Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

Advances in our understanding of the molecular events leading to cancer have facilitated the development of next-generation targeted therapies. Among the most promising new approaches is immuno-oncology, where therapeutic agents engage the immune system to fight cancer. One exciting strategy therein is the adoptive transfer of ex vivo cultivated tumor-specific T lymphocytes into a cancer patient. Tumor-specific T cells can be produced by engineering a patient’s own T cells with synthetic receptors (e.g. chimeric antigen receptors (CARs)) designed to redirect T cell cytotoxicity against a tumor target. CAR-engineered T cells (CAR-T cells) were expected to be a non-toxic cellular therapy which would seek out and specifically eliminate disseminated tumors. The clinical experience supports the promise of CAR-T cell therapy (striking efficacy has been observed in the treatment of hematological malignancies), while highlighting areas for improvement; CAR-T cell use has been associated with a host of toxicities and robust clinical efficacy has yet to be replicated in solid tumors. This thesis uses pre-clinical models to describe previously unappreciated aspects of CAR-T cell-associated toxicity and novel synthetic receptor strategies, including: i. The capacity of NKG2D-based CAR-T cells to mediate toxicity. ii. The utility of designed ankyrin repeat proteins as CAR antigen-binding domains. iii. The discovery that variables intrinsic to human CAR-T cell products contribute to toxicity. iv. A novel synthetic receptor capable of redirecting T cell specificity against a tumor target – the T cell antigen coupler (TAC). Unlike equivalent CAR-T cells, TAC-T cells are capable of mediating efficacy against a solid tumor in the absence of toxicity. We anticipate that these results will contribute towards the development of next-generation synthetic receptor-engineered T cell products that can deliver upon the promise of safe, systemic cancer therapeutics. / Thesis / Doctor of Philosophy (PhD) / The human immune system has the unique capacity to “seek and destroy” tumor cells throughout the body. A novel class of drugs, immuno-oncology agents, harness this ability to fight cancer. Within this class is a new cellular drug where genetic engineering is used to create killer immune cells (called T cells) capable of recognizing and eliminating tumors. Two of these cellular drugs have recently received FDA approval, supporting the feasibility of this approach. However, further research is needed to improve the safety of engineered-T cells and increase the number of patients whom can benefit from their use. This thesis uses laboratory investigations to better understand the side-effects associated with anti-cancer engineered-T cells and evaluate new engineering strategies. We anticipate that these results will contribute towards the development of next-generation engineered-T cell drugs which retain the ability to function systemically against cancer but offer an enhanced safety profile.

[EN] Lung cancer is the leading cause of cancer-related death worldwide, and is the third most common cancer type; it can be classified into two subgroups based on histology: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). The 5-year survival still remains poor and despite the existence of several distinct tumour phenotypes, therapeutic decisions are mainly based on clinical features such as stage or performance status. This highlights the need for new diagnostic and prognostic biomarkers in different types of samples (such as blood, fresh-frozen tissue or formalin-fixed, paraffin-embedded samples). The field of tumour immunology has changed in the last decade, and it is now accepted that the immune system plays a pivotal role in cancer. Although the immune cells that infiltrate the tumour microenvironment are potentially capable of eliminating tumour cells, they cannot prevent tumour development and progression. Tumours acquire mechanisms to regulate their immune microenvironment such as the release of a series of factors to subvert normal reaction mechanisms, the modulation of co-stimulatory pathways, also known as immune checkpoints, and the induction and attraction of suppressor cells (myeloid-derived suppressor cells, tumour-associated macrophages, and regulatory T cells). The potential effect of the patient's immune system on clinical outcome is important for the identification of prognostic markers as well as markers that predict treatment responses. The study of immune-related markers, especially those implicated in immunoregulatory processes, could provide valuable prognostic information that could help in many applications in future clinical practice. Thus, the objective of this thesis is to characterise cancer immunoregulation biomarkers and to evaluate the possible correlation between these biomarkers and clinicopathological and prognostic variables in patients with NSCLC by the use of well-tested and accurate techniques such as quantitative PCR and immunohistochemistry. Furthermore, this study will provide information about the immunological features of the tumour microenvironment in NSCLCs. / [ES] El cáncer de pulmón es una de las principales causas de muerte relacionada con cáncer en el mundo, siendo el tercer tipo de cáncer más común. El cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón y la supervivencia a los 5 años va desde el 50% en estadios IA hasta el 15% en estadios IIIA. Hasta el momento, no se han descubierto biomarcadores capaces de predecir la progresión de la enfermedad en pacientes tanto en estadios resecables como en estadios avanzados, por lo que existe una clara necesidad de realizar estudios centrados en la búsqueda de biomarcadores pronósticos y diagnósticos en los diferentes tipos de muestra disponibles, como por ejemplo sangre, tejido fresco y tejido parafinado. El campo de la inmunología tumoral ha cambiado en la última década y actualmente se sabe que el sistema inmune juega un papel clave en cáncer. Las células inmunes que infiltran el tumor son un componente más del microambiente tumoral. Pese a que son potencialmente capaces de eliminar los antígenos tumorales, estas células no pueden evitar la formación y progresión tumoral. Esto es debido a que el tumor adquiere diversos mecanismos de regulación del microambiente tumoral con el objetivo de escapar del ataque del sistema inmune, como por ejemplo liberación de factores que impiden el correcto funcionamiento de los mecanismos de reacción inmune, modulación de vías co-estimuladoras y reclutamiento y activación de células inmunoreguladoras como las células T reguladoras, las células mieloides supresoras y los macrófagos asociados a tumores. El estudio de marcadores relacionados con la respuesta inmune y concretamente con los procesos de inmunoregulación puede proporcionarnos información pronóstica y predictiva relevante sobre los pacientes con cáncer. Por todo ello, el principal objetivo de esta tesis doctoral es analizar la presencia de marcadores relacionados con la inmunoregulación y evaluar su posible correlación con las variables clínico-patológicas y pronósticas en pacientes con CPNM mediante el uso de técnicas fiables y aplicables en la práctica clínica como la PCR cuantitativa y la inmunohistoquímica. Así mismo, esto nos permitirá conocer en mayor profundidad las características inmunológicas del microambiente tumoral en pacientes con CPNM. / [CA] El càncer de pulmó és una de les principals causes de mort relacionades amb càncer al món, sent a més a més el tercer tipus de càncer més comú. El càncer de pulmó no microcític (CPNM) representa el 85% de tots els casos de càncer de pulmó aproximadament i la supervivència als 5 anys continua sent molt baixa. Fins el moment, no s'han descobert biomarcadors capaços de predir la progressió de la malaltia tant en pacients en estadis inicials com en estadis avançats. Per aquest motiu, existeix una clara necessitat de realitzar estudis centrats en la recerca de biomarcadors pronòstics i predictius en els diferents tipus de mostres disponibles, com per exemple sang, teixit fresc i teixit parafinat. El camp de la immunologia tumoural ha canviat en l'última dècada i actualment se sap que el sistema immune exerceix un paper clau en el càncer. Les cèl¿lules immunològiques que infiltren el tumour són un component més del microambient tumoural. Malgrat que aquestes cèl¿lules són potencialment capaces d'eliminar el antígens tumourals, s'ha evidenciat que no poden previndre la formació i progressió tumoural. Una de les raons per les quals s'observa aquest fenomen és que el tumour adquireix diversos mecanismes de regulació del microambient tumoural. Aquests mecanismes es basen en l'alliberació de factors que impedeixen el correcte funcionament del sistema immune, la modulació de vies coestimuladores i el reclutament i activació de cèl¿lules immunoreguladores com poden ser les cèl¿lules T reguladores, les cèl¿lules mieloides supressores i els macròfags associats a tumour. L'estudi de marcadors relacionats amb la resposta immune i més concretament amb els processos d' immunoregulació pot proporcionar informació pronòstica i predictiva rellevant sobre els pacients amb càncer. Per tot això, el principal objectiu d'aquesta tesi doctoral és analitzar la presència de marcadors relacionats amb la immunoregulació i avaluar la seva possible correlació amb les variables clinicopatològiques i pronòstiques de pacients amb CPNM mitjançant l'ús de tècniques fiables i aplicables a la pràctica clínica com són la PCR quantitativa i la immunohistoquímica. Així mateix, aquestes anàlisis ens permetran conèixer amb major profunditat les característiques immunològiques del microambient tumoural de pacients amb CPNM. / Usó Marco, M. (2015). ANALYSIS OF IMMUNOREGULATORY BIOMARKERS IN NON-SMALL CELL LUNG CANCER [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51283

Since their discovery, natural killer cells (NK) cells have been implicated as important players in cancer immunosurveillance. In recent years, researchers have taken advantage of this role by developing NK cell-based immunotherapies in the fight against cancer. While these treatments have been moderately successful against hematological malignancy, they are less effective against solid cancers. This lack of success partially results from the immunosuppressive effects of the tumor microenvironment (TME). While tumors use myriad processes to evade the immune system, the avid consumption of nutrients common to NK and cancer cell metabolism and the production of toxic waste products can have significant deleterious effects on NK cell anti-tumor function. However, it may be possible to avoid some of this tumor-induced inhibition of NK cell anti-tumor function by manipulating NK cell metabolism and/or environmental conditions. Recent studies have revealed that different activation regimens can affect the metabolic dependencies of different NK cell subsets. Furthermore, studies have identified potential targets in the TME that can make the environment less hostile for infiltrating NK cells. By considering the interrelationship of NK cell metabolism and function—especially in the TME—this thesis illuminates potential strategies to modulate immunometabolic suppression. Despite the promising work already done, many gaps in the knowledge of NK cell metabolism remain. Future work will need to investigate the specific molecular mechanisms linking metabolism and function, the role of tissue-resident NK cells in cancer immunosurveillance, and the influences of chronic disease and altered systemic metabolism on NK cell anti-tumor activity.

T lymphocytes engineered with chimeric antigen receptors (CARs) have shown remarkable success in the treatment of leukemias. Conventional CARs seek to recapitulate TCR and costimulatory signals through fusion of T cell signaling elements into a single receptor. The robust anti-tumor activity of CAR T cells is often accompanied by debilitating toxicities due to excessive T cell activation and cytokine production following infusion. Our lab has generated a novel chimeric receptor termed T cell antigen coupler (TAC), which is designed to engage native T cell signaling domains for cellular activation. In a murine xenograft model, we previously found that TAC T cells mediated rapid tumour regression in the absence of toxicities. Comparatively, CAR T cells elicited significant lethal toxicities to the mice due to reactivity against an unspecific antigen that resulted in excessive proliferation and cytokine production in vivo. Here, we report that TAC and CAR T cells have fundamentally different biology, both at rest, and during activation. TAC T cells were more sensitive to the context of stimulation compared to CAR T cells. Whereas TAC T cells can discriminate between antigen bound to a bead, or antigen present on a cell, CAR T cells do not make the same distinction and responds equally well to both. Compared to several different CAR constructs, TAC T cells are less prone to tonic signaling and T cell differentiation in the absence of antigen. These findings support that TAC T cells may pose a safety benefit as a cancer immunotherapy, due to its distinct biology from CAR T cells that enables them to require more stringent contexts for activation. / Thesis / Master of Science (MSc) / Cytotoxic T cells are also known as “resident killer” cells of the immune system, as they can seek and eliminate diseased or infected tissue, including cancer cells. However, cancer cells can evade elimination by T cells over time. Genetic engineering of T cells allows us to re-arm T cells against cancer cells. T cells isolated from a patient are genetically modified to recognize cancer cells specifically. So far, these modified T cells have been successful against several leukemias. However, the side effects of this treatment can be substantial and life-threatening, due to the massive reaction of the T cells against the cancer cells following infusion. We explore the biology of two different types of engineered T cells to better understand the interaction between T cell and tumour cell. Our results aim towards mitigating the side effects of T cell treatment, while investigating how we can improve its effectiveness for the future.

Abstract Macrophages found in the tumor microenvironment play a crucial role in initiating an immunosuppressive tumor microenvironment that negatively impacts immunotherapy efficacy and aids tumor progression and metastasis. Constituting the most abundant immune cell in tumor microenvironment (TME), tumor associated macrophages (TAMs) have emerged as an attractive approach for anti-cancer therapy. However, two major challenges need to be overcome for successfully utilizing macrophages for immunotherapy. First, tumors repolarize the TAMs predominantly to M2 tumor-aiding phenotype by secreting various immunosuppressive cytokines. Second, cancer cells overexpress a membrane protein CD47 that interacts with signal-regulating protein alpha (SIRPalpha) expressed on macrophages. This crosstalk provides a downregulatory signal in the form of activation of SHP1/2 that inhibits cancer cell phagocytosis, and CD47, therefore, functions as a “don’t-eat-me” signal. We rationalized that these challenges can be overcome by engineering a nanoparticle system that can deliver a rationale combination of immunomodulatory agents to the TAMs that can both repolarize the M2 macrophages to M1 phenotype efficiently and concurrently block CD47-SIRPalpha interactions by inhibiting SHP2 signaling. Herein, we designed a lipid nanoparticle (LNP) system loaded with amphiphilic R848-cholesterol in its hydrophobic lipid bilayer, while SHP099 gets encapsulated in the hydrophilic core. Our previous studies have shown that the conjugation of cholesterol to the inhibitor stabilizes the lipid bilayer at a high inhibitor concentration. The LNPs showed high optimal drug loading, size, and stability. In vitro studies showed that the M2 macrophages treated with the LNPs system repolarized to M1 phenotype and expressed co-stimulatory molecules while having enhanced phagocytic potential. In vivo efficacy studies in 4T1 tumor-bearing mice showed that LNPs exhibit superior anti-tumor efficacy compared to other treatments. We evaluated the effect of MARCO-targeted LPNs by the conjugating anti-MARCO antibody on the LPN surface. However, no comparable difference in treatment efficacy was observed between the targeted MARCO-LNPs and the non-targeted LNPs. These results demonstrate that the MARCO targeting system designed in this study is largely ineffective in the targeted delivery of its drug cargo specifically to TAMs. Thus, the lipid nanoparticle-mediated co-delivery of a rational combination of TLR7/8 agonist and SHP2 inhibitor in the TAMs increases M2 to M1 repolarization and phagocytosis potential of macrophages. Recommended Citation Malik, V., Ramesh, A. and Kulkarni, A.A. (2021), TLR7/8 Agonist and SHP2 Inhibitor Loaded Nanoparticle Enhances Macrophage Immunotherapy Efficacy. Adv. Therap., 4: 2100086. https://doi.org/10.1002/adtp.202100086

The kinetics of tumor growth and progression are governed by the interaction between tumor cells, the non-malignant stroma and both innate and adaptive immune cell lineages. Innate immunity has a critical role in the control of tumor cell growth and metastasis. The microenvironment of many tumors is populated with innate immune cells, including regulatory natural killer (NK) cells and dendritic cells (DCs), tumor associated macrophages, and myeloid derived suppressor cells, that suppress normal immune function. Much of our understanding of interactions between tumors and the innate immune system is based on experimental studies performed in mouse “syngenic” models. However, there is clear need for a mechanistic understanding of the human innate immune system within the tumor microenvironment. The goal of my thesis is to characterize the interactions between human innate immune cells and tumors and to define specific pathways and cell lineages that are targets for immune modulation. A central focus of my thesis is the use of cutting-edge humanized mouse models based on the immunodeficient NOD-scid IL2Rgnull (NSG) mouse strain to study human immuno-oncology. In the first section of my thesis I describe studies that evaluate the influence of inflammatory stimuli on innate immune control of tumors. Agents that induce inflammation have been used since the 18th century for the treatment of cancer. The inflammation induced by agents such as toll-like receptor (TLR) agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. While NSG mice lack murine adaptive immunity (T and B cells), these mice maintain a residual murine innate immune system that responds to TLR agonists. Here I describe a novel NSG mouse strain lacking TLR4 that fails to respond to lipopolysaccharide (LPS). NSG-Tlr4null mice support human immune system engraftment and enables the study of human specific responses to TLR4 agonists. My data demonstrate that specific stimulation of TLR4 activates human innate immune system and promotes regression of human patient derived xenograft (PDX) tumors. In the second section of my thesis I describe the development of an NSG mouse strain that constitutively expresses human Interleukin 15 (IL15) and supports the development of functional human NK cells. Using humanized NSG-IL15 transgenic mice (NSG-Tg(Hu-IL15), my data clearly demonstrate a critical role for human NK cells in limiting growth of a PDX melanoma. In the third section of my thesis I describe, the use of the bone marrow/liver/thymus (BLT) humanized mouse model to study the interactions between the human immune system and PDX melanoma and to evaluate the response of the melanoma to immunotherapy modalities. My results collectively suggest that mice engrafted with human immune systems and bearing human tumors can be harnessed as translational models, which are critically needed as tools to study tumor immunotherapy. These humanized mouse models are an ideal translational tool to advance our understanding of human immuno-oncology and for development and testing of novel immune therapies for the treatment of malignancies.

Stimuler la réponse immunitaire anti-tumorale constitue une voie d’avenir indiscutable pour le traitement des cancers. Aujourd'hui, les thérapies ciblées à base d'anticorps ont une place majeure dans l’immunothérapie des cancers du sein de par leur impact positif sur le pronostic des patientes. Cependant, les cancers du sein triple négatifs (TNBC) résistent aux innovations thérapeutiques actuelles, et, par défaut de traitement ciblé efficace, restent de sombre pronostic. Notre équipe développe des stratégies d’immunothérapie à base d'anticorps bispécifiques (bsFabs) conçus à partir de fragments d'anticorps de camélidés qui présentent la particularité de cibler simultanément les cellules immunitaires et tumorales. Ainsi, mon projet visait à évaluer le potentiel anti-tumoral de deux bsFabs sur des modèles précliniques de TNBC à travers leur capacité à activer et à rediriger le système immunitaire contre les cellules tumorales. La finalité du projet est de proposer un nouvel axe de thérapie ciblée susceptible d'améliorer le pronostic des patientes atteintes de TNBC. / Mounting evidence of the key contribution of NK cells in immunity against cancer has boosted the investigations on NK cell-based therapies. Among these strategies, monoclonal antibody-based therapeutics (mAbs) are currently the fastest growing segment of the medicine market. Despite therapeutic innovations, triple negative breast cancers (TNBC) remain insensitive to the current targeted or hormono-therapies. Our objective is to manipulate NK cell functions and tumor targets using an original format of nanobody-based bispecific antibodies (bsFab) to revert the dampened immune response for treating TNBC. Thus, we generate two bsFabs able to crosslink NK and tumor cells. NK antitumor effects driven by mAbs and bsFabs, alone or in combination, were investigated in vitro and in vivo on preclinical TNBC models. Here, we demonstrate the potential of bsFabs to enlarge the number of patients eligible for breast cancer immunotherapy and prompt to consider combination strategies.

CD73 est un ecto-enzyme qui a été associé à la suppression de l'immunité anti-tumorale. Ses valeurs pronostiques et thérapeutiques ont été mises de l'avant dans plusieurs types de cancer. La première hypothèse du projet est que l'expression de CD73 dans la tumeur prédit le pronostic des patients atteints du cancer de la prostate. L'expression de CD73 a été étudiée par immunofluorescence dans des échantillons de tumeur. Puis, des analyses univariées et multivariées ont été conduites pour déterminer si l'expression de CD73 permet de prédire la récidive biochimique des patients. Nous avons déterminé que CD73 prédit indépendamment le pronostic des patients atteints du cancer de la prostate. De plus, nous avons déterminé que son expression dans le tissu normal adjacent ou dans la tumeur prédit différemment la survenue de la récidive biochimique. La deuxième hypothèse est que l'inhibition de CD73 permet d'améliorer l'efficacité d'un vaccin thérapeutique contre le cancer de la prostate. L'effet d'un vaccin de type GVAX a été étudié dans des souris CD73KO ou en combinaison avec un anticorps ciblant CD73. Nous avons observé que l'efficacité du vaccin était augmentée dans les souris où CD73 était absent. Cependant, la combinaison avec l'anti-CD73 n'a pas permis d'améliorer l'efficacité. / The ectoenzyme CD73 has been associated with suppression of anti-tumor immunity. Its prognostic and therapeutic values have been highlighted in many types of cancer. The first hypothesis of this project is that tumor CD73 expression predicts the prognosis of prostate cancer patients. CD73 expression was determined by immunofluorescence in tumor tissues. Univariates and multivariates analysis were conducted to determine if CD73 expression could predict patients' biochemical recurrence. We determined that CD73 predicts independently the prognosis of prostate cancer patients. Moreover, we determined that CD73 expression in normal adjacent tissue or in the tumor predicts differently the occurrence of biochemical recurrence. The second hypothesis is that CD73 blockade enhances the efficacy of a therapeutic vaccine against prostate cancer. The GVAX-like vaccine was studied in CD73KO mice or in combination with an antibody against CD73. We observed that the vaccine was more efficient in mice where CD73 was absent. However, the combination with the anti-CD73 did not enhances the efficacy of the vaccine.

La reconnaissance de l’implication du système immunitaire dans le cancer a guidé l’industrie vers de développement d’immunothérapies nombreuses et prometteuses. Or, à l’ère de l’immuno-oncologie, on constate un manque criant de modèles précliniques capables de simuler les interactions immunitaires entre un patient et sa tumeur. Pour remédier à cette situation, nous avons développé des modèles de souris humanisées combinant la reconstitution immunitaire de souris immunodéficiente et l’injection de lignées tumorales issues d’un même donneur. L’utilisation de cellules souches pluripotentes induites (iPSC) a permis notamment le développement de multiples lignées tumorales à partir d’un seul donneur sain, facilitant ainsi l’accès aux cellules immunitaires nécessaires à l’humanisation des souris. La transformation des cellules primaires ou dérivées d’iPSC a été faite par la transduction lentivirale des proto-oncogènes de la télomérase (hTERT), de Ras oncogénique (HRASV12) et de la région précoce du viruse simen 40 (SV40ER) encodant les gros et petits antigènes T (LgT et SmT). Cette approche permis de générer des tumeurs de haut grade, agressives et peu différenciées à l’aide de fibroblastes primaires et de cellules hépatiques, de cellules souches neurales et d’astrocytes dérivés d’iPSC. Dans tous les cas, les tumeurs ainsi générées ont été efficacement reconnues, infiltrées et souvent rejetées par le système immunitaire autologue implanté. Le rejet partiel de la plupart de ces tumeurs ouvre toutefois la porte à l’évaluation préclinique d’immunothérapies diverses reposant sur les réactions immunitaires anti-tumorales de l’hôte. Par exemple, nous avons pu étudier l’impact d’un traitement d’inhibition du point de contrôle immunitaire PD-1 sur la croissance de tumeurs d’origine fibroblastique où une augmentation marquée du taux d’infiltration immunitaire humaine a été observé sans toutefois mener à une réduction significative du fardeau tumoral. Nous avons aussi pu produire, de façon autologue, des lymphocytes T exprimant un récepteur d’antigène chimérique (CAR) contre le ganglioside GD2, un antigène tumoral préalablement identifié et détecté sur les tumeurs de cellules souches neurales générées par notre approche. L’efficacité cytotoxique de ces CAR a ainsi pu être validée in vitro dans un système autologue. Finalement, nous avons utilisé le modèle de tumeurs fibroblastiques dans des contextes immunitaires autologues et allogéniques pour déterminer si le potentiel immunomodulateur des cellules stromales mésenchymateuses (MSC) pouvait affecter la croissance tumorale. Selon nos résultats, les MSC n’auraient aucun effet ni sur le taux d’émergence et de croissance tumoral, ni sur l’infiltrat immunitaire, suggérant que leur utilisation thérapeutique serait sécuritaire en ce qui concerne ce type de tumeurs ayant préalablement un microenvironnement tumoral immunosuppresseur. En somme, les modèles innovateurs décrits dans cette thèse visent à améliorer la qualité prédictive des modèles murins précliniques en immuno-oncologie en récapitulant certaines interactions immunitaires entre un patient et sa tumeur. La grande flexibilité de cette approche permettra d’adapter aisément le modèle aux problématiques d’intérêt, tant fondamentales que précliniques. / Identification of the human’s immune system implication in cancer has guided the biotech industry towards the development of numerous and promising cancer immunotherapies. However, in the era of immuno-oncology, a distinct lack preclinical models can simulate the interactions between a patient’s tumor and immune cells. To tackle this issue, we developed humanized mouse models combining immune reconstitution of immunodeficient mice and injection of tumor cells lines from the same human donor. The use of induced pluripotent stem cells (iPSC) allowed the generation of multiple tumorigenic cell lines from a single donor, facilitating access to autologous immune cells necessary for mouse immune humanization. The transformation of primary or iPSC-derived cell lines was done using lentiviral transduction of proto-oncogenes telomerase (hTERT), oncogenic Ras (HRASV12) and simian virus 40 early region (SV40ER) encoding large and small T antigens (LgT and SmT). This approach allowed to generate high grade, aggressive and undifferentiated tumors from primary fibroblasts and iPSC-derived hepatic cells, neural stem cells and astrocytes. In all cases, such tumors were efficiently recognized, infiltrated and often rejected by the implanted autologous immune system. However, partial rejection of most tumors allows for preclinical evaluation of targeted immunotherapies relying on the hosts’ pre-existing immune response. For instance, we could study the impact of PD-1 checkpoint blockade inhibition on tumor growth in fibroblastic tumors where a significant increase in tumor infiltration was observed, but without an associated decrease in tumor burden. We could also produce autologous chimeric antigen receptor (CAR)-expressing T lymphocytes against GD2 ganglioside, a previously described tumor antigen detected on our neural stem cell-derived tumor cells. Cytotoxic efficiency of these autologous CAR T cells could thus be validated in vitro. Finally, we used our fibroblast-derived tumor models in autologous and allogeneic settings to determine if mesenchymal stem cells’ (MSC) immunomodulatory potential could impact tumor growth. Our results showed that MSC had no effect neither on tumor emergence and growth nor on immune infiltration, suggesting therapeutic use of these cells should be safe regarding such tumors already harboring a strongly immunodeficient microenvironment. Overall, the novel models described in this thesis aim at improving the predictive capacity of mouse pre-clinical models in immuno-oncology by recapitulating some immune interactions between a patient and its tumor. The great flexibility of this approach will allow for easy adaptation to many research problematics both preclinical and fundamental.