efficacy are also described. Finally the early development of a chimeric tumor model to further characterize the potential of dual-specific T-cells to address tumor heterogeneity is presented. / Thesis / Master of Science (MSc)

Background: Despite the clinical severity and rising prevalence of peanut allergy, there is a marked absence of widespread, practical treatments available for peanut-allergic patients. Peptide immunotherapy, a disease-modifying treatment that uses short peptides recognized by T cells, has been shown to reduce allergic symptoms of allergic rhinoconjunctivitis. This project investigated the ability of peptides from the major peanut allergen Ara h 1 to protect against peanut-induced anaphylaxis and induce immunomodulatory changes in a mouse model. Methods: Mice transgenic for the human leukocyte antigen DRB1*0401 were sensitized to peanut epicutaneously and treated with two intraperitoneal injections of peptides from Ara h 1. Mice were then challenged with intraperitoneal whole peanut and observed for signs of anaphylaxis. Flow cytometry was used to isolate peanut-specific CD4+ T cells labelled with Ara h 1 peptide-loaded tetramers and additional Th1, Th2, and regulatory markers. Results: Peptide-treated mice were protected from severe peanut-induced anaphylaxis. Control mice treated with a sham peptide experienced a mean maximum temperature drop of 3.2°C, while mice treated with Ara h 1 peptides experienced a drop of 1.6°C (p=0.067 vs control). Maximum clinical score was 2.5 in control mice, and 1.4 in treated mice (p=0.0097). Mean hematocrit for control mice was 52.5%, and 47% for treated mice (p=0.013). PD-1+CD4+ T cells were significantly increased in the mesenteric lymph nodes (p = 2.28e-0.05) and spleens (p = 0.014) of peptide-treated mice. MIP1-a+CD4+ T cells were significantly decreased in the peritoneal lavage (p = 0.008). Conclusion: Ara h 1 peptide immunotherapy protected against severe peanut-induced anaphylaxis in a mouse model. Peptide-treated mice experienced significantly reduced drops in core body temperature, clinical signs of allergic reaction, and hemoconcentration. Clinical protection was associated with decreased expression of the pro-inflammatory chemokine macrophage 1-a and increased expression of the surface marker programmed cell death protein 1. / Thesis / Doctor of Philosophy (PhD) / Peanut allergy is a growing public health concern. Its prevalence has doubled in the past 10 years and currently stands at 2%. Reactions to peanut account for the majority of food-induced fatal allergic reactions, termed anaphylaxis. Currently, there are no treatments available for patients with peanut allergy. Healthcare workers can only offer peanut-allergic patients advice on peanut avoidance and rescue medications in case of accidental ingestion. This research project investigated the ability of a new treatment called peptide immunotherapy to prevent severe allergic reactions to peanut in a mouse model of peanut allergy. Peptide treatment uses small portions of the peanut allergen to shift the immune response from pro-inflammatory to anti-inflammatory. After peptide treatment, peanut-allergic mice were protected from severe allergic reactions in response to peanut and their immune cells produced lower levels of pro- inflammatory molecules.

Recent developments in the expansion and manipulation of primary NK cells has allowed this source of effective anti-tumour cells to be exploited for cell-based cancer immunotherapies. While ex vivo expanded primary NK cells are highly effective in the treatment of haematological malignancies, their efficacy against the solid tumour has been limited due to the presence of immune-regulatory factors in the tumour microenvironment. These factors can abrogate NK cell function by down regulating the expression of NK activating receptors, thus preventing these highly cytotoxic effector cells from activating in response to tumour challenge. Our work explores whether the expression of a tumour specific chimeric antigen receptor (CAR) on ex vivo expanded primary NK cells would allow the lost activatory signalling to be recouped, and regain their efficacy against the solid tumour. Unfortunately, the use of primary NK cells as effectors in CAR based cell immunotherapies has been hampered by the technical limitations of producing large numbers of CAR positive primary NK cells. This has led many researchers to utilise the NK-92 cell line instead of primary cells. We demonstrate that ex vivo expanded primary CAR NK cells can be produced efficiently and demonstrate higher anti-tumour functionality than CAR NK-92. Finally, due to the intricacies of NK cell biology, they are able to effectively discriminate between healthy and malignant targets thus preventing their cytotoxic function from being directed towards the incorrect target. This could be a key advantage in the use of primary NK cells over T cells as effectors of CAR as the off-tumour/on-target adverse effects seen with CAR T cells has severely hampered this clinical strategy. We have shown that CAR T cells but not CAR NK cells are reactive towards phenotypically non-malignant, clinically relevant, healthy cells expressing the CAR target. / Thesis / Master of Science (MSc)

Activating the immune system in the therapeutic treatment of cancer is rapidly growing and has demonstrated tremendous success. One such method is engineering T cells with chimeric antigen receptors (CARs) to specifically direct them in targeting tumours, however this has been associated with several toxicities that may be linked to the synthetic nature of the CAR. To address this, our laboratory created the T Cell Antigen Coupler (TAC), an alternative receptor that redirects T cells in a more natural TCR-dependent fashion. The TAC consists of three components: the antigen-binding domain that recognizes a tumour antigen, a TCR-recruitment domain that co-opts the native CD3-TCR complex and a CD4 co-receptor domain. The TAC displays unique biology, specifically in the increased antitumor infiltration and clearance of solid malignancies without any of the observed host toxicities seen with CARs. The functionality of the TAC was shown to be dependent on both the antigen binding and TCR-recruitment domains, however the co-receptor domain remains relatively uninvestigated despite evidence in the literature indicating its importance in endogenous T cell activation. This thesis seeks to better understand the biology of the TAC receptor by investigating the contributions of co-receptor domain. In Chapter 3, we replaced the CD4 co-receptor domain with CD8 variants and showed that the TAC retains functionality. In Chapter 4, we removed the cytosolic domain of the TAC in its entirety (creating a “tailless TAC”) and observed increased in vivo efficacy. In Chapter 5, we evaluated the tailless TAC in different cancer models and consistently observed increased in vivo efficacy compared to the full length TAC. These results demonstrate an increase in the in vivo functionality of the TAC receptor when the cytoplasmic tail is removed, giving us further insights into the mechanisms behind the unique biology of the TAC receptor. / Thesis / Doctor of Philosophy (PhD) / Cancer is the leading cause of death in Canada, and it is expected that 2 in 5 Canadians will develop some form of cancer in their lifetime. The immune system presents an intriguing alternative method to treat tumours since immune cells such as T cells can circulate through the body and seek and destroy harmful cells, including tumours. Here, we focus on the T cell Antigen Coupler (TAC), a genetically engineered receptor that our laboratory originally designed that directs T cells to recognize and destroy specific cancer cells. This thesis looks at the inner workings of the receptor, specifically a part called the inner tail, and how this feature contributes to how the TAC works. Our results show that removing the tail increases the T cell’s ability to safely clear different tumours in living organisms, bringing us a step closer in designing new and safe treatments for cancer patients.

Introduktion: I Sverige har det uppmätts att 27% av befolkningen har en luftvägsallergi. Dessa behandlas främst med symtomlindrande läkemedel men när detta inte räcker till kan allergenspecifik immunterapi (ASIT) övervägas. Denna behandling kan ges antingen sublingualt eller med subkutana injektioner. Den första studien om ASIT publicerades 1911 och sedan dess har många studier gjorts angående dess effekter. Syfte: Syftet med studien var att undersöka patienters upplevelser av subkutan immunterapi (SCIT). De områden som belystes var upplevelsen av information och kommunikation, psykisk och fysisk påverkan av biverkningar samt hur behandlingen inverkar på vardagen. Metod: En kvalitativ studiedesign användes och data samlades in med hjälp av semistrukturerade intervjuer. Ett bekvämlighetsurval gjordes på en allergimottagning i Mellansverige och tio personer intervjuades. Resultat: Behandlingen påverkade somliga delar av vardagen i viss mån, till exempel att det är tidskrävande och att den fysiska aktiviteten blir begränsad under behandlingsdagen. En del biverkningar förekom, främst allmänna och lokala. Dessa hade viss påverkan på vardagen relaterat till exempelvis trötthet och koncentrationssvårigheter. Kontakten med mottagningen upplevdes positivt och informationen som erhölls inför behandlingsstart upplevdes som tillräcklig. Dock framkom att informationen kring allvarligare biverkningar inte nått fram till alla. Slutsats: Både de upplevda biverkningarna och behandlingen som helhet hade en viss påverkan på patienternas vardag. Kommunikationen och informationen från vården upplevdes överlag positivt. Vidare forskning inom området behövs för att kunna avgöra om dessa resultat är överförbara till en bredare population. / Introduction: It has been measured that 27% of the population in Sweden has a respiratory allergy. Primarily this is treated symptomatically with medication but in cases where this do not work, allergen specific immunotherapy (ASIT) can be considered. ASIT can be administered either sublingually or subcutaneously. The first study acknowledging ASIT was published in 1911 and since then many studies have been performed regarding its effects.      Aim: The aim of this study was to examine patients experiences of subcutaneous immunotherapy (SCIT). This included how patients experienced information and communication, how side-effects affected patients physically and psychologically and how the treatment affected the patients’ everyday life.  Methods: A qualitative study design was used and data was collected with semi-structured interviews. A convenience sample was made at an allergy reception in central Sweden and ten people were interviewed. Results: Parts of the patients’ everyday life were affected to some extent, for example because the treatment is time consuming and that the physical activity has to be limited during the same day as the injection is given. Some side-effects occurred, primarily general and local ones. These affected the everyday life in the sense that the patients felt tired and experienced difficulty concentrating. The contact with the reception was described in a positive manner and the information received before starting treatment was described to be enough. Although the information regarding serious side-effects had not been reached by everyone. Conclusion: Both the experienced side-effects and the treatment in its whole had a certain impact on the patients’ everyday life. The patients described the communication and the information in a positive way. More studies need to be performed in this field to be able to determine whether these results are transferable to a broader population.

Lun Samantha Wei Man. / "August 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 181-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Adenoviral CD40L gene therapy (AdCD40L) is a strong inducer of anti-tumor immune responses via its activation of dendritic cells (DCs). Activated DCs can in turn activate T cells, which are key players in an efficient anti-tumor response. This thesis includes three papers that focus on different aspects of AdCD40L gene therapy. In the first paper, the infiltration of suppressive CD11b+Gr-1+ cells in orthotopic MB49 bladder tumors was investigated and found to be significantly reduced while activated T cells were increased when the tumors had been treated with local AdCD40L gene therapy. Further, AdCD40L could tilt the cells in the tumor microenvironment in favor of an efficient anti-tumor immunity (M1 macrophages and activated T cells) instead of an immunosuppressive environment (CD11b+Gr-1int/low myeloid cells and M2 macrophages). Immunotherapy combined with chemotherapy has shown promising results, and the second paper investigates the combination of AdCD40L gene therapy together with the chemotherapeutic drug 5-Fluorouracil (5-FU). A synergistic effect of the combination treatment on orthotopic MB49 bladder tumors could be demonstrated. The combination therapy resulted in decreased tumor growth, increased survival and systemic MB49-specific immunity, whereas AdCD40L or 5-FU therapy alone had a poor effect on tumor growth. Efficient AdCD40L therapy is dependent on high transduction efficiency in both cancer cells and cells present in the tumor microenvironment. In an attempt to enhance the transduction efficiency, and thereby the therapeutic efficacy, a modified adenovirus was developed for paper three. This modified Ad5PTDf35(mCD40L) could, in comparison with the unmodified Ad5(mCD40L), demonstrate increased transduction capacity of a variety of murine cells. Further, the ability of antigen presenting cells (APCs) to present antigens to T cells was improved after transduction with Ad5PTDf35(mCD40L).

The CD40 ligand (CD40L) molecule is central to innate and adaptive immunity. CD40L expression is very tightly regulated whereas its CD40 receptor is constitutively expressed by many different cell types. CD40L is expressed transiently on helper T cells (Th) only after activation by specific immune recognition molecules carried by professional antigen presenting cells, in particular, dendritic cells (DC). CD40L subsequently binds to CD40 on DC to enable full Th activation. CD40 ligated DC produce interleukin-12 (IL-12) and contribute both to the development of IFNγ-secreting natural killer cells, a vital component of innate immunity, and of IFNγ-secreting type 1 Th (Th1) cells. CD40 ligated DC also contribute to the development of IL-4- and IL-10-secreting Th2 cells. CD40L on Th cells also binds CD40 on macrophages to enhance their cytotoxic functions. CD40L-expressing Th cells provide the ‘help’ pivotally required to activate other components of adaptive immunity responsible both for clearing invading pathogens and generating the memory cells required to prevent re-infection. Th-supplied CD40L binds (i) B cell CD40 to switch production of antibodies to more potent effector molecules that have higher avidity for antigen, and (ii) DC CD40 to prime then expand antigen-specific cytotoxic T lymphocytes (CTL). Activated NK cells and CTL are required both to eradicate malignant cells and cells infected with viruses or other intracellular pathogens. Genetic CD40L deficiency causes the very rare HyperIgM Syndrome Type 1 (HIGM1), which is realistically modelled by genetically engineered CD40L-deficient mice. Neither CD40L-deficient patients nor mice make effective antibodies or mount cellular immune responses that would defend them against intracellular pathogens such as parasites. Consequently, the only potentially curative therapy is allogeneic stem cell transplantation or CD40L gene replacement. Here, we used a retroviral vector, which constitutively expressed CD40L, to genetically modify CD40L-deficient bone marrow cells, which were used to reconstitute partially the immunity of CD40L-deficient mice. The crucial importance of tight regulation of CD40L expression was revealed when these mice later developed lethal thymic T cell malignancy. Growing tumours escape immune vigilance by genetic alterations that reduce their sensitivity to IFNγ. Using murine tumour models, we incorporated transgenic CD40L expression in therapeutic tumour vaccines to show that CD40L gene transfer augmented the immunogenicity of the host’s tumour thus reducing its tumorigenicity. We translated this finding clinically to safety and immunogenicity testing of a transgenic CD40L- and IL-2- expressing leukaemia vaccine. Finally, the common viral respiratory pathogen, respiratory syncytial virus (RSV) mainly infects young infants and the elderly to cause potentially lethal pneumonia. Both groups have reduced cellular and humoral immunity, which predisposes them to re-infection with RSV. Using a murine model, we showed first that simultaneous adenoviral expression of CD40L augmented primary RSV-specific Th1 responses that were associated with accelerated pulmonary viral clearance. Second, we showed that expression of CD40L in RSV-F and RSV-G subunit DNA vaccines elevated antibody and cellular immune responses to RSV challenge four and eight months after the initial immunisation. These results demonstrate the potent ability of CD40L gene transfer to solve the absolute immune deficiency caused by genetic lesions of CD40L. However, physiological regulation of the transgene is required to prevent serious adverse consequences. In contrast, no adverse effects were observed after transgenic CD40L expression was used to overcome relative immune deficiencies imposed by malignancy and RSV infection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298200 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2007

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