Most patients with bladder cancer are in the stage of non-muscle-invasive bladder cancer (NMIBC), while 30% of patients progress to the life-threatening muscle-invasive bladder cancer (MIBC) stage because of distant metastases. The selection of treatment options depends on the bladder cancer stage. We established a relationship network for the proteins from the mice urine samples collected during the progression of bladder cancer based on biological pathways and developed population pharmacodynamic models for the proteins in the light of their relationship network. Models that can quantitatively describe changes in the protein profiles of IL1a, IL1b, Csf2, and Casp3 in mice urine samples over time during bladder cancer progression were developed with the consideration of gender differences and progressing age. Our results assist the identification of the early protein diagnostic biomarkers in urine for detecting bladder cancer at its early stages and apply appropriate treatments on patients.

Les métastases hépatiques compliquent l'évolution de 50% des cancers colorectaux (CCR). Plus de la moitié des patients présentent une récidive à distance avec métastases occultes pour lesquelles une chirurgie peut être réalisée dans moins de 20% des cas. L'ablation par radiofréquence (RFA) induit une réponse lymphocytaire T qui n'est pas évaluée après une intervention chirurgicale seule. L'immunothérapie combinée à la RFA pourrait potentialiser cet effet conduisant à une réponse tumorale à distance. Nous proposons une approche qui combine la RFA avec hydrogel thermoreversible libérant des agents immunomodulateurs (GMCSF et BCG) sur le site du traitementPremièrement, nous nous sommes intéressés à la sélection et à la caractérisation de la formulation optimale d’hydrogel par des techniques physicochimiques. Les propriétés de l'hydrogel ont été étudiées par rhéologie et des tests de muco-adhésion ont été mis en place. Le temps de résidence de l'hydrogel et de la protéine dans la zone tumorale a été démontré par imagerie optique. De plus, la cinétique de libération et l'intégrité du GMCSF encapsulé ont été déterminées. Ensuite, nous avons démontré l’efficacité de l’association de la RFA avec le dépôt local de l’hydrogel immunomodulatuer sur un modèle murin de cancer colorectal. En effet, nous avons observé une survie améliorée des animaux et régression complète des tumeurs distantes chez les animaux traités par la combinaison complète. Cette réponse est caractérisée par un niveau élevé de sécrétion de cytokines pro-inflammatoires par les cellules T CD4 et TCD8 et une augmentation de l’infiltrat lymphocytaire dans les tumeurs. Ceci a permis d'envisager une association avec l'immunothérapie anti-PD1 dans le traitement de macrométastases échappant au traitement combiné RFA avec l’hydrogel immunomodulateur. En effet, l’immunothérapie dans le traitement du cancer colorectal métastatique présente une efficacité limitée chez les patients. Notre travail propose a démontré que l’efficacité de l’immunomodulation locale dans l’amélioration des réponses immunitaires dans le cancer colorectal. Ces résultats permettent de reconsidérer l’utilisation de l’immunothérapie chez les patients atteints de CCR métastatique non MSI. / Liver metastases complicate the progression of 50% of colorectal cancers (CRC). More than half of the patients have recurrent remissions with occult metastases for which surgery can be performed in less than 20% of cases. Radiofrequency ablation (RFA) induces a T lymphocyte response that is not observed after surgery alone. Combined immunotherapy with RFA may potentiate this effect leading to a distant tumor response. We propose an approach that combines RFA with thermoreversible hydrogel releasing immunomodulatory agents (GMCSF and BCG) at the treatment site.First, we focused on the selection and characterization of the optimal hydrogel formulation by physicochemical techniques. The properties of the hydrogel were studied by rheology and mucoadhesion tests were set up. The residence time of the hydrogel and the protein in the tumor zone was demonstrated by optical imaging. In addition, the release kinetics and integrity of the encapsulated GMCSF were determined. Then, we demonstrated the effectiveness of the combination of RFA with the local deposition of the immunomodulatory hydrogel on a mouse model of colorectal cancer. Indeed, we observed improved survival of animals and complete regression of distant tumors the complete treatment group. This response is characterized by a high level of pro-inflammatory cytokine secreted by CD4 and TCD8 T cells and an increase Lymphocytes infiltrating tumors. The immune escape of large lesions was reversed by association with anti-PD1 immunotherapy Indeed, immunotherapy in the treatment of metastatic colorectal cancer has limited efficacy in patients. Our work has demonstrated the effectiveness of local immunomodulation in improving immune responses in colorectal cancer. These results make it possible to reconsider the use of immunotherapy in patients with non-MSI metastatic CRC.

Dans les tauopathies, telle la maladie d'Alzheimer, la protéine tau devient anormalement hyperphosphorylée ce qui conduit à son accumulation et à son agrégation intracellulaire. Ce processus aboutit progressivement à une perte neuronale et un déclin cognitif. L'immunothérapie anti-tau est de plus en plus considérée comme un traitement potentiel en vue de bloquer la progression des tauopathies.Récemment, l’entreprise UCB BioPharma a montré que l’anticorps D, anticorps ciblant la protéine tau en un épitope central (aa 235 à 250), était en mesure de bloquer, in vitro, l'agrégation intracellulaire de protéines tau, induite par des PHFs purifiés au départ de cerveaux Alzheimer. L’anticorps A, de même isotype, associé à des propriétés de liaison comparables mais reconnaissant la protéine tau en son extrémité N-terminale (aa 15 à 24), n’est pas en mesure de prévenir l’agrégation dans ce même modèle, ce qui souligne l’importance du choix de l’épitope en vue de neutraliser les amorces pathologiques issues de cerveaux Alzheimer.En vue d’étudier les propriétés de l’anticorps D in vivo, nous avons développé deux modèles murins de tauopathies. Premièrement, un modèle étudiant les phénomènes de recrutement et nucléation, basé sur l'injection unilatérale d’un homogénat de cerveau Alzheimer dans l'hippocampe de jeunes souris transgéniques (Tg30tau). Deuxièmement, un modèle permettant l’étude de la propagation intercellulaire de formes pathologiques de tau, par injection unilatérale de fibrilles P301L-K18 dans l’hippocampe de souris transgéniques (hTauP301L). Les tauopathies induites par ces injections intracérébrales ont été quantifiées dans l'hippocampe ipsi et controlatéral, en présence de traitements immunothérapeutiques utilisant les anticorps anti-tau A et D ou un anticorps témoin négatif de même isotype. La quantification des formes hyperphosphorylées et agrégées de tau a été réalisée par des approches immunohistochimique ou biochimique.Dans le modèle de nucléation, l’anticorps D est en mesure de prévenir significativement l’apparition des formes hyperphosphorylées et agrégées de tau à la fois dans l’hippocampe ipsilatéral (injecté avec l’homogénat Alzheimer) et contralatéral. A l’opposé, l’anticorps A n’est pas en mesure de prévenir l’apparition de la tauopathie dans ce modèle. Dans le modèle de propagation, basé sur l'injection unilatérale hippocampique de fibrilles P301L-K18, le traitement immunothérapeutique utilisant l’anticorps D réduit significativement la propagation d’espèces pathologiques de la protéine tau dans l'hippocampe controlatéral.De par l’utilisation de ces deux modèles murins de tauopathies, nous avons pu confirmer in vivo, la capacité de l’anticorps D à neutraliser les espèces pathologiques contenues dans un homogénat de cerveau Alzheimer et avons démontré sa capacité à s’opposer à la propagation intercellulaire de la tauopathie in vivo. Dans le modèle de nucléation, l’anticorps A n’a pas été en mesure de s’opposer à l’apparition de la tauopathie. Les résultats obtenus confirment ceux décrits par l’entreprise UCB BioPharma dans leur modèle d’agrégation in vitro et confirme l’importance considérable du choix de l’épitope en vue de prévenir efficacement le développement de tauopathies in vivo. / In tauopathies, such as Alzheimer's disease, tau protein becomes abnormally hyperphosphorylated which leads to its accumulation and intracellular aggregation. This process gradually leads to neuronal loss and cognitive decline. Anti-tau immunotherapy is increasingly considered as a potential treatment to block tauopathies’s progression.UCB BioPharma recently showed that antibody D, targeting an epitope in the central region of tau (aa 235-250), is able to block, in vitro, the intracellular seeding of tau proteins induced by PHFs purified from the brain of Alzheimer's patients. The antibody A, same isotype and associated with similar binding properties but recognizing the N-terminal region of tau (aa 15 to 24) is not able to prevent tau seeding in this cell based assay. This observation underlines the importance of the targeted epitope on tau protein in order to neutralize pathological species contained in Alzheimer's brains (Courade et al., 2018).In order to study the properties of antibody D in vivo, we developed two murine models of tauopathies. First, a seeding model based on a unilateral injection of Alzheimer's brain homogenate into the hippocampus of young Tg30tau mice. Secondly, a spreading model to study the propagation of pathological tau seeds, based on unilateral hippocampal injection of P301L-K18 fibrils in hTauP301L transgenic mice. Tauopathies induced in these models were quantified in the ipsi and contralateral hippocampus in the presence of immunotherapeutic treatments with anti-tau antibodies (D, A) or a negative control antibody. Quantification of hyperphosphorylated and aggregated tau was performed by immunohistochemical or biochemical analyses.In the seeding model, antibody D significantly reduces the appearance of hyperphosphorylated and aggregated tau both in the ipsi and contralateral CA1 regions of hippocampus. In contrast, antibody A is not able to prevent the appearance of pathological tau in this model. In the spreading model, immunotherapeutic treatments with antibody D significantly reduces the spread of pathological tau seeds in the contralateral hippocampus.From these two murine models of tauopathies, we confirmed in vivo the ability of antibody D to neutralize the pathological tau species contained in an Alzheimer's brains homogenate and demonstrated its capacity to reduce the intercellular propagation of tauopathies. In the seeding model, antibody A wasn’t able to affect the onset of tauopathy. These results confirm those described by UCB BioPharma based on their in vitro aggregation assay and confirm the importance of the targeted epitope in order to effectively prevent the development of tauopathies in vivo.

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Chimeric antigen receptors (CAR) are modular genetically modified receptors that consist of an extracellular antigen binding domain fused to intracellular T-cell signaling domains. CAR therapy broadly consists of engineering a patient’s own T-cells to express a CAR directed against a tumor cell surface antigen. This therapy has been extremely successful in treating B-cell neoplasms by targeting CD19 and is paradigm changing in developing personalized immunotherapy for oncology applications. Although impressive response rates are observed, the durability of therapeutic response remains a concern and relapse mechanisms frequently center around issues of antigen loss. In addition, heterogeneous disease and solid tumors present formidable barriers toward extending the applicability of CAR technology as a result of compounding issues of tumor microenvironment and cell trafficking. In this thesis we review the current thought on the state of CAR therapy and the challenges to therapeutic efficacy, therapeutic manufacture, and clinical safety in the context of each other with an overall emphasis on identifying the fundamental goal of making fit-for-purpose CARs for different diseases.

Monoclonal antibody-based immunotherapy has been widely used as a strategy to treat cancer. Successful treatment of B-cell lymphoma with the monoclonal antibody (mAb) Rituximab (RTX) in combination with chemotherapy has increased the survival of patients and minimized the side effects of the treatment. However, many patients do not react to the treatment with RTX or gain resistance quickly. Thus, strategies to enhance the tumor cell killing and improve the response rates of mAb-based immunotherapy are a fundamental goal. In this study, I use four different B-cell lymphoma cell lines grown into 3D structures, called spheroids, as a model organism. Those spheroids, which are closer to the in vivo situation of B-cell lymphoma in patients compared to conventional in vitro 2D cell cultures, in combination with RTX, are tested for the activation of effector functions to eliminate tumor cells and compared to experiments conducted in the same cell lines in 2D cell cultures. Moreover, the therapeutic mAb RTX is only approved by the FDA in an IgG1 isotype form. Here, I test different isotype forms of RTX on their efficacy to kill cancer cells by the complement system and also by the activation of monocytes to engulf them in the process of phagocytosis. Interestingly, the IgG3 isotype form of RTX can induce both effector functions most efficiently while the IgG1 isotype form, used in clinical approaches, is only second most efficient in eradicating cancer cells. In addition, when grown into spheroids, the efficacy of both effector functions is reduced compared to 2D cell cultures. Furthermore, the efficacy of the complement system to kill the different B-cell lymphoma cell lines was directly correlated with the expression of the complement regulatory surface protein CD59. By blocking CD59, the efficacy of the complement system could be partially enhanced when cells were treated in 2D cell cultures but not when grown into 3D spheroids. In addition, the antibody-dependent phagocytosis (ADP) of cancer cells by monocytes might correlate with the expression of the RTX target surface protein CD20. Also, the previous incubation of B-cell lymphoma cells with a chemotherapy agent can enhance the efficacy of ADP by presumably providing an “eat me” signal to the effector cells.  In summary, this work shows that the outcome of a treatment with RTX in B-cell lymphoma patients could be improved by the detection of the specific features of the cancer cells, for example the expression of CD59 and CD20 and the structure of the tumor. Moreover, the different isotypes of RTX can activate effector functions in different intensities. The IgG3 isotype form might be able to overcome resistance or lack of reaction to the treatment in B-cell lymphoma patients but further experiments will be needed to investigate these possibilities.

Cancer immunotherapy, including immune checkpoint blocking antibodies are important components for treatment of patients with various types of cancer as they enhance the ability of the immune system to fight tumours. However, tumor cells have the ability to develop resistance to a variety of transitional therapies such as chemotherapy. In this study, in vitro Tumour-Immune co-culture system (TICS) has been developed to evaluate the impact on the antitumor activity of the primary human lymphocytes and response to PD-1 (nivolumab) and PD-L1 (durvalumab) checkpoint blocking antibodies against acquired chemotherapy resistance cancer cell lines. Using paired ovarian and neuroblastoma cancer cell lines obtained prior to chemotherapy (naïve) and after chemotherapy resistance, the results show that resistant ovarian cancer cells have differential effect on activation of lymphocytes and respond poorly to nivolumab and durvalumab, compared to chemotherapy naïve cells. On the other hand, chemotherapy neuroblastoma resistance cells show to respond to PD-1/L1 blockade therapy in TICS. Furthermore, blocking important molecular interactions between cancer cells and human lymphocytes such as HLA-ABC, HLA-DR and IFN-γ receptor compromises response to immune checkpoint blockade. In accordance, deletion of programmed death ligand 1 (PD-L1) on cancer cells by the CRISPR/Cas9 system significantly increases antitumor activity of immune cells in TICS. Moreover, deletion of beta-2-microglobulin (B2M) on human cancer cells resulted in substantial downregulation of HLA-ABC, which influenced immune activation induced by PD-1 blockade. Together, these findings demonstrate that chemotherapy resistance in human cancer cells could limit efficient response of PD-1/L1 blockade and thus immune checkpoint therapy could be more effective in early stage cancers.

Breast cancer is the most common type of cancer among women worldwide. Multiple studies have reported the role of tumor-immune interactions and mechanisms that the immune system uses to combat tumor cells. Therapies based on the immune response are evolving by time, but more research is required to understand and identify the patterns and relationships within the tumor microenvironment. This study aims to characterize immune cell expression patterns using a multiplex method and to investigate the way different subpopulations in breast cancer patients’ tissue samples are correlated with clinicopathological characteristics. The results of this study indicate that there must be an association within immune cell composition and clinicopathological characteristics (Estrogen Receptor Status (ER+/ER-), Progesterone Receptor (PR+/PR-), Grade (I,II,III), which is a way to characterize the cancer cells on how similar they look to normal ones, Menopause, Tumor size, Nodal status, HR status, HER2) but validation in larger patient population is required in order to evaluate the role of the immune infiltration as a predictive / prognostic biomarker in early breast cancer.

S’appuyer sur le métabolisme pour lutter contre la prolifération tumorale est une démarche qui commence à montrer de nombreux résultats. Notamment, il a été montré que la privation en nutriments ou jeûne permettait de ralentir l’incidence et la croissance de plusieurs modèles tumoraux en préclinique, effet d’autant plus important quand il est combiné aux agents chimiothérapeutiques. Notre laboratoire a identifié des agents capables de mimer certains effets biochimiques du jeûne. Ces composés, nommés Caloric Restriction Mimetics, permettent également d’améliorer l’effet antitumoral de plusieurs chimiothérapies. De façon importante, le système immunitaire était nécessaire à l’efficacité de ces différentes combinaisons thérapeutiques. Mon projet de thèse avait donc pour objectifs de comprendre en quoi ces traitements impactaient les populations immunitaires infiltrées dans la tumeur et si ces modulations métaboliques pouvaient être mises à profit en combinaison d’autres thérapies anticancéreuses. / Taking advantage of metabolism to fight against tumor proliferation begin to show several results. Indeed, it has been demonstrated that fasting had the potential to reduce the incidence and the proliferation of differents preclinical tumor models. This effect could be further enhanced when combined with chemotherapeutics agents. Thus, our laboratory has pinpointed compounds that have the ability to mimic some of the biochemical properties of nutrient deprivation. Those compounds, named Caloric Restriction Mimetics, can also improve the antitumor effect of several chemotherapies. Importantly, immune system is necessary to the efficiency of those therapeutics combinations. Accordingly, my thesis project was to understand in which manner tumor immune infiltrated populations were impacted by those treatments, and if those metabolic modulators could benefit to others anticancer therapies.