Metastatic breast cancer is a leading cause of cancer-related mortality worldwide. While existing interventions are effective at treating localized tumors, disseminated malignancies remain incurable. Vaccine-induced anti-tumor immunity is a promising approach for treating disseminated tumors, as immune responses are systemic, have antigen-restricted cytotoxicity, and generate protective immune “memory” populations. Our group has developed a novel heterologous prime/boost vaccine protocol that treats established 4T1 murine mammary tumors. Briefly, this approach entails a vaccine prime consisting of tumor lysate antigens encapsulated within poly(lactic-co-glycolic) acid (PLGA) microparticles (MPs). The vaccine prime was followed by a vaccine boost consisting of tumor lysates plus adjuvants. Spontaneous 4T1 lung metastasis was evaluated at a pre-determined endpoint in vaccinated versus untreated mice. Vaccinated mice demonstrated significant, but incomplete, reductions in metastatic tumor burdens relative to untreated control mice. Encouraged by these results, we evaluated additional vaccine variations with the goal of improving therapeutic responses. The addition of immunomodulatory chemotherapy or checkpoint blockade immunotherapy failed to significantly improve the initial vaccine’s efficacy. Conjugation of streptavidin/biotin complexes to the PLGA MP significantly improved vaccine efficacy, with vaccinated mice demonstrating 88% less metastatic tumor burdens than their untreated counterparts. These findings illustrate that vaccines based upon PLGA MP-mediated delivery of tumor lysates can form the basis of an effective treatment for metastatic breast cancer and suggest that similar approaches may be both efficacious and well-tolerated in the clinic.

Abstract not available

Pseudomonas aeruginosa (P. aeruginosa) is a frequently isolated pathogen that causes septicaemia and chronic respiratory infection. It exhibits a higher mortality rate than other gram-negative bacteria and the need for effective immunotherapy is emphasised by the frequency of antibiotic resistance associated with this organism. Mucosal immunisation with a whole killed cell P. aeruginosa vaccine has previously demonstrated a significant immune response in both rodent studies and human trials. This study is a continuation of that research, with the major goal being the identification of a purified protein antigen that could form the basis of a mucosal vaccine against P. aeruginosa. Specifically, the aims of this study were the development of purification protocols for the isolation of previously untested protein antigens, assessment of the efficacy of these antigens to enhance bacterial clearance in an animal model of acute respiratory infection, determination of the immune parameters that are associated with the resolution of P. aeruginosa respiratory infection and finally, cloning of an identified antigen which demonstrated vaccine efficacy. Protocols were established to isolate proteins for use as antigens in immune response studies. The proteins purified in this study were Pa 13, Azurin, acyl carrier protein (ACP), Amidase, Aminopeptidase, KatA and Pa70. These proteins were used to immunise rats by intestinal intra-Peyer's patch (IPP) inoculation and intratracheal (IT) boost. The immunisation protocol employed was designed to target mucosal antigen-specific immune responses where the route of immunisation, Peyer's patch (PP) intestinal inoculation, is akin to the oral delivery of antigens to the gut-associated lymphoid tissue (96). Investigations of a previously uncharacterised antigen, Pa60, later identified this protein as the P. aeruginosa catalase, KatA. This study demonstrated enhanced bacterial clearance of both homologous and heterologous challenge following immunisation with KatA. The level of clearance demonstrated by KatA was promising when compared to that of killed whole cell immunisation. KatA was cloned and studies with the recombinant protein showed enhanced bacterial clearance commensurate with that of the native protein. Immunisations with other proteins identified four additional antigens which enhanced bacterial clearance; Pa13, Pa40, Pa45 and Pa70. Amino acid sequence analysis indicated that Pa13 may be a novel protein, whereas Pa40 was determined to be amidase and Pa45, aminopeptidase. Pa70 was not successfully sequenced. These proteins were effective in significantly enhancing bacterial clearance of homologous P. aeruginosa challenge. For KatA, Pa13 and Pa70, clearance was associated with a marked phagocytic cell recruitment. In contrast, amidase and aminopeptidase demonstrated clearance with a minimal cellular response. Proteins; azurin and ACP were non-protective, failing to clear a live P aeruginosa challenge. Analysis of the antigen-specific responses of these nonprotective proteins and comparison with those antigens which enhanced bacterial clearance were used to determine factors that may contribute to the resolution of an acute pulmonary infection. The study has demonstrated that mucosal immunisation using purified protein antigens can enhance the clearance of pulmonary infection with P. aeruginosa. It has also contributed to the understanding of immune responses to newfound antigens of P. aeruginosa and identified antigen-specific responses which confirm their potential as vaccine candidates.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

No description available.

Distant metastases are a limiting factor in cancer patient survival as they are least accessible to conventional therapies. Effective therapy should treat primary tumours and metastatic disease. Use of image-guided radiation therapy (IGRx) enables high doses of radiation to be delivered for better tumour control while minimizing toxicity to healthy tissues. Systemic effects on distant non-irradiated tissues have been observed following IGRx. This phenomenon, termed the abscopal effect, is hypothesized to be mediated by the immune system. The inflammatory milieu generated following IGRx may activate immune cells to mount specific anti-tumour responses. The work described in this thesis aims to develop a model to study the abscopal effect, and evaluate the potential of combining IGRx and immunotherapy to enhance such distant tumour killing. Results from these studies may have clinical implications, where a combined IGRx and immunotherapy approach may prove useful in eliciting regression of local tumours and distant metastases.

Distant metastases are a limiting factor in cancer patient survival as they are least accessible to conventional therapies. Effective therapy should treat primary tumours and metastatic disease. Use of image-guided radiation therapy (IGRx) enables high doses of radiation to be delivered for better tumour control while minimizing toxicity to healthy tissues. Systemic effects on distant non-irradiated tissues have been observed following IGRx. This phenomenon, termed the abscopal effect, is hypothesized to be mediated by the immune system. The inflammatory milieu generated following IGRx may activate immune cells to mount specific anti-tumour responses. The work described in this thesis aims to develop a model to study the abscopal effect, and evaluate the potential of combining IGRx and immunotherapy to enhance such distant tumour killing. Results from these studies may have clinical implications, where a combined IGRx and immunotherapy approach may prove useful in eliciting regression of local tumours and distant metastases.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

Milk fat globule - epidermal growth factor - factor 8 protein (MFG-E8) is an important mediator of the tolerogenic functions of GM-CSF, and a dominant-negative RGE mutant augments the therapeutic potential of irradiated, GM-CSF-secreting tumor vaccines (GVAX) in the MFG-E8-negative B16 melanoma model. The frequent expression of MFG-E8 in various solid and hematological malignancies, however, prompted us to investigate the effect of the RGE mutant in a MFG-E8-positive transplantable breast tumor model. Here, we report that MFG-E8 blockade augmented anti-tumor humoral responses and modulated immune infiltrates at vaccination sites, which was associated with defective phagocytosis and clearance of apoptotic tumor cells. The RGE mutant enhanced the therapeutic potential of two irradiated, GM-CSF-secreting vaccines and improved protection correlated with augmented tumor-specific IgG1 and IgG2a antibody responses as well as increased ratios of T effectors to Tregs in TILs. These findings are consistent with the notion that MFG-E8 blockade potentiates anti-tumor responses through the preferential expansion of effector over regulatory T cells. Our data also validate the use of the RGE mutant to achieve therapeutically effective MFG-E8 blockade even in the context of tumors and vaccines that express high levels of endogenous MFG-E8.

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine