There is increasing evidence that the effect of chemotherapy on tumor rejection is not cell autonomous but relies on the immune system. Indeed, several reports have shown that human and murine tumors respond to chemotherapeutic agents more efficiently when the host immune system is intact. In particular, we have shown that cyclophosphamide treatment of DBA/2 mice bearing P815 mastocytoma induces rejection and long term protection in a CD4- and CD8-dependent manner. We used this tumor model, as it is poorly immunogenic, expresses tumor-associated P1A and tumor-specific P1E antigens, encoded by germline and mutated genes, respectively, and allows the identification of some tumor-specific CD8+ T cells.We have previously reported that tumor regression correlates with selective infiltration of CD8+ T cells specific for P1E/H-2Kd antigen in tumor bed upon cyclophosphamide treatment. Unexpectedly, the proportion of CD8+ T cells specific for the tumor-associated antigen P1A in the context of H-2Ld decreases concomitantly, indicating that cyclophosphamide alters the repertoire of CD8+ T cells recognizing tumor antigens. Using P1A KO mice, we found that preferential activation of CD8+ T cells to P1E is not solely due to thymic negative selection. The major role of “mutated” antigens in tumor resistance has been recently highlighted in humans and raises an interesting question about the immune mechanisms of tumor rejection. Additionally to its effect on the specific immune response, cyclophosphamide promotes tumor infiltration by effector memory (P1E/H-2Kd)+ CD8+ T cells which are characterized by higher expression of KLRG1 and Eomes. Our data point to a role of IL-15 and type 1 IFNs for their development, as increased levels of IL-15 and IRF7 were measured in tumor after cyclophosphamide. IFNAR1 blockade interferes with the tumor rejection in 50% of mice and decreases the (P1E/H-2Kd)+ CD8+ T cell infiltration induced by cyclophosphamide, suggesting a role of this cytokine in the expansion and/or recruitment of (P1E/H-2Kd)+ CD8+ T cells in vivo.Altogether, our results suggest that type 1 IFNs and IL-15 induced after cyclophosphamide promote the reprogramming of CD8+ T cells specific for the “mutated” P1E/H-2Kd antigen into effector memory lymphocytes. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

De nouveaux traitements comme l’immunothérapie ont été proposés en oncologie. Ils sont basés sur les mécanismes de régulation du système immunitaire. Cependant tous les patients ne répondent pas à ces nouveaux traitements. Afin de pouvoir les identifier, on mesure l’association des marqueurs immunologiques exprimés à la réponse au traitement ainsi qu’à la toxicité à l’instaurationdu traitement et leur évolution sous traitement. En situation observationnelle, l’absence de tirage au sort empêche la comparabilité des groupes et l'effet mesuré est juste une mesure d'association. Les méthodes d’inférence causalepermettent dans certains cas, après avoir identifié les sources de biais de par la construction de diagrammes acycliques dirigés (DAG), d'atteindre l’interchangeabilité conditionnelle entre exposés et non exposés etpermettent l’estimation d’effets causaux. Dans les cas les plus simples où le nombre de variables est faible, il est possible de dessiner leDAG à partir d’expertise. Dans les situations où le nombre de variables explosent, des algorithmes d’apprentissage ont été proposés pour retrouver la structure de ces graphes. Néanmoins ces algorithmes font d’une part l’hypothèse qu’aucune information n’est connue et n’ont été développés que dans les cas où les covariables sont mesurés à un seul temps. L’objectif de cette thèse est donc de développer ces méthodes d’apprentissages de graphes à des données répétées, puis d’intégrer des connaissances a priori pour améliorer l’estimation de ceux-ci. Une fois les graphes appris les modèles causaux peuvent être appliqués sur les biomarkers immunologiques répétés pour détecter ceux qui sont associés à laréponse et/ou la toxicité. / In oncology, new treatments such as immunotherapy have been proposed, which are based on regulation of the immune system. However, not all treated patient have a long-term benefit of the treatment. To identify those patients who benefit most, we measured markers of the immune system expressed at treatment initiation and across time. In an observational study, the lack of randomization makes the groups not comparable and the effect measured is just an association. In this context, causal inference methods allow in some cases, after having identified all biases by constructing a directed acyclic graph (DAG), to get close to the case of conditional exchangeability between exposed and non-exposed subjects and thus estimating causal effects.In the most simple cases, where the number of variables is low, it is possible to draw the DAG with experts’ beliefs. Whereas in the situation where the number of variables rises, learning algorithms have been proposed in order to estimate the structure of the graphs. Nevertheless, these algorithms make the assumptions that any a priori information between the markers is known and have mainly been developed in the setting in which covariates are measured only once. The objective of this thesis is to develop learning methods of graphs for taking repeated measures into account, and reduce the space search by using a priori expert knowledge. Based on these graphs, we estimate causal effects of the repeated immune markers on treatment response and/or toxicity.

Le projet de thèse à pour but de produire et caractériser un nouvel allergène de pollen d'ambroisie. Le projet est décliné en cinq axes: production d'une forme recombinante mature et en conformation native, caractérisation structurale, étude de la fonction enzymatique, étude de l'allergenicité et l'immunogénicité et évaluation du potentiel thérapeutique. / The goal of the thesis project is to product and caracterize a novel ragweed pollen allergen.The project have five axes: production of recombinant mature and native form,structurale caracterization, study of enzymatique function, study of allergenicity and immunogenicity and evaluation of therapeutic potential.

abstract: Glioblastoma (GBM) is a highly invasive and deadly late stage tumor that develops from abnormal astrocytes in the brain. With few improvements in treatment over many decades, median patient survival is only 15 months and the 5-year survival rate hovers at 6%. Numerous challenges are encountered in the development of treatments for GBM. The blood-brain barrier (BBB) serves as a primary obstacle due to its innate ability to prevent unwanted molecules, such as most chemotherapeutics, from entering the brain tissue and reaching malignant cells. The GBM cells themselves serve as a second obstacle, having a high level of genetic and phenotypic heterogeneity. This characteristic improves the probability of a population of cells to have resistance to treatment, which ensures the survival of the tumor. Here, the development and testing of two different modes of therapy for treating GBM is described. These therapeutics were enhanced by pathogenic peptides known to improve entry into brain tissue or to bind GBM cells to overcome the BBB and/or tumor cell heterogeneity. The first therapeutic utilizes a small peptide, RVG-29, derived from the rabies virus glycoprotein to improve brain-specific delivery of nanoparticles encapsulated with a small molecule payload. RVG-29-targeted nanoparticles were observed to reach the brain of healthy mice in higher concentrations 2 hours following intravenous injection compared to control particles. However, targeted camptothecin-loaded nanoparticles were not capable of producing significant treatment benefits compared to non-targeted particles in an orthotopic mouse model of GBM. Peptide degradation following injection was shown to be a likely cause for reduced treatment benefit. The second therapeutic utilizes chlorotoxin, a non-toxic 36-amino acid peptide found in the venom of the deathstalker scorpion, expressed as a fusion to antibody fragments to enhance T cell recognition and killing of GBM. This candidate biologic, known as anti-CD3/chlorotoxin (ACDClx) is expressed as an insoluble protein in Nicotiana benthamiana and Escherichia coli and must be purified in denaturing and reducing conditions prior to being refolded. ACDClx was shown to selectively activate T cells only in the presence of GBM cells, providing evidence that further preclinical development of ACDClx as a GBM immunotherapy is warranted. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019

Endemic Burkitt lymphoma (eBL) is the leading pediatric cancer in sub-Saharan Africa and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Current treatment options in Africa are combination chemotherapy with a survival rate hovering around 50%. Relapsed or refractory eBL patients have failed to receive any targeted treatments in the clinic. Our focus was to delineate immune responses in eBL, interrogate the tumor variation in responses to targeted treatments and develop mouse models that can be used to target essential mediators of tumor pathogenesis. Immune-based treatments including immune checkpoint inhibition have recently become an effective therapeutic modality in oncology. However, some B cell lymphomas such as Hodgkin Lymphoma (HL), are more receptive to checkpoint inhibition than others suggesting a need to understand the efficacy of checkpoint inhibition on different lymphoma subtypes. Checkpoint inhibitors act by blocking inhibitory receptors on T cells and improving anti-tumor responses. One of the goals of this thesis was to characterize checkpoint inhibitors on Tumor-infiltrating lymphocytes (TILs) in eBL tumors and to identify T cell subsets that exhibit increased expression of inhibitory receptors, poor cytokine production, poor proliferation and express transcription factors associated with exhaustion. Using scRNA seq, we identified T cell clusters that co-expressed inhibitory receptors, poor proliferative markers but also sustained costimulatory signals, as well as cytokine expression suggesting a pre dysfunctional state and not terminally exhausted state. Furthermore, we quantified the dominant co-inhibitory receptors PD1 and TIGIT that are upregulated in the tumor microenvironment via immunohistochemistry (IHC) and in peripheral blood of eBL patients via flow cytometry. We compared eBL patients with healthy pediatric cohorts with a history of persistent malaria exposure to those who had little to no malaria infections, to understand uniquely T cell mediated responses in BL children. Tumors had high co-expression of PD1 and TIGIT but fewer PD1 only populations, suggesting that both ligands may play a role in restraining immune activation via IHC. Next, we investigated if PD1 ligands or TIGIT ligands were overexpressed in eBL tumors. Nectin-2, TIGIT ligand was highly expressed in eBL tumors but was not highly correlated with TIGIT expression. These studies provide insights for PD1/ TIGIT blockade in Burkitt lymphoma patients. Additionally, we established new patient-derived cell lines from eBL tumors to study tumor variation and to study targeted treatments. We established five new patient-derived eBL lines BL717, BL 719, BL720, BL725, and BL740 that were interrogated for their inter-patient variation by studying their gene expression profiles. Further, we developed a patient cell-line derived xenograft (CDX) mouse model by injecting newly patient-derived BL cell lines in immunodeficient mice (NSG BL) and studying BL tumorigenesis. Having successfully established NSG BL tumors, we observed differences in tumor growth sensitivity and survival. We tested rituximab efficacy, one of the most established treatments for B cell lymphomas in our mouse model. We also identified pathways associated with unfolded protein response (UPR) and the mammalian target of rapamycin (mTOR) signaling, as well as apoptosis in one of the cell line xenografts, BL740, in response to rituximab. BL717, BL720 cell line xenograft failed to control tumor growth and was enriched in IFN-ɑ signature genes. This mouse model will prove to be useful to study combination therapy against eBL tumors as well as mechanisms of resistance to drug targets. Collectively, these studies provide insights into intratumoral variation including subtypes during tumor progression and expression profiles of TILs in eBL tumors. This will be important in designing new therapeutic strategies as well as help pose novel therapeutic targets.

No description available.

T cells can fight cancer, but an immunosuppressive tumor microenvironment (TME) disallows them from carrying out their function over time. Upregulation of inhibitory checkpoint molecules such as programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can lead to such an immunosuppressive TME. Despite their widespread use, immune checkpoint blockade (ICB) antibodies targeting checkpoint molecules remain ineffective in most cancer patients. We do not understand why some patients respond to ICB better than others. To understand the heterogeneity of ICB response, we must understand the heterogeneity of the T cell subsets acted upon by such therapies. Here, we ask how T cell subsets change in the presence and absence of ICB. We track T cell clones through their T cell receptor sequences and link phenotypes with T cell receptor specificities. Through multiplexed single cell TCR sequencing, single cell RNA sequencing, and the use of cell- surface CITE-seq antibodies, coupled with surgical biopsy, we longitudinally tracked the fate of individual T cell clones within tumors at baseline and in response to ICB in an immunogenic mouse tumor model. Furthermore, computational clustering of T cells solely based on their gene expression profiles may ignore upstream regulatory mechanisms that control T cell gene expression. Hence, we employed Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to cluster CD8+ and CD4+ T cell phenotypes. VIPER leverages inference of gene regulatory networks to allow full quantitative characterization of protein activity for transcription factors, co-factors, and signaling molecules by assessing the enrichment of their transcriptional targets cell-by-cell among expressed genes. This gave us a window into the transcriptional states and their inferred protein activity. We next developed a computational analysis toolkit to study TCR clonality incorporating sub-sampling of TCR clonotypes, forward and back tracing of shared clones between timepoints, and in turn, inferred shared clonal evolution. We employed the above workflow to MC38 tumor-infiltrating and tumor-draining lymph node-derived CD8+ and CD4+ T cells. We found that T cell phenotypes are highly dynamic within tumors at baseline, in the absence of ICB, particularly within the window that they are responsive to therapy. In the absence of ICB, effector phenotype of CD8+ T cells diminished, while the exhaustion phenotype was enhanced as tumors progressed. Within the CD4+ population, a heterogenous subset of regulatory CD4+ T cells (Tregs) changed phenotype over time, and CD4+ Th1 like effectors, along with stem like progenitor CD4+ showed distinct dynamism. Next, by analyzing responses to therapy within his context, we found that both anti-PD1 and anti-CTLA4 act through distinct mechanisms on CD8+ and CD4+ T cells. Anti-PD1 acted upon intra-tumoral effector CD8+ T cells to slow their progression to terminally differentiated exhausted cells, i.e., increased their persistence within tumors. Anti-CTLA4 therapy increased recruitment of novel effector CD8+ T cell clones to tumors from lymph nodes while diminishing tumor-infiltrating Tregs. ICB also potentiated CD4+ Th1 like phenotype. These results uncovered a behavior pattern of CD8+ and CD4+ T cells within tumors at baseline tumor progression, and then in the presence of ICB. We believe these findings have added to our understanding of the subtleties of T cell phenotypes in tumors, specifically in response to ICB. This will provide a practical framework for designing and validating novel checkpoint blockade therapies in the future.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal cancer, accounting for 3.2% of new cancer cases yearly but nearly 8% of all yearly cancer mortalities. Over the past twenty years, our understanding of cancer biology has greatly improved which has resulted in vastly improved prognoses for many cancers. However, the prognosis of pancreatic ductal adenocarcinoma has not improved despite the advance in cancer treatments. This is especially apparent with cancer immunotherapies, a newer therapeutic strategy that utilizes the innate defense mechanism of the body to target malignancies. Immune checkpoint inhibitors are a type of cancer immunotherapy that act by inhibiting the PD-1/PD-L1 and CTLA-4 immune checkpoint pathways and allowing T lymphocytes to proliferate and generate an antitumor response. They have greatly improved the prognosis for many types of malignancies, but clinical studies show that immune checkpoint inhibition has had a limited effect on the prognosis of PDAC. Recent studies have demonstrated that the immune microenvironment of PDAC is highly immunosuppressive, which is a probable factor in limiting the therapeutic efficacy of immune checkpoint inhibitors. Myeloid derived suppressor cells (MDSCs) are a main component of the immune microenvironment in PDAC. They are immature cells of myeloid origin that express CD11b+Gr-1+ on their surface, making them phenotypically distinct from mature dendritic cells. Their infiltration of the PDAC microenvironment early on in the course of the disease is promoted in a large part by the cytokine GM-CSF. MDSCs are believed to contribute to the limited efficacy of immune checkpoint inhibitor therapy both directly and indirectly. Indirect mechanisms are mediated by promoting the activity of other immunosuppressive cells in the PDAC microenvironment such as tumor associated macrophages and regulatory T lymphocytes. MDSCs induce the transformation of naïve CD4+ T lymphocytes into protumorigenic regulatory T lymphocytes. They also promote the polarization of macrophages to the tumor associated macrophage phenotype (IL-10high IL-12low) by secreting IL-10, which decreases IL-12 synthesis by macrophages present in the tumor microenvironment. On top of mediating immunosuppression through other cell types, MDSCs directly mediate immunosuppression by decreasing the amounts of amino acids necessary for anti-tumor immunity in the tumor microenvironment and disrupting the activity of antigen presenting cells and the signaling needed to initiate a cytotoxic T lymphocyte response. The decreased amount of arginine limits the ability of T cells to proliferate, resulting in a weaker cytotoxic response. These mechanisms limit the antitumor response against pancreatic ductal adenocarcinoma, resulting in the decreased response to immune checkpoint inhibitor therapy observed in clinical trials. Future attempts to strengthen the anti-tumor immune response must be combinatorial therapies that incorporate therapeutic strategies that seek to alleviate MDSC-mediated immunosuppression of T lymphocytes from the tumor microenvironment in addition to the more widely available immune checkpoint inhibitor therapy. Such therapeutics are currently being studied in murine models and have shown promising preliminary results but have yet to have been examined in clinical trials. These therapies are an ideal avenue to explore in a search for more effective therapy for this highly lethal disease.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19668号 / 薬科博第56号 / 新制||薬科||7(附属図書館) / 32704 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 橋田 充, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...