Pseudomonas aeruginosa is an opportunistic pathogen capable of infecting the human cornea. Such infections are difficult to treat, and are often fulminative, in that the infected eye is lost, or severely scarred. The use of alternative therapeutic agents has been necessitated by the frequent failure of conventional antibiotic therapy. Equine hyperimmune antilipopolysaccharide plasma (Anti-LPS) was obtained by the plasmapheresis of suitably immunized horses. The plasma contained 1,O- 1 ,5g/ml of LPS-precipitible IgG antibodies. Topical administration of Anti-LPS as a lavage was shown to be effective against Pseudomonas keratitis in rabbits and guinea pigs. Subsequent use of topical corticosteroids was found to further reduce corneal pathology. The improvement noted in these experimental infections involved all three parameters measured, area of keratitis, depth of lesion, and degree of vascularization. In vitro , Anti-LPS was shown to be rapidly bactericidal for Gram negative bacteria. The plasma can therefore be said to have a dual mechanism of action: antitoxic, and antibacterial. Ocular administration of Anti-LPS, by both the topical and subconjunctival routes, was well tolerated by both rabbits and baboons. In conclusion, Anti-LPS is a potentially useful immunotherapeutic agent with many applications in both veteriary and human medicine, particularly in the treatment of surface infections involving antibiotic-resistant Gram negative bacteria / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in

No description available.

Triple negative breast cancer (TNBC) is an aggressive subtype of the disease with dismal clinical outcome. Immune checkpoint blockade (ICB), which blocks inhibitory pathways on T cells, has surged to the forefront of cancer therapy with clinical success in a variety of cancer types. However, ICB for TNBC only benefits 10-20% of patients. Thus, a deeper understanding of the immune landscape in TNBC is required to develop efficacious therapies and delineate prognostic biomarkers of disease. We have developed combination therapy platforms that sensitize TNBC tumors to ICB. Using a clinical chemotherapy (FEC) combined with oncolytic virotherapy (oHSV-1) we show enhanced tumor-infiltrating lymphocytes (TILs), upregulation of B cell receptor signaling pathways, suppression of myeloid-derived suppressor cells (MDSCs) and improved survival. In vivo depletion studies revealed that B cells were required to achieve cures with treatment. Furthermore, the absence of B cells resulted in the expansion of MDSCs. This crucial finding of the importance of B cells for mediation and downregulation of MDSCs is a novel and significant contribution to the field. RNA sequencing revealed that two of the top upregulated genes in mice treated with FEC + oHSV-1 were S100A8 and S100A9, calcium binding proteins highly expressed in myeloid cells. These genes have controversial findings in the literature with both pro- and antitumorigenic functions being reported. Investigation of data from the Cancer Genome Atlas revealed that high levels of S100A8 and S100A9 correlate with improved prognostic outcomes in breast cancer patients. In line with the clinical data, our data suggests that increased levels of S100A8 and S100A9 results in improved responses to immunotherapy treatments and that this increased expression is involved in macrophage-mediated epigenetic reprogramming of the tumor microenvironment. Our second therapeutic platform used a radiolabeled biomolecule containing the beta-emitting radioisotope, lutetium-177. We found that two doses of radiotherapy, combined with ICB improved overall survival in murine TNBC tumors, increased TILs and suppressed circulating MDSCs. These findings offer insight into the newly explored field of combination radioimmunotherapy and again highlight the importance of suppressing MDSCs to alleviate tumor immunosuppression. / Thesis / Doctor of Philosophy (PhD) / Triple-negative breast cancer (TNBC) has poor prognostic outcomes due to lack of expression of targets for therapy. As such, patients routinely undergo aggressive treatment regimens with many harsh side effects, including high levels of toxicity. Immunotherapy, a form of therapy that boosts the immune system to fight cancer cells, has gained increasing prominence largely due to its safety and low toxicity to the patient. In the work within this dissertation, we have developed therapeutic platforms and studied them in a murine model of TNBC. The completed studies show the use of clinical therapies, in combination with immunotherapy and investigate the fundamental biology associated with therapeutic outcomes. These findings contribute knowledge to progress clinical regimens for TNBC patients as well as to better identify patients that will respond to therapy. Although this proposal is specific to breast cancer, the underlying concepts can be applied to many other forms of cancer.

Cancer immunotherapy has revolutionized the field of oncology by giving the possibility to ligands (e.g., antibodies) to selectively target tumor antigens and accumulate at the site of the disease while sparing normal tissues. During the past years, the number of patient eligible for immune-based cancer treatments has seen an exponential increase as these therapies are becoming first line treatment for many cancer indications. A promising anticancer strategy consists in the targeted delivery of bioactive compounds (e.g., cytokines) to the tumor microenvironment with high-affinity ligands specific for tumor-associated antigens. This approach improves the efficacy of the drug by reducing related side effects and increasing the therapeutic index of the payload. Currently, antibodies represent one of the most successful class of ligands used for this purpose as they can be generated against virtually any antigen. Many methodologies have been described for the generation and isolation of antibodies with high antigen-binding specificity. Among these, phage display technology has emerged as a powerful and versatile tool for the in vitro discovery of antibodies and peptides. Since it was invented in mid 1980s, phage-display has paved the way to the generation of more than 70 phage–derived monoclonal antibodies (mAbs) that entered clinical studies, and 14 of which have been approved in the market. Cytokines are proteins capable of modulating the activity of the immune system and some cytokine-products have gained marketing authorization for the treatment of cancer. In order to increase the therapeutic index of cytokine payloads, the generation of fusion proteins with tumor-homing antibodies has been proposed. These so-called “immunocytokine” products constitute a class of “armed” antibody products, in which a tumor-targeting immunoglobulin is fused with a cytokine. In this thesis, we present the generation and characterization of antibodies specific for two tumor microenvironment-associated antigens: Tenascin C and Fibroblast Activation Protein. Both antigens are undetectable in healthy tissues but abundantly expressed in the tumor stroma. In the first part of the thesis, we have isolated antibodies specific for the spliced domain D of Tenascin C from the synthetic phage library “ETH2Gold”. Antibodies were affinity matured randomizing key residues of CDR1 of heavy and light chains. The highest affinity clone, R6N, was characterized in vitro and in vivo showing selective accumulation at the tumor site in mouse models of cancer. An immunocytokine featuring IL12 as payload has been generated and its therapeutic activity evaluated in tumor bearing mice. R6N-IL12 exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. In the second part of this thesis, a monoclonal antibody has been isolated against Fibroblast Activation Protein. After affinity maturation of the CDR2 of heavy and light chains of the parental antibody C5, the selected 7NP2 antibody showed improved affinity and excellent tumor targeting properties in SKRC52-hFAP tumor bearing mice. When fused to IL12, 7NP2 was able to induce tumor growth retardation and tumor remission in mouse models of cancer. Collectively, in this thesis we have isolated and validated two monoclonal antibodies selective for tumor microenvironment-associated antigens. Both antibodies when fused to IL12 induced tumor growth retardation and remission in immunodeficient and immunocompetent mouse models providing a rationale for possible future applications of R6N and 7NP2 antibodies for the treatment of cancer patients.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults which is characterized by extensive cellular and genetic heterogeneity. Even with surgery, chemotherapy with temozolomide, and radiation, tumor re-growth and patient relapse are inevitable. The extensive inter- and intra-tumoral heterogeneity (ITH) of recurrent GBM emerges from dysregulation at multiple -omic levels of the tumor. ITH exits at the cellular level due to a small subpopulation of chemo- and radio-resistant cells, called brain tumor initiating cells, which may drive GBM treatment resistance. Although a wealth of literature describes the biology of primary GBM (pGBM), we currently lack an understanding of how GBM evolves through therapy to become a very different tumor at recurrence, which may explain why therapies against primary GBM fail to work in recurrent GBM (rGBM). Thus, understanding the tumor evolution from a multi-omic perspective is critical for the development of effective therapeutic approaches. The current work focuses on identification and validation of novel predictive and prognostic biomarkers for rGBM using proteomics analysis on a large cohort of patientmatched pGBM-rGBM samples. This work allowed for detailed characterization of rGBM and its cognate TIME toward a better understanding of the molecular players driving recurrence which can be further used for instructing effective targeted and personalized therapies for the treatment of therapy-resistant GBM. In another part, we developed a novel immunotherapeutic modality called dual antigen T cell engager, to target Carbonic Anhydrase 9, a highly enriched hypoxia-inducible enzyme in GBM. We demonstrated that this immunotherapeutic strategy which allows for targeting tumor cells while recruiting and triggering T cells through simultaneously, is highly effective in eliminating tumor cells and can be a complementary component of combinatorial therapy for GBM patients. Altogether, this study provided key data for instructing novel and rational combinatorial polytherapeutic approaches for the treatment of therapy-resistant GBM. / Thesis / Doctor of Philosophy (PhD) / Cancer is the leading cause of death in Canada and Glioblastoma Multiforme (GBM) is the most common type of malignant adult brain tumor which is one of the difficult human cancers to treat. In spite of the multi-model therapy including surgery, chemotherapy, and radiation, tumor re-growth and patient relapse are inevitable. A wealth of literature describes the biology of treatment-naïve or primary GBM, but we currently lack an understanding of how GBM evolves through therapy to become a very different tumor at recurrence, which may explain why therapies against primary GBM fail to work in recurrent GBM (rGBM). Clinical trials have not shown significant survival advantages for GBM patients, due not only to the lack of biological characterization of the distinct landscape of GBM recurrence, but also due to our poor understanding of the tumor immune microenvironment (TIME), the immune cells surrounding the tumor that may somehow fail to attack it due to GBM cells’ ability to suppress the immune system and evade detection. To understand how GBM cells and the TIME evolve under therapeutic pressure, we performed proteomics analysis on a large cohort of primary-recurrent GBM patient samples, to further understand treatment failure and develop effective and empirical combinatorial poly-therapies for the treatment of therapy-resistant GBM. Besides, in the next part of this study we showed existence of few cells within the tumor, termed brain tumor initiating cells (BTICs) can alone drive tumor growth and cause therapy resistance. To be able to target these population of cells, we identified treatment resistant tumor associated markers (highly expressed cell surface proteins) on these cells and developed novel treatments using a new class of biologics, Dual Antigen T cell Engagers (DATEs), to target these tumor associated markers. DATEs act like a “molecular glue” that specifically binds the patient’s own naturally circulating T-cells (soldier cells of the immune system) to cancer cells. Once bound, the T-cells attack and kill the patient’s cancer cells. In this strategy, the abnormal expression of tumor surface proteins can be used as a handle to drive T cell-mediated cell death. We predict that the dual specific antibodies through this study could be used alone or in combination with existing drugs to treat recurrent GBM.

No description available.

This thesis describes a general platform for the synthesis of radiolabelled antibody recruiting small molecules (R-ARMs) for combination radio and immune recruitment therapies. The novel trifunctional ARM was synthesized and radiolabelled with beta (lutetium-177) and alpha (actinium-225) emitting radionuclides in high yield. Biodistribution of the lutetium-ARM revealed rapid renal clearance and minimal uptake in non-target tissues with all organs and tissues containing less than 0.3 %ID/g by 24 hours post-injection. Having determined the pharmacokinetic properties of the ligand, a biodistribution study was performed to determine the targeting potential of the platform. Through the use of a validated bone targeted bisphosphonate, uptake in the arm and leg bones was achieved. Flow cytometry studies successfully demonstrated ARM and antibody dependent immune cell recruitment. Based on the promising results of the ARM in vitro and in vivo, the next step was to perform therapy studies. In order to validate the novel R-ARM, an intratumoral (i.t.) strategy was developed through the preparation of a TCO-bovine serum albumin (BSA) derivative. This new chemical entity was used in both an aggregated and non-aggregated form to retain the R-ARM in the tumour after i.t. administration. Biodistribution showed high retention of the aggregated and non-aggregated BSA out to 120 hours with 167 ± 94 and 81 ± 32 %ID/g respectively remaining in the tumour. An autoradiography study revealed the after i.t. administration the aggregated material was localized in specific regions within the tumour compared to the non-aggregated material which diffused throughout. The aggregated material was used in a single and multi-dosing radiotherapy study in which the latter induced a statistically significant survival advantage compared to the control. One additional multi-dosing study was performed with the non- aggregated material which resulted in the largest survival advantage to date. Intratumoral administration of TCO-BSA linked to the trifunctional tetrazine showed promising radiotherapy results and future work on dose optimization with lutetium and actinium is required prior to the combination R-ARM therapy. In parallel, the efficacy of the unlabelled ARM linked to TCO-BSA was interrogated in preclinical models. The compound was administered i.t. three times per week in a breast cancer tumour model, and response to therapy monitored. The immunized group showed no survival advantage compared to the control group comprised of naïve animals. Biodistribution studies were performed to determine if TCO-BSA was accessible to the bloodstream following i.t. administration in both the aggregated and non-aggregated forms. Saline, aggregated or non-aggregated TCO-BSA were administered i.t. followed by the R-ARM. The results showed very low uptake in the tumour for all three groups, with minimal change in distribution from that of the native R-ARM. This suggests that after i.t. administration, the TCO-BSA was not available to molecules in the bloodstream or the concentration was insufficient to promote in vivo coupling. Further work on this component of the platform is needed before further ARM studies are performed. / Thesis / Doctor of Science (PhD)

Cancer is considered to be one of the most significant public health issues in the world. For the last century, cancer therapy has mainly relied on surgery, radiotherapy, and chemotherapy. Radiotherapy and chemotherapy preferentially kill rapidly proliferating cells. Unfortunately, most of these treatments do not efficiently localize at the tumor site; they are characterized by several undesired toxicities and low therapeutic activity. A real breakthrough in oncology was the advent of cancer immunotherapy. Antibodies are molecules that can specifically target tumors sparing healthy organs, reducing toxicity, and improving the therapeutic window. Cytokines are small potent immunomodulatory proteins that can be used both to stimulate or to suppress the immune system. As a result, cytokines find utility in the development of drugs for the treatment of several malignancies and anti- immune diseases. Some products based on recombinant cytokines (e.g., IL2, TNFα and, INFα) have gained marketing authorization. However, when administered systemically, they are characterized by small dosage tolerability and severe side effects (e.g., pulmonary, gastrointestinal, hepatic, cardiovascular, hematological, and neurological problems). The fusion of cytokines to antibodies capable of binding tumor associate antigens was demonstrated to be a very effective strategy. These so called “immunocytokines” can deliver the active payloads directly to the tumors improving cytokine therapeutic index and sparing healthy tissues. In the first part of this thesis, we describe the development of novel IL15-based immunocytokines. IL15 is a potent pro-inflammatory cytokine, closely related to IL2, that can stimulate the proliferation of T cells, promote the synthesis of immunoglobulins, and preserve the survival of Natural Killer cells. IL15 has been considered for cancer therapy due to its ability to activate CD8+ memory T cells and non-activating regulatory T cells. Attempts to deliver IL15-based immunocytokines to tumors have already occurred in the past, showing a poor targeting ability to the tumor site. In this work, a novel fusion protein composed of IL15 linked to a single chain diabody F8 antibody showed a preferential localization in neoplastic lesions, as well as a potent anti-cancer activity in immunocompetent murine tumor models. Moreover, a potentiated version of the novel anti-cancer prototype was generated by incorporating the Sushi Domain of the IL15Rα. The fusion protein was able to eradicate a lung metastasis in a mouse model of the disease. Immunocytokines administered as monotherapy are rarely capable of inducing complete responses in tumor-bearing mice and in cancer patients. For this reason, combination partners are constantly studied to improve the efficacy, usually using cytotoxic drugs or other immunocytokines. The second part of this thesis is focused on finding the best combination partner for L19-TNF for the treatment of sarcoma malignancies. Notably, the combination of L19-TNF with Dacarbazine has shown complete eradication of solid tumors in all the treated animals. This result provided the rationale to start a new clinical trial in patients to treat advanced or metastatic soft-tissue sarcoma.

Among women worldwide, breast cancer is the second leading cause of death. Despite improved screening techniques and new treatments such as immunotherapies and targeted hormone therapy, survival rates of advanced or metastatic disease are at 27%. Current treatments focus on the primary tumor and are generally ineffective against metastases due to the location and heterogeneity of the metastatic tumor cells. However, all cells rely on the proteostasis network (PN), a collective term for protein synthesis, folding, and degradation, consisting of both chaperones and chaperonins. Chaperonin-containing TCP-1 (CCT) consists of 16 heterologous subunits (CCT1-8) and folds ~10% of the known proteome. However, breast cancer cells rely heavily on CCT clients including oncogenes and mutated tumor suppressors as well as essential cytoskeletal proteins. The objective of my research is to investigate the role of CCT in cancer by overexpressing or depleting a single CCT subunit, CCT2 (2nd subunit of CCT) in both breast cancer and normal breast epithelial cells. We found increasing CCT2 in a 2D setting leads to a more invasive phenotype in breast cancer cells characterized by increased proliferation and migration. Additionally we observed increased tubulin, one of CCT's obligate clients as well as increasing other CCT subunits. Loss of CCT2 leads to cell death, reduction of other CCT subunits, and in vivo prevents tumors from forming. To further understand the role of CCT, we grew CCT2 overexpressing breast cancer lines as spheroids, an in vitro tumor analogue. The CCT2 overexpressing spheroids grew faster and larger than control cells. Additionally, they express increased actin, another CCT obligate client, as well as show significantly increased attachment with both cell-to-cell and cell-to-substrate connections, which could be suggestive of a metastatic phenotype. Finally, we used a CCT inhibitor, CT20p, to treat breast cancer cells and found it induces immunogenic cell death (ICD). ICD stimulates the immune system to attack the tumor cells and mice vaccinated with dying CT20p-treated cells did develop protective immunity against challenge with live cancer cells. Investigating the role of CCT in breast cancer advances both a novel marker and potential target for new cancer treatments, meeting an urgent need to reduce the mortality of advanced and metastatic breast cancer.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy