Introduction: Manipulation of the immune system to eliminate cancer, known as cancer immunotherapy, is an emerging field that has shown impressive clinical success and promise. Adoptive transfer of T cells engineered for tumor reactivity is an avenue of therapy for patients with previously untreatable disease. Our lab has developed a novel chimeric receptor, called a T cell antigen coupler (TAC), which redirects T cell cytotoxicity towards a tumor target. Although considerably effective, this receptor does not provide T cell costimulation necessary for optimal anti-tumor effectiveness. Methods: We explored two methods to deliver costimulation to TAC-engineered T cells. First, we designed a receptor to be utilized in conjunction with the TAC in a dual receptor system. This chimeric costimulatory receptor (CCR) was generated by fusion of the T cell TIGIT and CD28 receptors. In our second approach, we investigated direct incorporation of costimulatory domains into the TAC design. To do so, we substituted in regions from the CD28 or 4-1BB costimulatory receptors. Results: Three TIGIT/CD28 chimeras were successfully generated. Of these, two were well surface-expressed on primary human T cells. Despite testing of these receptors in several biological assays, we were unable to confirm functionality of these receptors in transmitting CD28 signals. We next generated the 4-1BB and CD28TAC variants. The 4-1BBTAC was poorly surface-expressed M.Sc Thesis – Arya Afsahi McMaster University – Medical Sciences iv and was difficult to introduce into T cells at high efficiency. The CD28TAC-variant was virtually absent from the T cell surface membrane. Further analysis indicated that the CD28TAC was retained in the endoplasmic reticulum (ER) and the 4-1BBTAC was produced at an extremely low amount. Conclusions: Our investigation into delivery of costimulation through a novel CCR or TAC receptor was inconclusive. We recommend several optimizations to both receptor design and experimental analysis to further elucidate the potential of these receptors. / Thesis / Master of Science (MSc)

Tumors induce immune dysfunction through the production of soluble factors that subvert macrophage (Mf) function to favor tumor growth. Previous studies suggested that tumor-induced immune cell dysfunction may be reversible through regimens that disrupt tumor cell suppressor mechanisms and concurrently promote tumoricidal activities. Because the antineoplastic agent paclitaxel (TAXOL) activates Mf function, we studied mechanisms of paclitaxel-mediated cytotoxic and immunostimulatory responses by tumor-induced Mfs. Although tumor-derived factors, including interleukin-10 and transforming growth factor-b1, modulate Mf response to activation signals, paclitaxel partly reverses tumor-induced Mf-mediated suppression of T-cell reactivity through enhanced production of the immunostimulatory cytokine interleukin-12 (IL-12). Concurrently, paclitaxel induces Mf cytotoxic and proinflammatory molecule production, including tumor necrosis factor-a and interleukin-1b. In contrast to its apparent immunotherapeutic effect on Mf populations, paclitaxel's cytostatic mechanisms suppress lymphocyte proliferation and function. We showed that IL-12 can reverse paclitaxel-mediated suppression of T-cell responses in vitro, establishing the foundation for a novel antitumor therapy using paclitaxel in combination with IL-12. We show that the administration of paclitaxel as a chemotherapeutic agent, followed by IL-12 as an immunotherapeutic agent to alleviate paclitaxel-mediated immunosuppression, prolongs survival, reduces tumor progression, and activates immune effector populations in a murine tumor model. These results are the first experimental evidence to suggest that paclitaxel and IL-12 are an effective antitumor modality. Collectively, these studies show that paclitaxel induces multiple antitumor mechanisms that can be enhanced with proper ancillary administration of immunotherapeutic cytokines. / Ph. D.

Cancer is a multifaceted and intricate disease that poses a significant global health burden and impacts millions of individuals worldwide. Among the diverse subtypes of cancer, sarcoma stands out as a rare yet highly aggressive malignancy originating from connective tissues such as bone, cartilage, and muscle, and presents challenges in diagnosis and treatment. Despite remarkable progress in cancer research and therapy, the prognosis for sarcoma patients remains low, requiring development of novel therapeutic avenues. Cancer immunotherapies focused on generating tumor-specific responses are emerging as promising alternatives to traditional cancer treatments. T cell-based immunotherapies, such as cancer vaccines and CAR-T cells, are designed to target tumor antigens and generate long term immune memory capable of constant surveillance against recurrence. Therefore, the objective of this study is to establish the groundwork for a novel T cell-based immunotherapeutic approach tailored specifically to sarcoma. Throughout the study, we explored various critical aspects associated with the development of immunotherapy. First, we conducted a proof-of-concept study, evaluating a novel prime-boost vaccine combination employing anti-DEC205 and oncolytic rhabdoviruses targeting a model antigen in a pre- clinical model of melanoma. This study showed that using the DEC205 dendritic cell-targeting antibody as a vector for antigen delivery is a promising alternative to other prime-boost strategies being evaluated in the clinic (NCT02285816). To facilitate the translation of this therapeutic approach to clinical applications, a comprehensive understanding of the human sarcoma tumor immune microenvironment and the identification of a suitable target antigen are essential. Therefore, we conducted an in-depth immune profiling of a high grade and aggressive dedifferentiated liposarcoma (DDLS) using gene expression profiling and immunohistochemistry. We gained valuable insights into the tumor biology and the complex immunological mechanisms within the tumor immune microenvironment. Notably, we identified a novel antigen that is highly expressed in human DDLS and absent in normal tissues, that could be used as a potential antigenic target for immunotherapy. Finally, we evaluated a range of prime and boost vaccine vectors targeting the newly discovered target antigen in pre-clinical murine sarcoma models. Ultimately, we found that an oncolytic rhabdovirus prime and a modified vaccinia Ankara virus boost targeting the sarcoma antigen generates strong antigen specific cellular and humoral responses and protects against tumor growth in a prophylactic model of sarcoma. Altogether, this study lays the foundations for the development of a T cell-based immunotherapy employing an oncolytic rhabdovirus and targeting a novel antigen for the treatment of sarcoma.

A thesis submitted in partial fulfilment of tine requirements for the Degree of Master of Technology: Biomedical Technology, Durban University of Technology, 2007. / In 2004, it was reported that KwaZulu-Natal had the greatest number of HIV infected people, approximately 1.8 million people, of whom an estimated 450 000 were in need of antiretroviral drug therapy based on their Cluster of Differentiation 4 (CD4) counts and clinical status. Studies on the success of antiretroviral drugs in improving the quality of life in HIV infected individuals have been extensively performed and published. However, there are no published data on the effect that immune boosters have in improving the quality of life in such persons. Considering the side effects, toxicity, multi-drug regimens and drug resistance problems associated with antiretroviral therapy, alternative or supplementary therapies may play an important role in improving the quality of life in HIV infected people. Such therapy might help in situations where some patients who qualify for antiretroviral treatment are unable to access them because of several reasons such as long waiting lists, travelling costs, unwilling to take antiretroviral drugs, etc. Some patients have reservations in taking antiretroviral drugs. The stigma associated with the disease may be a major factor. The aim of this study was to investigate the change in the immune status of HIV infected patients that were on the Inochi New Medicine immune booster, as well as, to assess the safety and efficacy of this immune booster in improving the patients’ quality of life. / M

Despite expressing antigens that can induce immune surveillance and immune eradication, tumours demonstrate the capacity to evade anti-tumour immunity. Recently, this has been attributed to the ability of tumours to induce a local immunosuppressed micro-environment, which is a major obstacle to successful natural and vaccine induced anti-tumour immunity. Soluble factors such as transforming growth factor beta (TGFβ), and interleukin 10 (IL-10), released by cancer and stromal cells, are thought to play a significant role in this local immunosuppression. In order to assess the influence of antagonising these soluble factors locally on tumour biology and tumour immunity, a murine CT26 colorectal carcinoma model that can express cytokine antagonists under Doxycycline (Dox) control was engineered. Two stable CT26 cell lines expressing Dox-inducible soluble extracellular domain of TGFβ receptor II (STGFβRII) or soluble extracellular domain of IL-10 receptor (SIL-10R), were established. Expression of STGFβRII in vitro and in vivo was only evident after Dox treatment. When Dox was administered directly following subcutaneous (s.c.) inoculation of STGFβRII-expressing CT26 cells into Balb/c mice, tumour growth was significantly suppressed. Interestingly, inducing STGFβRII in well-established tumours showed less suppression of tumour growth. To assess the effect of expressing STGFβRII on tumour immunity the RNA expression of 22 common cytokines was measured in the tumours of mice receiving Dox and control mice. The levels of some of these cytokines were modulated by STGFβRII expression (e.g TGFβ,Tnfsf9, IL-2). We also tested for any additive effect between expressing STGFβRII, and the administration of anti-CTLA-4 antibody on tumour growth, and tumour immunity. The model described here could help address various limitations seen previously in studies of TGFβ blockade. It provides means of effective local antagonism, and it addresses immunological endpoints which have been limited in previous studies. Because of its tight regulation, the model also allows identification of the best timing of TGFβ blockade alone or in combination with other immunotherapeutics.

No description available.

Submitted in partial fulfillment of the requirements for the degree of Doctor of Technology: Clinical Technology, Durban Institute of Technology, 2005. / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They exert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancerpromoting metabolites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunornodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL -1, IL-6, IL-8, IL-10 and TNF-a) were determined using ELISA kits. / D

<p>Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.</p> / Dissertation

Neuroblastoma is the third most common pediatric cancer. Dinutuximab is a recently approved monoclonal antibody targeting GD2, a ganglioside ubiquitously present on neuroblastoma. Recent studies have shown that αGD2 therapy activates PD1-PDL1 signalling, resulting in the inhibition of its full therapeutic potential. The PD1-PDL1 signalling axis is a cellular checkpoint that inhibits immune responses. The blocking of this interaction has been successful in the treatment of numerous cancers, including in combination with anti-GD2 therapy. The Inhibitor of apoptosis (IAP) proteins are commonly upregulated in cancers and prevent cell death through the inhibition of caspases and through the control of NF-κB activity. Smac mimetic compounds (SMCs) have been designed to target IAP activity, thereby promoting cancer cell death. Here, I used the SMC, LCL161, to improve αGD2 antibody treatment against a GD2+ syngeneic neuroblastoma mouse model. I found that murine cell lines NXS2 and N2a were resistant in vitro to LCL161-mediated apoptosis, despite expressing apoptotic components often silenced in neuroblastoma. In vivo, I observed a slight delay in tumour growth induced by LCL161 and I confirmed an in vivo anti-angiogenic effect of LCL161 through ultrasound imaging and necropsy evaluation. I then combined LCL161 and αGD2 antibody (clone ME361-S2a) treatment and reported a delay in NXS2 subcutaneous tumour growth, which was further potentiated with the addition of an αPD-L1 antibody. With optimization, there is potential for SMCs to be used in combination with αGD2 therapy in GD2+ cancers like neuroblastoma.

by Cho Chi Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / Acknowledgements --- p.I / Table of Contents --- p.II / Abbreviations --- p.VII / Aim and Scope of This Dissertation --- p.X / Abstract --- p.XII / Chapter Chapter One --- General Introduction --- p.1 / Chapter 1.1 --- An Overview of the Immune System --- p.1 / Chapter 1.1.1 --- Humoral antibody immune responses --- p.2 / Chapter 1.1.2 --- Cell-mediated immune responses --- p.3 / Chapter 1.2 --- Immunomodulation --- p.4 / Chapter 1.3 --- An Overview of the Anti-tumour Strategies --- p.6 / Chapter 1.3.1 --- Immunological defense mechanisms against tumours --- p.8 / Chapter 1.3.1.1 --- T and B lymphocytes --- p.9 / Chapter 1.3.1.2 --- The monocytes/macrophages --- p.9 / Chapter 1.3.1.3 --- Non-specific killer cells --- p.10 / Chapter 1.3.2 --- Adoptive immunotherapy against tumours --- p.11 / Chapter 1.3.3 --- Induction of tumour cell differentiation --- p.12 / Chapter 1.4 --- Traditional Chinese Medicines as Potential Immunomodulators and Anti-tumour Agents --- p.13 / Chapter 1.5 --- General Properties of Astragalus membranaceus --- p.16 / Chapter Chapter Two --- Materials and Methods --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.1.1 --- Animals --- p.20 / Chapter 2.1.2 --- Astragalus membranaceus --- p.20 / Chapter 2.1.3 --- "Buffers, culture media and chemicals" --- p.20 / Chapter 2.1.4 --- Cell lines --- p.25 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Extraction and fractionation of Astragalus membranaceus --- p.28 / Chapter 2.2.2 --- Characterization of Astragalus membranaceus --- p.32 / Chapter 2.2.3 --- In vivo drug treatment --- p.33 / Chapter 2.2.4 --- Isolation and preparation of cells --- p.34 / Chapter 2.2.5 --- Assays for the immunomodulatory activities of Astragalus membranaceus --- p.36 / Chapter 2.2.6 --- Assays for the immunorestorative properties of Astragalus membranaceus --- p.42 / Chapter 2.2.7 --- Assays for the anti-tumour activities of Astragalus membranaceus --- p.43 / Chapter 2.2.8 --- Statistical analysis --- p.47 / Chapter Chapter Three --- "Extraction, Fractionation and Characterization of Bioactive Components from Astragalus membranaceus" --- p.49 / Introduction --- p.49 / Results --- p.50 / Chapter 3.1 --- Extraction and Fractionation of Astragalus membranaceus --- p.50 / Chapter 3.2 --- Lack of Cytotoxicity of A.M. to Mouse Splenocytes --- p.51 / Chapter 3.3 --- Mitogenic Effect of A.M. Fractions on Mouse Splenocytes --- p.51 / Chapter 3.4 --- AP and AI Fractions Did Not Exhibit Lectin-like Activity --- p.52 / Chapter 3.5 --- Heat Stability of AP and AI Fractions --- p.52 / Chapter 3.6 --- Chemical Destruction of the Mitogenic Activity of AI by Sodium Periodate But Not by Acetic Acid Treatment --- p.53 / Discussion --- p.54 / Chapter Chapter Four --- The Immunomodulatory Activities of Astragalus membranaceus --- p.63 / Introduction --- p.63 / Results --- p.65 / Chapter 4.1 --- Effect of Astragalus membranaceus on the Specific and Nonspecific Immunity --- p.65 / Chapter 4.1.1 --- Mitogenic effect of AI on mouse splenocytes in vivo --- p.65 / Chapter 4.1.2 --- Effect of AI on lymphocyte sub-populations --- p.66 / Chapter 4.1.3 --- Co-mitogenic effect of AI on mouse splenocytes in vitro --- p.66 / Chapter 4.1.4 --- Enhancement of the mitogen-induced lymphocyte transformation in vitro by oral administration of AI --- p.67 / Chapter 4.1.5 --- Mitogenic and co-mitogenic effects of AI on human cord blood lymphocytes in vitro --- p.67 / Chapter 4.1.6 --- Primary humoral immune response to SRBC in AI-treated mice --- p.68 / Chapter 4.1.7 --- Effect of AI on interleukin-2 production --- p.68 / Chapter 4.1.8 --- Effect of AI on interleukin-2 receptor expression on mouse splenocytes --- p.69 / Chapter 4.1.9 --- Immunopotentiating effects of AI on macrophage functions --- p.70 / Chapter 4.1.9.1 --- Effect of AI on the cytostatic activity of macrophages in vitro --- p.70 / Chapter 4.1.9.2 --- In vivo migration and phagocytic activity of macrophages in AI-treated mice --- p.70 / Chapter 4.1.9.3 --- Cytostatic activity of macrophages in AI-treated mice --- p.71 / Chapter 4.1.9.4 --- Effect of AI on the Fc receptor expression on mouse resident peritoneal exudate cells --- p.71 / Chapter 4.2 --- Immunorestorative Properties of Astragalus membranaceus --- p.72 / Chapter 4.2.1 --- Effect of AI on lymphocyte blastogenesis in aging mice --- p.72 / Chapter 4.2.2 --- Effect of AI on lymphocyte blastogenesis in tumour-bearing mice --- p.72 / Chapter 4.2.3 --- Effect of AI on lymphocyte blastogenesis in cyclophosphamide- treated mice --- p.73 / Discussion --- p.74 / Chapter Chapter Five --- The Anti-tumour Activities of Astragalus membranaceus --- p.94 / Introduction --- p.94 / Results --- p.95 / Chapter 5.1 --- Lack of Direct Cytotoxicity of AI to Murine and Human Tumour Cell Lines In Vitro --- p.95 / Chapter 5.2 --- Cytostatic Effect of AI on Various Murine and Human Cell Lines In Vitro --- p.96 / Chapter 5.3 --- Effect of AI on the Growth of Transplantable Tumour Cells In Vivo --- p.97 / Chapter 5.4 --- Effect of AI on TNF Production in Tumour-bearing Mice --- p.97 / Chapter 5.5 --- In Vitro Induction of Lymphokine-activated Killer Cell Activity by AI --- p.98 / Chapter 5.6 --- Tumour Cell Differentiation-inducing Activity of AI --- p.99 / Discussion --- p.100 / Chapter Chapter Six --- General Discussion and Future Perspectives --- p.120 / References --- p.130