京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21303号 / 医博第4392号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13557号 / 論医博第2286号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 伊藤 能永, 教授 森信 暁雄 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Treatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising. Materials and Methods: Primary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable. Results: PD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS. Conclusion: High tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

<p dir="ltr">Immunotherapy has revolutionized the field of oncology. While a lot of antibodies and small molecule inhibitors have been developed for this, a lot of targets remain undruggable in humans.</p><p dir="ltr">Targeted protein degradation has opened a new horizon in drug discovery where we can target these undruggable proteins. Proteolysis targeting chimeras using the ubiquitin-proteasomal system is one of the most popular TPD strategies that complement lysosomal degradation strategies to degrade intracellular proteins, typically using bifunctional small molecule degraders. Recently, large biomolecular and antibody conjugates have been developed for degrading membrane and extracellular proteins in cells, such as lysosomal targeting chimeras (LYTACs) and genetically encoded LYTACS, among several others. However, larger molecules have limitations in penetrating solid tumors. This dissertation work focused on the development of bifunctional small molecule degraders for programmed death-ligand 1 (PD-L1), a transmembrane protein ligand for the immune checkpoint programmed cell death 1 (PD-1). PD-L1 is highly expressed on several tumors, such as triple-negative breast cancer (TNBC), non-small cell lung carcinoma, and renal cancer, and is known to suppress cancer-killing immune cells via interaction with PD-1 on T-cells. In addition, PD-L1 is also present on macrophages in the tumor microenvironments leading to further immune suppression and acquired resistance to anti-PD-1 therapy is associated with the upregulation of alternative immune checkpoints, thereby reducing anti-tumor efficacy. We have designed and synthesized bifunctional small molecules as PD-L1 degraders with different recruiters and linkers guided by computational studies with known PD-1/PD-L1 structures to show both cell surface and total protein degradation in human TNBC cells. In a separate project, we also developed small molecule conjugates to degrade an intracellular integral membrane protein of the endoplasmic reticulum with an unknown 3D structure, namely Diglyceride acyltransferase 2 (DGAT2). Recently, our lab identified DGAT2 as a new target for combating Alzheimer’s disease. Specifically, DGAT2 catalyzes triacylglycerol (TAG) synthesis using diacylglycerol and fatty acyl CoA as substrates. The accumulation of TAGs, mechanistically linked to DGAT2, results in “fat” or lipid droplets (LDs) inside the cells. Our lab showed that microglial cells (resident immune cells in the brain) accumulate LDs in the postmortem brains of human patients and mouse models (5xFAD) of Alzheimer’s disease and that the LD accumulation is driven by amyloid-beta (Ab) – a hallmark of Alzheimer’s disease – via DGAT2 pathway. Further, these LD-laden microglia have phagocytic defects and are spared Aβ thereby affecting plaque accumulation and clearance. Inhibiting DGAT2 reduces the amount of TAG in the brain, which in turn reduces LDs and restores microglial ability to phagocytose Ab. However, commercially available DGAT2 inhibitors were unable to reduce LD load in older 5xFAD mice. Using AlphaFold’s models of DGAT2, we designed and identified sites to synthesize bifunctional DGAT2 degraders that resulted in reduced LDs in mouse primary microglial cells and enhanced phagocytosis of Aβ plaques in vivo in aged 5xFAD mice. Our approach shows a framework to develop bifunctional small molecule degraders for membrane proteins to potentially combat immune dysregulation in chronic diseases.</p>

Melanoma is a highly malignant neoplasia with high rates of metastasis in humans and dogs. Regardless of being considered a highly immunogenic neoplasm, the function of the immune system is hampered by the expression of immune checkpoint molecules by the cancer cells. In contrast, soft tissue sarcomas are poorly immunogenic, as Tumor infiltrating Lymphocytes are lacking, or when present they are usually at the periphery of the tumor. Still, soft tissue sarcomas are considered immunosuppressed. Checkpoint molecules from the PD-axis are overexpressed in numerous human malignant neoplasia and have recently gained attention with a few reports in canine tumors. Immunotherapies against these checkpoint molecules have shown great efficacy in humans, but in order to determine translational approaches into canine patients, more research is needed. Here we determined the gene expression of Programed Death receptor-1, and its ligands PD-L1 and PD-L2 in canine tumors with two distinct immune profiles. Our results show that regardless of their immune profiles, melanoma versus soft tissue sarcoma, checkpoint molecules expression was higher in malignant tumors with a higher grade. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal ultrasound focused therapy that induces mechanical stress to the cells, leading to liquefactive necrosis. Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of CD3+ T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasia, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia. / Doctor of Philosophy / Melanoma is a highly malignant tumor and very resistant to therapy for humans and dogs. At the same time, this neoplasia is usually highly infiltrated by cells from the immune system. However, this immune infiltration is often inhibited by molecules expressed by the melanoma cells. In contrast, soft tissue sarcoma is considered poorly immunogenic, as they often contain low levels of immune cell infiltrates but are still considered immune suppressed. In this study, we determined the expression of molecules that inhibit the effect of T lymphocytes, specifically Programed cell death receptor-1, PD-Ligand 1, and PD-Ligand 2 for these neoplasms with distinct immune profiles. We encounter that despite their immune profiles, the expression of these molecules is higher in malignant tumors. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal focused ultrasound therapy that induces mechanical stress to the cancerigenous cells, leading to its death (necrosis). Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasms, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia.

Le virus d’Epstein-Barr (EBV) est le premier virus transformant à avoir été identifié chez l’Homme. Il infecte plus de 90% de la population adulte mondiale, persistant sous forme épisomale dans le compartiment B mémoire tout au long de la vie de l’hôte. Lors de la primo-infection et lors de phases de réactivation du virus, les cellules B immortalisées sont en programme de latence III, aussi appelée phase de prolifération, où l’ensemble des protéines de latence sont exprimées. Lorsque les hôtes sont immunocompétents, un équilibre entre hôte et virus s’établit et la plupart des cellules B infectées sont éliminées par le système immunitaire de l’hôte, principalement par les lymphocytes T cytotoxiques. En cas de déficit immunitaire, il peut y avoir émergence de lymphomes, tels que les désordres lymphoprolifératifs des patients immunodéprimés (PTLDs), les lymphomes non-Hodgkiniens (LNH) et Hodgkiniens (LH). Les travaux antérieurs du laboratoire ont permis de révéler que l’immuno-inhibiteur PD-L1/B7-H1/CD274 est surexprimé à la surface des lymphocytes B en latence III de l’EBV. L’interleukine-10 (IL-10) est également sécrétée par ces cellules. Ces caractéristiques sont communes aux cellules B régulatrices (Bregs). Le but de ma thèse était d’interroger les caractéristiques immuno-modulatrices des cellules B en latence III de l’EBV, dans le cadre des propriétés des Bregs. Nous montrons que les cellules B en latence III de l’EBV possèdent les déterminants antigéniques communs aux Bregs immatures (CD24High CD38High PD-L1High), associée à une surexpression des cytokines immunosuppressives cardinales des Bregs (IL-10, TGF-β1 et IL-35). Nous montrons que les cellules B en latence III de l’EBV peuvent conduire à la mort des cellules T CD4 autologues, ainsi qu’à l’inhibition de la prolifération des lymphocytes T CD4 et CD8, au profit de l’expansion de lymphocytes T régulateurs (Tregs). Nous avons trouvé que cette expansion est médiée par l’axe PD-1/PD-L1. Ces travaux mettent en évidence un nouveau mécanisme de l’EBV concernant le détournement du système immunitaire de l’hôte, augmentant ses capacités oncogéniques. / The Epstein-Barr virus (EBV) is the first transforming virus discovered in humans. It infects more than 90% of the global adult population, persisting in an episomal form in the memory B-cell compartment throughout the life of the host. During primo-infection and during phases of viral reactivation, immortalized B-cells are in latency III, also called the proliferation program, in which the full range of latency proteins are expressed. In immunocompetent subjects, a balance between virus and host is established, and most infected B-cells are eliminated by the host’s immune system, mainly by cytotoxic T lymphocytes. Deficit of the immune system may lead to lymphomagenesis, such as post-transplantation lymphoproliferative disorders (PTLDs), non-Hodgkin’s (NHL) or Hodgkin’s lymphomas (HL). Previous studies in the lab revealed that the immuno-inhibitor PD-L1/B7-H1/CD274 was overexpressed on the surface of EBV latency III B-cells. Interleukin-10 (IL-10was also secreted by these cells. These features are shared with regulatory B-cells (Bregs). Our objective was to examine the immunomodulatory features of EBV latency III B-cells, in the frame of Bregproperties. We found that EBV latency III B-cells possessed the antigenic determinants common to immature Bregs (CD24High CD38High PD-L1High), associated with overexpression of Breg immunosuppressive cytokines (IL-10, TGF-β1 and IL-35). EBV latency III B-cells led to death of autologous CD4 T-cells, as well as inhibition of CD4 and CD8 T-lymphocyte proliferation, favoring regulatory T-cell (Treg) expansion. We found that this expansion was mediated by the PD-1/PD-L1 axis. This study highlights a new mechanism of EBV for t diversion of the host immune system thereby increasing its oncogenic properties.

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

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Les cellules du système immunitaire sont programmées pour reconnaître et éliminer les cancéreuses, pourtant les cellules tumorales ont la capacité de mettre en œuvre les divers moyen pour échapper à la mort induite par les effecteurs du système immunitaire. Au cours de cette thèse, nous avons étudié plus particulièrement PD-L1, qui est impliqué dans la protection des cellules tumorales contre une attaque du système immunitaire et induit au cours de la TEM. / EMT foster cancer progression by acting on mechanisms allowing tumors to exide immune surveillance. Recent clinical advances immunotherapy demonstrated that some cancer with established lymphocyte infiltrates, express immune checkpoint inhibitory molecules, such as PD-L1, to allow their progression. During this thesis, another link between EMT and immune escape, through the regulation of PD-L1 in non-smal cell lung caricinoma was established. A new role of IKKƐ in the regulation of PD-L1 during EMT was also been shown.