Improving immunotherapy for high grade glioma

Glioblastoma multiforme (GBM) is a malignant primary brain tumour that is almost always fatal. Conventional treatment modalities are limited by toxicity. T cell-based immunotherapy is a promising alternative that has the potential to specifically target tumour cells. The author of this thesis was a principal investigator for a recently completed Phase I clinical trial in which patients with recurrent GBM were treated with surgery, dendritic cell-based immunotherapy and chemotherapy. In addition to conducting the trial in collaboration with others, the author used peripheral blood mononuclear cells from trial participants to assess anti-tumour immune responses before and after treatment. A broad correlation was observed between clinical outcome and anti-tumour immunity, with sustained progression-free survival occurring in two patients with baseline responses that persisted or increased after treatment. However, the overall clinical benefit was modest. For progress to be made, there is a need to develop a more potent vaccine. With this in mind, a novel “Glioma-Gal” vaccine was devised and tested in an orthotopic mouse model of glioma, This simple vaccine consisted of irradiated autologous tumour cells pulsed with the glycolipid alpha-galactosylceramide, an immunoadjuvant that induces invariant Natural Killer T cells to licence endogenous dendritic cells. The vaccine was shown to be effective in a therapeutic setting when accompanied by depletion of regulatory T cells. Mechanistically, vaccine efficacy was dependent on CD4 T cells and the mediastinal lymph node was shown to be an important site of T cell priming. It was further shown that components of the immune system necessary for the vaccine to work were present and competent in a cohort of GBM patients. The final chapters explore the idea of enhancing the therapeutic benefit of this vaccine by targeting certain tumour cell subsets or phenotypes. Cancer stem cells (CSC) are proposed to be a subset of tumour cells with a unique capacity for initiating and maintaining tumours. Eliminating these cells may therefore be both necessary and sufficient to achieve cure. Using the same mouse model, a variety of methods were assessed for their ability to isolate or enrich for a CSC subset. Of these, culture in serum-free medium in the presence of certain growth factors was shown to enrich for a more stem cell-like phenotype. However, a vaccine constructed from these stem cell-like cells was not more effective than the standard vaccine. Next, the author tested the hypothesis that a vaccine manipulated to target chemoresistant cells would be more effective than standard vaccine when used in combination with chemotherapy. However, the modified vaccine showed no advantage over standard vaccine in this model. In the course of these experiments, synergy was observed between the vaccine and the chemotherapy agent doxorubicin. The mechanism responsible for this supra-additive effect remains undetermined but is most likely due to an immunomodulatory effect of low dose doxorubicin. The Glioma-Gal vaccine design holds promise but more studies are needed to realise the full potential of this approach. The data presented in the thesis did not support targeting CSC or chemoresistant cells as ways of achieving this. In contrast, combining the vaccine with immunomodulation was effective and merits further exploration

Biology of GD2 ganglioside: implications for cancer immunotherapy

Part of the broader glycosphingolipid family, gangliosides are composed of a ceramide bound to a sialic acid-containing glycan chain, and locate at the plasma membrane. Gangliosides are produced through sequential steps of glycosylation and sialylation. This diversity of composition is reflected in differences in expression patterns and functions of the various gangliosides. Ganglioside GD2 designates different subspecies following a basic structure containing three carbohydrate residues and two sialic acids. GD2 expression, usually restrained to limited tissues, is frequently altered in various neuroectoderm-derived cancers. While GD2 is of evident interest, its glycolipid nature has rendered research challenging. Physiological GD2 expression has been linked to developmental processes. Passing this stage, varying levels of GD2, physiologically expressed mainly in the central nervous system, affect composition and formation of membrane microdomains involved in surface receptor signaling. Overexpressed in cancer, GD2 has been shown to enhance cell survival and invasion. Furthermore, binding of antibodies leads to immune-independent cell death mechanisms. In addition, GD2 contributes to T-cell dysfunction, and functions as an immune checkpoint. Given the cancer-associated functions, GD2 has been a source of interest for immunotherapy. As a potential biomarker, methods are being developed to quantify GD2 from patients’ samples. In addition, various therapeutic strategies are tested. Based on initial success with antibodies, derivates such as bispecific antibodies and immunocytokines have been developed, engaging patient immune system. Cytotoxic effectors or payloads may be redirected based on anti-GD2 antibodies. Finally, vaccines can be used to mount an immune response in patients. We review here the pertinent biological information on GD2 which may be of use for optimizing current immunotherapeutic strategies

Activation of Natural Killer T cells and Dendritic cells with Caulobacter crescentus: Implications for developing tumour immunity

Cancer remains a leading cause of mortality worldwide. Efforts to develop immuno-therapies to control the growth of cancer, while limiting host cell damage, have focused on targeting specific tumour associated antigens. These treatments have yielded some clinical success however; the limited targeting of tumour antigens potentially allows the tumour to escape the treatment through antigen mutation or down-regulation of expression. In this thesis, we focused on the ability of non-pathogenic, Gram negative bacteria, Caulobacter crescentus to stimulate innate immunity to generate a response capable of controlling the growth of syngeneic tumours. We evaluated the ability of C.crescentus to activate natural killer T cells (NKT) and dendritic cells (DCs) as both cell populations affect the continued development of the inflammatory process. The activation of NKT cells was determined using Ja18-/- or CD1d-/- mice which lacked either a subset or all CD1d-dependent NKT cells respectively. NKT cell activation was determined through measurements of the early activation marker CD69 and various cytokines such as IFN-gamma. DC activation by C.crescentus was characterized through observations made with bone marrow derived DCs and their ability to express co-stimulatory markers such as CD40, CD54, CD80, and CD86. The interaction of C.crescentus stimulated NKT cells and DCs revealed that C. crescentus stimulated NKT cells through a contact dependent pathway which may not require the recognition of the CD1d-lipid complex. Additionally, the interaction of C.crescentus activated NKT cells and DCs resulted in an enhanced expression of factors that are known promoters of Th1 cellular immunity such as IL-12p70 and CD40. The immunity stimulated by C.crescentus was shown to slow the growth of EL4 subcutaneous tumours. Interestingly, through the course of our studies we revealed a role for a subset of NKT cells, type 1 NKT cells, absent in Ja18-/- mice to support the growth of syngeneic tumours. We found that Ja18-/- mice bone marrow derived DCs expressed increased Th1 promoting factors. This novel observation indicates a role for NKT cells in the development and maintenance of DC homeostasis in the wild-type animal

Exosomes : immunomodulators in cancer and therapy

Exosomes are nano-sized membrane vesicles derived from the late endosomal compartment. They are capable of transferring proteins, lipids and RNA between cells. B cell and dendritic cell (DC)-derived exosomes express major histocompatibility complex (MHC) class I and II, as well as costimulatory molecules (CD80/86) and can initiate T cell responses. Several clinical trials have shown DC-derived exosome-based cancer immune therapy to be safe but limited in inducing antigen-specific T cells. In contrast, tumour cell-derived exosomes can express immune inhibitory molecules and play an important role in spreading oncogenic activity by carrying tumour antigens, inducing angiogenesis at distant sites and preparing tissues for metastasis. This thesis aimed at I) analysing how to enhance the immunogenicity of exosomes for therapy, II) investigating whether MHC complexes on exosomes are needed to induce an anti-tumour immune response, III) comparing microvesicles and exosomes side by side for their immunogenic capacity, IV) understanding the metastatic process induced by tumour-derived exosomes from bladder cancer patients and whether certain exosomal proteins can be used as markers for diagnosis and prognosis. Study I reveals that exosomes loaded with the NKT cell ligand alpha-galactosylceramide (αGC) and the model antigen ovalbumin (OVA) activate NKT cells, induce strong NK and γδ T cell innate immune responses, and induce OVA-specific T and B cell responses far better than only OVA-loaded exosomes. Exosomes loaded with αGC/OVA decreased tumour growth and increased median survival compared to exosomes loaded with OVA only or soluble αGC + OVA alone in a B16 melanoma model. This study demonstrates how to increase the immunogenicity of DC-derived exosomes for cancer treatment. Study II demonstrates that exosomal MHC class I is dispensable for the induction of antigen-specific T cell responses if whole OVA is present. We show that OVA-loaded DC-derived exosomes from MHCI-/- mice induce antigen-specific T cells to the same extent as wild type exosomes. Even exosomes with MHC class I and II mismatch induced tumour-infiltrating CD8+ T cells and increase survival in a B16 melanoma model. This study provides new opportunities for the design of allogeneic exosome-based vaccines and therapies. Study III compares microvesicles (MV) and exosomes from OVA-exposed DCs side by side for their capacity to induce OVA-specific immune responses in vivo. MV and exosomes express similar surface markers but only exosomes induced OVA-specific CD8+ T cells and OVA-specific IgG antibodies. In contrast, MV induced a higher number of plasma cells. Finally, we found that exosomes contain more OVA compared to MV. We conclude that exosomes from DCs are superior in inducing antigen-specific immune responses in vivo compared to MVs, while MVs might activate the immune system unspecifically. Study IV evaluates the proteomic profile of exosomes from tumour tissue explants and urine from urinary bladder cancer patients. We show that exosomes from malignant or benign tissue can be distinguished by the proteomic profile and are involved in platelet, metabolic and immune signalling networks. We show that, even if no tumour is left, exosomes can express a metastatic memory phenotype which might be involved in cancer progression. In summary, this thesis gives new insights into how to design vesicle-based cancer vaccines and provide new opportunities for the use of allogeneic DC-derived exosomes in patients. In addition, we demonstrate that exosomes isolated from the urine of urinary bladder cancer patients express specific markers for malignancy, which provides new possibilities for diagnostic strategies

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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Characterisation of anti-glycan monoclonal antibodies

The aims of this thesis are to establish the therapeutic value of two anti-glycan mAbs produced in-house, to develop an immunisation protocol with the aim of improving the immunogenicity tumour-associated glycolipids with the intention of producing therapeutically valuable mAbs and to determine the implication of a mAb with the ability to induce apoptosis in colorectal cancer. The anti-glycan mAbs 692/29 and 505/4 have previously been produced in-house and this study aimed to determine their fine specificity using a glycan array. 692/29 displayed binding predominantly to Lewis b as well as Lewis y-containing glycans. 505/4 was discovered to bind to sialyl Lewis a as well as sialyl di-Lewis a, with no cross-reactivity with other blood group antigens. This was compared to other anti-Lewis mAbs, with differences in specificity being observed. Characterisation of 505/4 mAb distribution showed binding to 80% of colorectal tumours and low levels of binding to normal tissues by IHC, suggesting it may be therapeutically useful. This thesis aimed to assess the ability of 505/4 and 692/29 to meditate immune mediated and non-immune mediated cell death as well as to determine whether non-immune-mediated cell death would be a desirable therapeutic property. Resistance to apoptosis is one of the hallmarks of cancer cells and mAbs stimulating apoptosis may not be very effective. Alternatively, cancer cells are driven to initiate apoptosis by genomic and other aberrations thus if pro-apoptotic pathways are stimulated these cells may be more susceptible to death than normal cells. To investigate the significance of apoptosis in cancer a large tissue microarray of colorectal tumours was assessed for apoptosis and its relationship to patient prognosis. Cleaved caspase-3 is a good marker of apoptosis as it is the executioner caspase for both the extrinsic and intrinsic pathways. Immunohistochemical analysis of colorectal tumour samples revealed that a high expression of cleaved caspase-3 in tumour was associated with good prognosis in colorectal cancer. This suggested that some tumours were still susceptible to apoptotic death but some are resistant and an alternative mechanism of cell death may be an advantage in these tumours. High expression of cleaved caspase-3 in the tumour-associated stroma was also an independent marker of good prognosis in colorectal cancer. This may be because apoptosis of the tumour-associated stroma reduces the level of pro-tumour signals originating from tumour-associated immune cells and stromal cells. As the tumour microenvironment can act in an immunosuppressive and pro-tumour manner, the ability of a mAb to induce direct cell death without the need for effector cells or complement would be an advantage. Lewis y and Lewis b are blood group antigens commonly overexpressed on the surface of a range of cancers. Characterisation of effector functions of 505/4 and 692/29 demonstrated that both mAbs have the ability to mediate apoptosis by antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and cause direct cell death in an oncosis-like manner. Comparison with other anti-Lewis mAbs demonstrated that a number of anti-Lewis mAbs can induce direct cell death independently of apoptosis. Thus, they could effectively target apoptotic sensitive and resistant colorectal cancers. Tumours aberrantly express glycolipids and these molecules may be involved in a number of cellular pathways. In addition a large proportion of anti-glycan mAbs, including 505/4 and 692/29 in this thesis, have displayed the ability to induce direct cell death. Therefore this thesis aimed to develop an immunisation protocol capable of increasing the immunogenicity of tumour-associated glycolipid for the production of anti-tumour glycolipid mAbs directed against ovarian cancer. This study suggests that the incorporation of tumour glycolipid into liposomes and their immunisation along with the iNKT cell adjuvant α-galactosylceramide, elicits an anti-tumour glycolipid immune response, which can yield IgG mAbs capable of binding a high proportion of ovarian cancers. In summary, this thesis confirmed specificity of 692/29 to Lewis y and Lewis b and 505/4 to sialyl Lewis a and sialyl di-Lewis a. Furthermore, this thesis demonstrated a promising tissue distribution of 505/4 in vitro. Characterisation of mAb effector functions suggest that both Lewis y and sialyl Lewis a directed mAbs have the ability to cause direct cell death, independently of apoptosis in antigen positive cells, as well as the ability to cause immune-mediated cell death. This may be an important factor in the immune-suppressive tumour microenvironment. Furthermore, this thesis provides the basis for the production of new anti-glycolipid antibodies that may also be able to induce direct cell death

Human γδ T cell-based immunotherapy for breast cancer

Scientific background. The inherent resistance of breast cancer stem cells (CSCs) to existing therapies has largely hampered effective treatments for advanced breast cancer. My research aimed at establishing novel immunotherapy approaches efficiently targeting CSCs by harnessing human γδ T cells as non-MHC-restricted killer cells and simultaneously as APCs to induce tumour-specific CD8+ T cell responses. Approach. An experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with γδ T cells and CD8+ T cells. FluM1 and CMVpp65 viral epitopes were used as surrogates for yet-to-be-discovered CSC-associated antigens. Results. Stable sublines with characteristics of CSCs and non-CSCs were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their (i) distinct expression profiles of CD24, CD44 and GD2, (ii) mesenchymal- and epitheliallike characteristics, (iii) differential growth patterns in mammosphere culture and (iv) distinct tumourigenicity, self-renewal and differentiation in NSG mice. The resistance of both CSCs and non-CSCs to γδ T cells could be overcome by inhibition of FPPS through pretreatment with zoledronate or FPPS-targeting shRNA, resulting in increased cytotoxicity and APC function of γδ T cells. CSCs presenting FluM1 or CMVpp65 exhibited stronger resistance to antigen-specific CD8+ T cells as compared to their non-CSC counterparts. Of note, pretreatment of Flu M1- or CMVpp65-presenting CSCs with γδ T cell conditioned supernatant significantly increased surface expression of MHC class I and ICAM-1 by both CSCs and non-CSCs as well as their susceptibility to CD8+ T cellmediated killing. Moreover, using the humanised anti-GD2 monoclonal antibody,Hu14.18K322A, a specific direction of γδ T cell responses against CSCs could be achieved. In addition to their direct cytotoxicity and ability to modulate the susceptibility of CSCs and non-CSCs to CD8+ T cell-mediated killing, γδ T cells concomitantly functioned as APCs to initiate de novo tumour-specific cytotoxic CD8+ T cell responses. Conclusions. My findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of both CSCs and non-CSCs, thus establishing a powerful positive feedback loop for the eradication of residual cancer cells survived from killing by γδ T cells. My research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T cell and CD8+ T cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses

Characterisation of anti-glycan monoclonal antibodies

The aims of this thesis are to establish the therapeutic value of two anti-glycan mAbs produced in-house, to develop an immunisation protocol with the aim of improving the immunogenicity tumour-associated glycolipids with the intention of producing therapeutically valuable mAbs and to determine the implication of a mAb with the ability to induce apoptosis in colorectal cancer. The anti-glycan mAbs 692/29 and 505/4 have previously been produced in-house and this study aimed to determine their fine specificity using a glycan array. 692/29 displayed binding predominantly to Lewis b as well as Lewis y-containing glycans. 505/4 was discovered to bind to sialyl Lewis a as well as sialyl di-Lewis a, with no cross-reactivity with other blood group antigens. This was compared to other anti-Lewis mAbs, with differences in specificity being observed. Characterisation of 505/4 mAb distribution showed binding to 80% of colorectal tumours and low levels of binding to normal tissues by IHC, suggesting it may be therapeutically useful. This thesis aimed to assess the ability of 505/4 and 692/29 to meditate immune mediated and non-immune mediated cell death as well as to determine whether non-immune-mediated cell death would be a desirable therapeutic property. Resistance to apoptosis is one of the hallmarks of cancer cells and mAbs stimulating apoptosis may not be very effective. Alternatively, cancer cells are driven to initiate apoptosis by genomic and other aberrations thus if pro-apoptotic pathways are stimulated these cells may be more susceptible to death than normal cells. To investigate the significance of apoptosis in cancer a large tissue microarray of colorectal tumours was assessed for apoptosis and its relationship to patient prognosis. Cleaved caspase-3 is a good marker of apoptosis as it is the executioner caspase for both the extrinsic and intrinsic pathways. Immunohistochemical analysis of colorectal tumour samples revealed that a high expression of cleaved caspase-3 in tumour was associated with good prognosis in colorectal cancer. This suggested that some tumours were still susceptible to apoptotic death but some are resistant and an alternative mechanism of cell death may be an advantage in these tumours. High expression of cleaved caspase-3 in the tumour-associated stroma was also an independent marker of good prognosis in colorectal cancer. This may be because apoptosis of the tumour-associated stroma reduces the level of pro-tumour signals originating from tumour-associated immune cells and stromal cells. As the tumour microenvironment can act in an immunosuppressive and pro-tumour manner, the ability of a mAb to induce direct cell death without the need for effector cells or complement would be an advantage. Lewis y and Lewis b are blood group antigens commonly overexpressed on the surface of a range of cancers. Characterisation of effector functions of 505/4 and 692/29 demonstrated that both mAbs have the ability to mediate apoptosis by antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and cause direct cell death in an oncosis-like manner. Comparison with other anti-Lewis mAbs demonstrated that a number of anti-Lewis mAbs can induce direct cell death independently of apoptosis. Thus, they could effectively target apoptotic sensitive and resistant colorectal cancers. Tumours aberrantly express glycolipids and these molecules may be involved in a number of cellular pathways. In addition a large proportion of anti-glycan mAbs, including 505/4 and 692/29 in this thesis, have displayed the ability to induce direct cell death. Therefore this thesis aimed to develop an immunisation protocol capable of increasing the immunogenicity of tumour-associated glycolipid for the production of anti-tumour glycolipid mAbs directed against ovarian cancer. This study suggests that the incorporation of tumour glycolipid into liposomes and their immunisation along with the iNKT cell adjuvant α-galactosylceramide, elicits an anti-tumour glycolipid immune response, which can yield IgG mAbs capable of binding a high proportion of ovarian cancers. In summary, this thesis confirmed specificity of 692/29 to Lewis y and Lewis b and 505/4 to sialyl Lewis a and sialyl di-Lewis a. Furthermore, this thesis demonstrated a promising tissue distribution of 505/4 in vitro. Characterisation of mAb effector functions suggest that both Lewis y and sialyl Lewis a directed mAbs have the ability to cause direct cell death, independently of apoptosis in antigen positive cells, as well as the ability to cause immune-mediated cell death. This may be an important factor in the immune-suppressive tumour microenvironment. Furthermore, this thesis provides the basis for the production of new anti-glycolipid antibodies that may also be able to induce direct cell death