Tesis sobre el tema "Mesothelin targeted cancer immunotherapy"

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Integrins are a family of heterodimeric cell surface receptors that are functionally important for cell adhesion, migration and proliferation. Certain integrins, especially those that are known to recognize the arginine-glycine-aspartate (RGD) motif, are heavily overexpressed in many cancers relative to healthy tissue, making them attractive targets for therapeutic intervention. However, prior attempts to antagonize these integrins as a cancer therapy have all failed in the clinic. In this thesis, we instead exploit integrins as a target tumor antigen in the context of immunotherapy. The engineered cysteine knot peptide, 2.5F, is highly crossreactive and capable of recognizing multiple RGD-binding integrins. Our initial attempts to utilize this binder as a targeting moiety for delivering IL-2 as an immunocytokine failed. Mathematical modeling results indicated that immunocytokines, unless adhering to specific design criteria, are unlikely to benefit from targeting and may actually exhibit limited efficacy. Therefore, we "deconstructed" this immunocytokine into its functional parts: extended half-life IL-2 and 2.5F-Fc, the antibody-like construct directed against RGD-binding integrins. This combination immunotherapeutic approach was able to synergistically control tumor growth in three syngeneic murine models of cancer, including durable cures and development of immunological memory. Contrary to prior attempts at integrin-targeting, the mechanism of action was independent of functional integrin antagonism, including effects on angiogenesis and tumor proliferation. In fact, efficacy of this therapy depended solely upon the adaptive and innate arms of immunity, specifically CD8+ T cells, macrophages, and dendritic cells. Furthermore, checkpoint blockade, the gold standard for immunotherapy to date, can further enhance the efficacy of this therapeutic approach. This signifies that the combination of IL-2 and 2.5F-Fc exerts a distinct, yet complementary immune response that opens the door for clinical translation.
by Byron H. Kwan.
Ph. D.

SMAC mimetic compounds (SMCs) are small molecule antagonists of the Inhibitor of Apoptosis (IAP) family of proteins. Binding of SMCs to the IAPs results in the sensitization of cancer cells to apoptosis in the presence of death ligands, such as tumour necrosis factor alpha (TNFα). I hypothesize that type I interferon (IFN) stimulation in cancer cells and in immune cells leads to the production of TNFα, which can then synergize with SMCs to kill cancer cells. The combined treatment of SMC and IFNα induces tumour regression in mice, and this effect is completely abrogated upon treatment with TNFα-neutralizing antibody. The synergistic effects are mediated by tumour cells and by contribution of immune cells, particularly macrophages and dendritic cells, as the systemic depletion of phagocytic innate immune cells results in an increase in tumour volume following combination treatment. The characterization of immune cell contribution will aid in the translation of the SMC combination therapy into clinical applications for the treatment of cancer.

Das Ziel der vorliegenden Arbeit bestand darin, ein Vektorsystem zu entwickeln, dass den simultanen Transfer verschiedener Transgene in CD8+ und CD4+ T-Zellen und dadurch die Herstellung eines immunotherapeutischen T-Zell-Produkts ermöglicht, welches aus zwei unterschiedlich modifizierten T-Zell-Subtypen besteht. Im ersten Teil der Arbeit wurde die Targeting-Technologie von lentiviralen auf γ-retrovirale Vektoren übertragen. Anschließend wird die Herstellung von Vektoren beschrieben, die spezifisch für murines CD4 oder CD8 sind. Deren Spezifität wurde zum einen durch die exklusive Expression von GFP in CD4+ oder CD8+ Zellen und zum anderen durch den Dosis-abhängigen Verlust des GFP-Signals nach Inkubation dieser Zellen mit CD4- und CD8-blockierenden Antikörpern nachgewiesen. Im dritten Teil der Arbeit wird gezeigt, dass MVm8 und MVm4 primäre T-Zellen spezifisch transduzieren. MVm8-vermittelter Transfer des Ovalbumin (OVA)-reaktiven TZRs OT-I führte zu T-Zellen, die OVA+ Tumor-Zelllinien erkannten und Interferon-γ sezernierten. Der vierte Teil dieser Arbeit beschäftigt sich mit der in vivo Transduktion primärer T-Zellen mithilfe von MVm8, welches den OT-I-TZR und eine Luciferase transferiert (MVm8/OT-I-luc). Durch systemische Applikation von MVm8/OT-I-luc wurden T-Zellen in vivo transduziert. Durch Immunisierungen konnten antigen-spezifisches Homing, Expansion und eine anschließende Kontraktion in vivo transduzierter T-Zellen gezeigt werden. Mäuse mit starker OT-I-luc-Expression waren gegenüber einer Infektion durch OVA-transgene listeria monocytogenes geschützt. Zusammenfassend lässt sich sagen, dass das in dieser Arbeit entwickelte Vektorsystem in der Lage ist zwischen Subtypen von T-Zellen zu unterscheiden und sie simultan mit unterschiedlichen Transgenen auszustatten. Für MVm8 konnte gezeigt werden, dass es T-Zellen direkt in vivo transduzieren kann.
The aim of this thesis was to generate a vector system that allows the simultaneous transfer of different transgenes into CD8+ and CD4+ T cells, allowing the generation of a immunotherapeutic T cell product comprised of two differently engineered T cell subsets. The first part of the thesis describes the transfer of the measles virus (MV) envelope-based targeting technology from lentiviral (LV) to γ-retroviral (gRV) vectors. The second part reports the generation of two targeting vectors specific for murine CD4 or CD8. The exclusive specificity of MVm4 and MVm8 was proven by expression of GFP in CD4+ and CD8+ reporter cells, respectively, but not in CD4-CD8- cells after transduction, and by a dose-dependent loss of GFP signal after incubation of reporter cells with CD4 or CD8 blocking antibodies before transduction. The third part shows that MVm8 but not MVm4 transduced primary T cells. MVm8-mediated transfer of the ovalbumin (OVA)-reactive TCR OT-I resulted in T cells secreting interferon-γ (IFNγ) upon recognition of OVA+ tumor cell lines. The final part of this thesis describes the in vivo transduction of primary T cells using MVm8 transferring OT-I and a luciferase (MVm8/OT-I-luc). To this end, B6 mice deficient for Rag2 have been repopulated with either polyclonal (B6) or monoclonal T cells derived from P14-TCR transgenic mice (P14). One day later the transferred T cells were transduced in vivo by systemic application of MVm8/OT-I-luc. Upon immunization in vivo-transduced T cells homed, expanded and contracted repeatedly in an antigen-dependent manner. Finally, mice exhibiting strong luc-signals showed improved protection against infections by OVA-transgenic listeria monocytogenes (LM-OVA). In conclusion, the viral vector system developed within this thesis is able to discriminate between the two main T cell subsets and to equip them with distinct transgenes simultaneously.

Pas de résumé
Recently targeted therapies appeared as attractive alternatives to classical antitumoral treatments. The approach, developed on the concept of targeting drug to cancer cells, aims to spear normal tissues and decrease the side effects. This doctoral dissertation focuses on developing new anticancer targeted treatments in the field of chemotherapy and cancer immunotherapy by exploiting an original targeting moiety, the B subunit of Shiga toxin (STxB). Its specific properties, such as, recognition with its receptor Gb3 overexpressed in cancer cells or in antigen-presenting cells, its unconventional intracellular trafficking, guided the choice of this protein as targeting carrier. This project is based in the use of copper-free Huisgen [3+2] cycloaddition as a coupling method, which led to successful preparation of various conjugates for their respective applications. The concept was first validated by STxB-biotin conjugate. The high yield of the reaction and the compatibility between the targeting carrier and the chemical ligation promoted the design of conjugates for chemotherapy and immunotherapy. Two therapeutical optimizations of previously developed strategy in STxB drug targeting delivery were investigated: synthesis of multivalent drug-conjugates and synthesis of conjugates containing a highly potent anticancer agent. Both approaches exploited three anticancer agents: SN38, Doxorubicin and Monomethyl auristatin F. The disulfide spacer, combined with various self-immolative systems, insured drug release. Two cytotoxic conjugates STxB–doxorubicin (STxB-Doxo) and STxB-monomethyl auristatin F (STxB-MMAF) were obtained in very high yield and demonstrated strong tumor inhibition activity in the nanomolar range on Gb3-positive cells. Based on the results the STxB-MMAF conjugate was investigated on a mouse model. The project aimed also to develop STxB bioconjugates for vaccine applications. Previous studies used B subunit as a targeting carrier coupled to an antigenic protein in order to induce a more potent immune response against cancer. The conjugates were prepared using a commercial linker, requiring modifying the antigen at first place, or by oxime ligation, where slightly acidic conditions promoted the coupling. Thus, the work presented herein proposed an alternative ligation via copper-free click chemistry especially for more sensitive antigenic proteins. Various types of conjugates were synthesised and investigated for their immune stimulation properties. The STxB targeting strategy was also applied to the development of a new vaccine based on coupling the targeting carrier to alpha-GalCer, one of the most potent immune stimulating agents known. The work focused on the synthesis of functionalised alpha-Galcer with an azide handle

Mestrado em Biomedicina Farmacêutica
Cellular immunotherapies, or Advanced Therapy Medicinal Products (ATMPs), are emerging as novel and specific therapeutic approaches to treat diseases, such as certain types of leukemias, which are difficult or impossible to treat with today’s biopharmaceutical products. Breakthroughs in basic, preclinical, and clinical science spanning cellular immunology, and cellprocessing technologies has allowed clinical applications of chimeric antigen receptor–based therapies. A recent example is CTL019, a lentivirus-based gene therapy for autologous T cells, acquired by Novartis in 2012 through a global alliance with the University of Pennsylvania. Although this technology is still in its infancy, clinical trials have already shown clinically significant antitumor activity in chronic lymphocytic leukemia and acute lymphocytic leukemia. Trials targeting a variety of other adult and pediatric malignancies are under way. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. The regulatory environment for these Advanced Therapies Medicinal Products is complex and in constant evolution. Many challenges lie ahead in terms of manufacturing process, non-conventional supply chain logistics, business models, intellectual property, funding and patient access.

La photothérapie dynamique à visée vasculaire utilisant le WST11 (VTP) induit la destruction rapide des tissus ciblés et constitue un traitement prometteur pour le cancer de la prostate. Cependant, la réponse immunitaire qui en résulte, qui peut jouer un rôle important dans la potentialisation ou l’atténuation des effets de la VTP, n’a toujours pas été comprise. Les cellules myéloïdes, telles que les MDSC et les macrophages, sont souvent présentes dans les tumeurs et sont largement associées à l'angiogenèse, au remodelage tissulaire et à l'immunosuppression. On sait également que ces cellules jouent un rôle essentiel dans la cicatrisation des plaies, induite lors de la destruction rapide des tissus. Nous avons étudié les effets de la VTP sur le recrutement de cellules myéloïdes infiltrant la tumeur (TIM), en particulier les MDSC et les macrophages associés aux tumeurs (TAM), dans les modèles de cancer de la prostate murin Myc-Cap et TRAMP C2. Nous rapportons que la VTP augmentait à la fois l'infiltration de cellules myéloïdes dans les tumeurs, mais aussi l’expression de CSF1R ; CSF1R étant un récepteur nécessaire à la différenciation, à la prolifération et à la migration des cellules myéloïdes. Comme un traitement anti-CSF1R avait déjà été utilisé pour diminuer l’infiltration de cellules myéloïdes dans d'autres modèles murins de cancer de la prostate, nous avons émis l'hypothèse que l'association d'un anti-CSF1R à un traitement par VTP entraînerait une diminution de la repousse tumorale et une amélioration de la survie. Nous avons constaté que le ciblage des cellules myéloïdes en utilisant un anti-CSF1R en association avec la VTP diminuait le nombre de MDSC et de TAM, et en particulier les macrophages M2. De plus cette association induisait une infiltration accrue de cellules T CD8+, diminuait la croissance tumorale et prolongeait la survie globale. Ces résultats suggèrent que le ciblage des cellules myéloïdes via le récepteur CSF1R est une stratégie prometteuse pour potentialiser les effets anti-tumoraux de la VTP
Vascular-targeted photodynamic therapy (VTP) induces rapid destruction of targeted tissues and is a promising therapy for prostate cancer. However, the resulting immune response, which may play an important role in either potentiating or blunting the effects of VTP, is still incompletely understood. Myeloid cells such as myeloid-derived suppressor cells (MDSCs) and macrophages are often found in tumors and are widely reported to be associated with cancer angiogenesis, tissue remodelling and immunosuppression. These cells are also known to play a critical role in wound-healing, which is induced by rapid tissue destruction. In this study, we investigated the effects of VTP on the recruitment of tumor infiltrating myeloid cells, specifically MDSCs and tumor-associated macrophages (TAMs), in the Myc-Cap and TRAMP C2 murine prostate cancer models. We report that VTP increased the infiltration of myeloid cells into the tumors, as well as their expression of CSF1R, a receptor required for myeloid differentiation, proliferation and tumor migration. As anti-CSF1R treatment has previously been used to deplete these cells types in other murine models of prostate cancer, we hypothesized that combining anti-CSF1R with VTP therapy would lead to decreased tumor regrowth and improved survival. Importantly, we found that targeting myeloid cells using anti-CSF1R in combination with VTP therapy decreased the number of tumor MDSCs and TAMs, especially M2 macrophages, as well as increased CD8+ T cell infiltration, decreased tumor growth and improved overall survival. These results suggest that targeting myeloid cells via CSF1R targeting is a promising strategy to potentiate the anti-tumor effects of VTP

Le cancer broncho-pulmonaire (CBP) le 4ème cancer le plus répandu au niveau mondial après les cancers de la prostate, du sein et du côlon. Diagnostiqués à des stades tardifs, il est la première cause de cancer par décès. Cependant, une meilleure compréhension des mécanismes moléculaires sous-jacents au cancer a permis de développer des thérapies personnalisées pour chaque patient. L’émergence des thérapies ciblées et de l’immunothérapie a révolutionné la prise en charge thérapeutique, permettant d’améliorer la survie globale, la survie sans progression et les effets secondaires des patients en comparaison avec les traitements de chimiothérapies conventionnelles. La prescription des thérapies personnalisées est basée sur les caractéristiques moléculaires de la tumeur et, par conséquent, nécessite des analyses moléculaires innovantes. Néanmoins, entre 10 et 30% des analyses moléculaires des patients atteints de CBNPC sont non contributives et l’accès aux thérapies ciblées est compromis. De plus, même si l’analyse anatomo-pathologique reste utile pour l’évaluation du stade ou encore de l’histologie, elle reste peu adaptée en cas d’actes répétitifs, tout au long de la maladie. La « biopsie liquide », est un concept émergeant, correspondant à l’analyse des acides nucléiques circulants mais aussi des cellules tumorales circulantes (CTC), issus de la tumeur. Cette méthode faiblement invasive, basée sur un prélèvement sanguin, permet d’analyser le patrimoine tumoral circulant et donne accès aux informations moléculaires de la tumeur primaire. Le développement de nouvelles activités de diagnostic est donc primordial pour répondre à ses nouvelles demandes cliniques. Depuis 2015, les Hospices Civils de Lyon (HCL) ont déployé un programme de recherche translationnelle, appelé CIRCAN « CIRculating Cancer » dans lequel s’inscrit ce travail de thèse. De nombreuses méthodes de détection des biomarqueurs pertinents en oncologie thoracique dans l’ADNcir ont été développées et été validées au laboratoire pour plus de 1500 patients actuellement, leur permettant de bénéficier de thérapies ciblées. L’optimisation et la validation des technologies de biologie moléculaire telles que le séquençage à haut débit et la PCR digitale ultra-sensible ont été réalisées durant ce travail de thèse et valorisées dans des journaux internationaux. Au-delà des thérapies ciblées, les immunothérapies représentent les nouveaux traitements prometteurs pour ces patients dont le taux d’expression PD-L1 sur biopsie tissulaire est le biomarqueur de choix. Etant donné les contraintes qu’occasionne la biopsie tissulaire, nous avons développé un protocole de caractérisation phénotypique de PD-L1 dans les CTC. De plus, de nombreuses études montrent la pertinence clinique de l’utilisation de la charge mutationnelle (TMB) comme marqueur prédictif de réponse à l’immunothérapie. En parallèle, nous avons développé des outils moléculaires en cours de validation pour le calcul du TMB dans l’ADNcir et dans les CTC en comparaison avec celui calculé dans le tissu, et le taux de PD-L1 évalué par immunohistochimie. Toutefois, pour environ 50% des patients atteints de CBP, aucun biomarqueur n’est retrouvé, bloquant l’accès à des thérapies personnalisées et réduisant les chances de survie du patient. A la recherche de nouveaux biomarqueurs, nous avons développé un protocole permettant d’accéder à la signature transcriptomique des CTC à un niveau « single-cell » afin de caractériser l’hétérogénéité tumorale de ces cellules et de mieux comprendre les mécanismes de résistance mis en place. Les échantillons cliniques de patients sont en cours d’analyse, avec ce protocole validé avec une lignée cellulaire modèle. En effet, les résultats de la validation de méthode mettent en exergue la possibilité d’évaluer l’hétérogénéité tumorale et les voies de signalisation impliquées dans la dissémination métastasique, telles que la transition épithélio-mésenchymateuse
Broncho-pulmonary cancer (PBC) is the 4th most common cancer worldwide after prostate, breast and colon cancer. Diagnosed at late stages, it is the leading cause of cancer death. However, a better understanding of the molecular mechanisms underlying cancer has led to the development of personalized therapies for each patient. The emergence of targeted therapies and immunotherapy has revolutionized the therapeutic management, improving the overall survival, progression-free survival and side effects of patients compared to conventional chemotherapy treatments. The prescription of personalized therapies is based on the molecular characteristics of the tumor and, therefore, requires innovative molecular analyzes. Nevertheless, between 10 and 30% of the molecular analyzes of NSCLC patients are non-contributory and access to targeted therapies is compromised. Moreover, even if the pathological analysis remains useful for stadium evaluation or histology, it remains unsuitable for repetitive actions throughout the illness. The "liquid biopsy", is an emerging concept, corresponding to the analysis of circulating nucleic acids but also circulating tumor cells (CTC), derived from the primary tumor. This low-invasive method, based on a blood sample, makes it possible to analyze the circulating tumor inheritance and gives access to the molecular information of the primary tumor. The development of new diagnostic activities is therefore essential to meet its new clinical demands. Since 2015, Hospices Civils de Lyon (HCL) has deployed a translational research program, called CIRCAN "CIRculating Cancer" in which this thesis. Many methods for detecting relevant biomarkers in thoracic oncology in circulating free DNA (cfDNA) have been developed and validated in the laboratory for more than 1500 patients currently, allowing them to benefit from targeted therapies. The optimization and validation of molecular biology technologies such as high-throughput sequencing and ultra-sensitive digital PCR were performed during this thesis work and published in international journals. Beyond targeted therapies, immunotherapies represent promising new treatments for these patients whose PD-L1 expression level on tissue biopsy is the biomarker of choice. Given the constraints of tissue biopsy, we have developed a phenotypic characterization protocol for PD-L1 in CTCs. In addition, numerous studies show the clinical relevance of the use of mutational load (TMB) as a predictive marker of response to immunotherapy. In parallel, we have developed molecular tools undergoing validation for the calculation of TMB in cfDNA and in CTC compared with the value calculated in tissue, and the PD-L1 level evaluated by immunohistochemistry. However, for about 50% of patients with CBP, no biomarker is found, blocking access to personalized therapies and reducing the patient's chances of survival. In search of new biomarkers, we have developed a protocol allowing access to the transcriptomic signature of CTCs at a "single-cell" level in order to characterize the tumor heterogeneity of these cells and to better understand the resistance mechanisms implemented. The clinical samples of patients are being analyzed, with this protocol validated with a model cell line. Indeed, the results of the method validation highlight the possibility of evaluating tumor heterogeneity and the signaling pathways involved in metastatic spread, such as the epithelial-mesenchymal transition

Il est largement admis qu'un cancer se développe lorsque les cellules cancéreuses échappent au contrôle du système immunitaire et que l'exploitation des défenses immunitaires afin de réactiver les cellules T endogènes anti-tumorales pourrait être une option thérapeutique pour obtenir des réponses complètes et durables.L'interféron de type I est connu pour sa puissante activité antitumorale dans les tumeurs expérimentales de la souris. En outre, il s'est avéré être une cytokine clé nécessaire à l'efficacité de nombreux agents anticancéreux ciblant non seulement les cellules cancéreuses (radiations ionisantes, produits chimiques cytostatiques, AcM...) mais aussi le système immunitaire (vaccination, cellules CAR-T...). Cependant, son utilisation n'est plus envisagée par le clinicien en raison des effets secondaires subis par les patients. Pour répondre à cette préoccupation, une technologie très prometteuse permettant la conception de molécules d'interféron ciblées et spécifiques aux cellules a été développée et l'objectif de notre travail actuel est de générer et d'évaluer pré-cliniquement des composés principaux. Pour cela, il faut s'attaquer à un certain nombre de frontières de la recherche, notamment répondre aux questions fondamentales "où" et "quand" l'interféron doit agir pour exercer son activité antitumorale, seul ou en combinaison avec les stratégies thérapeutiques susmentionnées.La question "quand" est importante car on soupçonne fortement que le moment relatif de l'action de l'interféron et de la stimulation du TCR détermine si l'effet de l'interféron est immunostimulant ou immunosuppresseur. La question "où" est évidente car elle détermine le choix de la partie ciblée des interférons modifiés. Nous savons que l'action de l'interféron sur les cellules dendritiques est nécessaire à son activité antitumorale, mais est-elle suffisante ? Une action sur les cellules T est-elle également obligatoire ? L'action de l'interféron sur les cellules tumorales ou les cellules du stroma est-elle nécessaire pour attirer les cellules immunitaires effectrices ?
It is widely accepted that a cancer develops when cancer cells escape from the control of the immune system and that harnessing the immune defences in order to reactivate endogenous anti-tumor T cells could be a therapeutic option for full and durable responses.Type I interferon is known for its potent antitumor activity in experimental mouse tumors. Furthermore, it has been shown to be a key cytokine necessary for the efficacy of many anticancer agents targeting not only cancer cells (ionising radiations, cytostatic chemicals, mAbs…) but also the immune system (vaccination, CAR-T cells…). However, its use is no longer considered by the clinician owing to the side effects experienced by the patients. To address this concern, a highly promising technology allowing the design of cell-specific targeted interferon molecules has been developed and the objective of our present work is to generate and pre-clinically evaluate lead compounds. For this, a number of research frontiers must be tackled, these include to answer to the fundamental questions 'where' and 'when' interferon must act in order to exert its antitumor activity either alone or in combination with the above-mentioned therapeutic strategies.The question 'when' is important because it is highly suspected that the relative timing of interferon action and TCR stimulation determines whether the effect of interferon is immunostimulant or immunosuppressive. The question 'where' is evident since it determines the choice of the targeting moiety of the engineered interferons. We know that the action of interferon on dendritic cells is necessary for its antitumor activity but is it sufficient? Is an action on T cells also mandatory? Is an interferon action on tumor cells or stroma cells necessary for attracting effector immune cells?

Le carcinome pulmonaire est une affection grave et fréquente dont la prise en charge a été bouleversée ces dernières années, tant sur le plan diagnostique que pronostique ou « théranostique » avec l’avènement des « thérapies ciblées ». Ces dernières permettent une nette amélioration de la survie et du confort des patients éligibles, mais ne sont pas sans compliquer le travail médical, depuis le diagnostic de la maladie jusqu’au suivi régulier du patient, sans oublier le choix des traitements ou les problèmes techniques posés par la multiplication arborescente des altérations moléculaires à rechercher à partir d’un tissu tumoral souvent peu abondant dans ce contexte particulier de l’oncologie thoracique. Ce travail de thèse collige 5 travaux de recherche selon deux angles d’approche : les marqueurs moléculaires pronostiques et « théranostiques » du cancer pulmonaire, et les procédures de diagnostic in vivo de cette pathologie. Le premier axe comporte 4 articles. Les deux premiers concernent l’évaluation d’une nouvelle technique moléculaire, la LD-RT-PCR, dans la détection des translocation géniques du cancer pulmonaire : la première étude est une étude de faisabilité, la deuxième est un travail de validation. Le troisième article explore l’association entre la présence d’une mutation STK11 dans les carcinomes pulmonaires et l’expression de PDL1. Enfin, le quatrième article est une étude de cas illustrant l’importance de l’approche morphologique du cancer pulmonaire. Le second axe est représenté par un travail comparant une technique d’imagerie in vivo par voie endoscopique utilisant la micro-endoscopie confocale par laser avec l’approche microscopique conventionnelle
Lung cancer is a serious and frequent condition for which the management strategies have been dramatically modified in recent years, from a diagnostic, prognostic and “theranostic” perspective, most notably with the introduction of “targeted therapies”. The latter have demonstrated dramatic improvement in both quality of life and survival rates of eligible patients, yet consequently highlight new complications in diagnosis, treatment options or technical considerations which can be attributed to the growing number of molecular alterations to be detected from limited tissue samples frequently encountered in thoracic oncology. This work combines 5 different research papers from 2 different angles: prognostic and “theranostic” molecular markers of lung cancer, as well as in vivo diagnostic procedures of lung cancer. The first angle encompasses 4 articles. The first two evaluate a new molecular technique, LD-RT-PCR, to detect gene translocation in lung cancer. The third article explores the association between STK11 mutations in lung cancer and the expression of PDL1. Finally, the fourth article is a case report illustrating the importance of a morphological approach to lung cancer. The second angle compares in vivo imaging techniques by endoscopy using confocal laser microendoscopy alongside a conventional microscopic approach

Une élévation majeure de la charge mutationnelle (hypermutation) est observée dans certains gliomes. Néanmoins, les mécanismes de ce phénomène et ses implications thérapeutiques notamment concernant la réponse à la chimiothérapie ou à l'immunothérapie sont encore mal connus. Sur le plan du mécanisme, une association entre hypermutation et mutations des gènes de la voie de réparation des mésappariements de l'ADN (MMR) a été rapportée dans les gliomes, cependant la plupart des mutations MMR observées dans ce contexte n'étaient pas fonctionnellement caractérisées, et leur rôle dans le développement d’hypermutation restait de ce fait incertain. De plus, l'impact de l'hypermutation sur l'immunogénicité des cellules gliales et sur leur sensibilité au blocage des points de contrôles immunitaires (par exemple par traitement anti-PD-1) n’est pas connu. Dans cette étude, nous analysons de manière exhaustive les déterminants cliniques et moléculaires de la charge et des signatures mutationnelle dans 10 294 gliomes, dont 558 (5,4%) tumeurs hypermutées. Nous identifions deux principales voies responsables d'hypermutation dans les gliomes : une voie "de novo" associée à des déficits constitutionnels du système MMR et de la polymérase epsilon (POLE), ainsi qu'une voie "post-traitement", plus fréquente, associée à l'acquisition de déficits MMR et de résistance secondaire dans les gliomes récidivant après chimiothérapie par temozolomide. Expérimentalement, la signature mutationnelle des gliomes hypermutés post-traitement (signature COSMIC 11) était reproduite par les dommages induits par le témozolomide dans les cellules MMR déficientes. Alors que le déficit MMR s'associe à l'acquisition de résistance au témozolomide, des données cliniques et expérimentales suggèrent que les cellules MMR déficientes conservent une sensibilité à la nitrosourée lomustine. De façon inattendue, les gliomes MMR déficients présentaient des caractéristiques uniques, notamment l'absence d'infiltrats lymphocytaires T marqués, une hétérogénéité intratumorale importante, une survie diminuée ainsi qu’un faible taux de réponse aux traitements anti-PD-1. De plus, alors que l'instabilité des microsatellites n'etait pas détectée par des analyses en bulk dans les gliomes MMR déficients, le séquençage du génome entier à l'échelle de la cellule unique de gliome hypermuté post-traitement permettait de démontrer la presence de mutations des microsatellites. Collectivement, ces résultats supportent un modèle dans lequel des spécificités dans le profil mutationnel des gliomes hypermutés pourraient expliquer l’absence de reconnaissance par le système immunitaire ainsi que l’absence de réponse aux traitements par anti-PD-1 dans les gliomes MMR déficients. Nos données suggèrent un changement de pratique selon lequel la recherche d’hypermutation par séquençage tumoral lors de la récidive après traitement pourrait informer le pronostic et guider la prise en charge thérapeutique des patients
High tumor mutational burden (hypermutation) is observed in some gliomas; however, the mechanisms by which hypermutation develops and whether it predicts chemotherapy or immunotherapy response are poorly understood. Mechanistically, an association between hypermutation and mutations in the DNA mismatch-repair (MMR) genes has been reported in gliomas, but most MMR mutations observed in this context were not functionally characterized, and their role in causing hypermutation remains unclear. Furthermore, whether hypermutation enhances tumor immunogenicity and renders gliomas responsive to immune checkpoint blockade (e.g. PD-1 blockade) is not known. Here, we comprehensively analyze the clinical and molecular determinants of mutational burden and signatures in 10,294 gliomas, including 558 (5.4%) hypermutated tumors. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and MMR genes, and a more common, post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas recurring after temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas (COSMIC signature 11) was recapitulated by temozolomide-induced damage in MMR-deficient cells. While MMR deficiency was associated with acquired temozolomide resistance in glioma models, clinical and experimental evidence suggest that MMR-deficient cells retain sensitivity to the chloroethylating nitrosourea lomustine. MMR-deficient gliomas exhibited unique features including the lack of prominent T-cell infiltrates, extensive intratumoral heterogeneity, poor survival and low response rate to PD-1 blockade. Moreover, while microsatellite instability in MMR-deficient gliomas was not detected by bulk analyses, single-cell whole-genome sequencing of post-treatment hypermutated glioma cells demonstrated microsatellite mutations. Collectively, these results support a model where differences in the mutation landscape and antigen clonality of MMR-deficient gliomas relative to other MMR-deficient cancers may explain the lack of both immune recognition and response to PD-1 blockade in gliomas. Our data suggest a change in practice whereby tumor re-sequencing at relapse to identify progression and hypermutation could inform prognosis and guide therapeutic management

La mesotelina è una glicoproteina di superfice cellulare sintetizzata a partire da un precursore di 71 kDa che viene processato in un frammento di 40 kDa legato alla membrana ed un fattore solubile potenziante i megacariociti di 31 KDa. La mesotelina rappresenta un promettente biomarcatore tumorale; in condizioni fisiologiche è espressa dalle cellule mesoteliali delle sierose dell’organismo (pleura, pericardio e peritoneo) mentre è overespressa sulla superficie cellulare di diversi istotipi tumorali tra cui: il mesotelioma maligno, il carcinoma ovarico, pancreatico e polmonare. La sua funzione è sconosciuta ma recentemente è stato ipotizzato un suo ruolo nei meccanismi di adesione cellulare attraverso il legame all’antigene tumorale CA125. La limitata espressione della mesotelina a livello dei tessuti fisiologici e, di contro, l’elevata espressione in molti tumori, rendono questo antigene un possibile target nella immunoterapia anti-tumorale. Il presente lavoro descrive la generazione di un nuovo anticorpo monoclonale anti-mesotelina ottenuto mediante la tecnologia degli ibridomi e la caratterizzazione delle sue proprietà di riconoscimento. Tale anticorpo monoclonale è stato testato per valutare un suo potenziale utilizzo terapeutico e diagnostico. L’anticorpo anti-mesotelina ha dimostrato, in analisi condotte mediante citofluorimetria, una notevole specificità, essendo in grado di riconoscere cellule che esprimono l’antigene mesotelina costitutivamente (OVCAR-3) e per trasfezione (HEK293-mesotelina); allo stesso tempo l’anticorpo ottenuto non possiede la capacità di legare cellule mesotelina negative. La specificità di riconoscimento in vitro è paragonabile all’anticorpo monoclonale commerciale K1 da noi utilizzato come controllo positivo. Studi di binding, inoltre, hanno permesso di evidenziare la migliore affinità del nostro anticorpo rispetto all’anticorpo monoclonale commerciale K1. Basandoci su questi dati abbiamo sviluppato uno immunotossina chimica con le capacità di riconoscimento del nostro anticorpo e l’attivita citotossica della ricina (una potente tossina appartenente alla famiglia delle proteine inattivanti i ribosomi) con lo scopo di poter utilizzare tale costrutto nella immunoterapia passiva di tumori overesprimenti la mesotelina. Le frazioni raccolte e purificate dell’immunotossina, ottenute mediante la produzione di un ponte disolfuro tra l’anticorpo monoclonale e la ricina, risultano capaci di inibire del 50% la proliferazione cellulare (IC50). Tale attività citotossica si manifesta su cellule mesotelina positive ad una concentrazione di 0,03 nM e 0,09 nM rispettivamente a 36 h e 72 h di incubazione. Inoltre il nostro costrutto dimostra una elevata specificità di riconoscimento in quanto non sono stati raggiunti valori di IC50 su cellule mesotelina-negative anche dopo aggiunta di concentrazioni pari o superiori a 78 nM dell’immunotossina. Si è infine osservato che l’attività citotossica di una concentrazione pari a 0,03 nM dell’immunotossina è completamente neutralizzata dalla contemporanea aggiunta, nel terreno di coltura, dell’ anticorpo anti-mesotelina da noi generato ad una concentrazione 1000 volte maggiore rispetto a quella dell’immunotossina. Questo ultimo dato supporta i precedenti ottenuti potenziando l’evidenza di una attività citotossica specifica e anticorpo mediata da parte della nostra immunotossina chimica.
BACKGROUND: Mesothelin is a tumor differentiation antigen (Ag) that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is, however, highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma. The normal biologic function of mesothelin is unknown but recent studies have shown that it binds to CA-125 and may play a role in the peritoneal spread of ovarian cancer. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer immunotherapy. RESULTS: In this study we have developed a monoclonal antibody (mAb) that is specific for the Ag mesothelin. It was produced by hybridomas technologies and we have performed Fluorescence Activated Cell Sorting (FACS) analysis to evaluate the specificity and affinity of this antibody for the Ag of interest. The mAb binds mesothelin-positive cell lines expressing the Ag constitutively (OVCAR-3) and transfected cells (HEK293-mesothelin). The same mAb not recognizes mesothelin-negative cell lines demonstrating an high specificity in vitro. Binding studies have demonstrated that our mAb has a better affinity with respect to mAb K1, a commercially available anti-mesothelin mAb. Our anti-mesothelin mAb was chemically linked to ricin A chain (RTA) toxin obtaining a powerful immunodelivered drug (immunotoxin, IT) with specific cytotoxic activity on mesothelin positive cells; in a cytotoxic assay on HEK293-mesothelin transfected cells the anti-mesothelin mAb-RTA IT shows an IC50 of 0.03 nM and 0,09 nM after 36 hrs and 72 hrs of incubation respectively; no cytotoxic activity was observed against mock-not transfected one and other mesothelin negative cells. As a further proof of specificity we observed that the cytotoxic activity of 0,03 nM of the above-mentionated IT on HEK293-mesothelin cells, is fully prevented by addition of whole molecule mesothelin-specific antibody at a concentration 1000 fold over IT. CONCLUSION: Our mAb holds great potential to be used as a research reagent and diagnostic tool in research laboratories and in the clinics because of its high quality and versatility. This antibody is also a strong candidate to be investigated for further in vivo passive immunotherapy studies. Moreover, the discovered of this new mAb anti-mesothelin enable us to develop in vivo diagnostic approaches using radionuclide, fluorescence trackers or nanoparticles. Its conjugation with therapeutic molecules allows a better distribution of the drug to the tumor sites; this ability increase the antitumor efficacy and reduce the specific toxicity at the same time.

碩士
長庚大學
生物醫學研究所
105
Cancer can be treated by various methods such as surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Studies shows that immunization with tumor cell based vaccines may effectively induce tumor antigen-specific T cell activation and proliferation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been broadly used as adjuvant with DNA vaccines and tumor vaccines. Immunizations with codon-optimized GM-CSF plasmid DNA-vaccine result in an effective cytotoxic T lymphocyte response. In previous studies, we used the high GM-CSF-producing TC-1 cells as a tumor cell vaccine that simultaneously expresses the HPV E6 and E7 oncoproteins. It significantly promotes the proliferation of CD8+ T cells. In this study, we hypothesized that the GM-CSF-secreting tumor cell vaccine was able to improve the antigen presentation and help the chemotherapy or targeted therapy to reduce tumor growth. We were combining tumor cell vaccine and chemotherapy drug, cisplatin, or targeted therapy drug, gefitinib, for cervical cancer in murine models. The frequencies of CD8+ T cells and Treg cells from the spleen and lymph node were analyzed via flow cytometry. Our result shows that cisplatin and gefitinib treatment suppress tumor growth better than the combination treatment with GM-CSF-secreting tumor cell vaccine. Combining GM-CSF-secreting tumor cell vaccine with cisplatin or gefitinib further enhance the tumor growth. However, flow cytometry data shows that the percentage of CD8+ T lymphocytes was reduced and the percentage of Treg cell was also higher. The data suggest that GM-CSF tumor cell vaccine may promote CD4+CD25+Foxp3+ Treg cells population and Treg cells suppress the effective T lymphocytes which may lead to tumor growth in established TC-1 tumor models. Combining immunotherapy with chemotherapeutic drugs seems to be a novel treatment. Few studies have investigated the options for combining chemotherapy and immunotherapy, because the two forms of treatment are considered to be antagonistic. According to our tumor growth curves and survival analysis, combining immunotherapy did not show retarded tumor growth in B6 mice. First, GM-CSF-secreting tumor cell vaccine induces the differentiation of CD4+CD25+Foxp3+Treg cells which suppress the CD8+ T cell. Second, most chemotherapies kill target cells by triggering a process of programmed cell death, or apoptosis, and this mode of cell death has been regarded as non-stimulatory or tolerogenic. Third, lymphocyte depletion (lymphopenia) is a common side effect of many anti-cancer drugs, and this too has been assumed to be detrimental to any potential immune response. Some targeted therapies and cytotoxic agents can modulate immune responses, which raises the possibility that these treatment strategies might be effectively combined with immunotherapy to improve clinical outcomes in the near future.

Cancer cells frequently escape from immune surveillance by down-regulating two important components of the immune defence: antigen-presenting MHC and costimulatory molecules. Therefore several novel anti-tumour compounds that aim to assist the immune system in recognising and fighting cancer are currently under development. Recombinant bispecific antibodies represent one group of such novel therapeutics. They target two different antigens and recruit cytotoxic effector cells to tumour cells. For cancer immunotherapy, bispecific T cell-engaging antibodies are already well characterised. These antibodies target a tumour-associated antigen and CD3ε, the constant molecule of the T cell receptor complex. On the one hand, this study presents the development of a bispecific antibody targeting CD3ε and the rhabdomyosarcoma-associated fetal acetylcholine receptor. On the other hand, it describes a novel two-part trispecific antibody format for the treatment of leukaemia and other haematological malignancies in the context of haematopoietic stem cell transplantation (HSCT). For HSCT, an HLA-identical donor is preferred, but very rarely available. In an HLA-mismatched setting, the HLA disparity could be exploited for targeted cancer treatment. In the present study, a two-part trispecific HLA-A2 × CD45 × CD3 antibody was developed for potential cases in which the patient is HLA-A2-positive, but the donor is not. This holds true for about half the cases in Germany, since HLA-A2 is the most common HLA molecule found here. Combinatorial targeting of HLA-A2 and the leucocyte-common antigen CD45 allows for highly specific dual-antigen restricted tumour targeting. More precisely, two single-chain antibody constructs were developed: i) a single-chain variable fragment (scFv) specific for HLA-A2, and ii) a scFv against CD45, both linked to the VL and the VH domain of a CD3ε-specific antibody, respectively. It turned out that, after the concomitant binding of these constructs to the same HLA-A2- and CD45-expressing cell, the unpaired variable domains of a CD3ε-specific antibody assembled to a functional scFv. In a therapeutic situation, this assembly should exclusively occur on the recipient’s blood cancer cells, leading to T cell-mediated cancer cell destruction. In this way, a relapse of disease might be prevented, and standard therapy (radiation and chemotherapy) might be omitted. For both approaches, the antibody constructs were periplasmically expressed in E. coli, purified via His tag, and biochemically characterised. Their binding to the respective targets was proven by flow cytometry. The stimulatory properties of the antibodies were assayed by measuring IL-2 release after incubation with T cells and antigen-expressing target cells. Both the bispecific antibody against rhabdomyosarcoma and the assembled trispecific antibody against blood cancer mediated T-cell activation in a concentration-dependent manner at nanomolar concentrations. For the trispecific antibody, this effect indeed proved to be dual antigen-restricted, as it could be blocked by prior incubation of either HLA-A2- or CD45-specific scFv and did not occur on single-positive (CD45+) or double-negative (HLA-A2- CD45-) target cells. Furthermore, antibodies from both approaches recruited T cells for tumour cell destruction in vitro
Krebszellen entgehen der Immunüberwachung oftmals dadurch, dass sie zwei wichtige Komponenten der Immunabwehr, nämlich antigenpräsentierende MHC- und kostimulatorische Moleküle, herunter regeln. Zurzeit befindet sich daher eine Reihe neuartiger Anti-Krebs-Substanzen in der Entwicklung, die darauf abzielen, das Immunsystem beim Erkennen und Bekämpfen von Krebs zu unterstützen. Rekombinante bispezifische Antikörper stellen eine Gruppe solch neuartiger Therapeutika dar. Sie erkennen zwei unterschiedliche Antigene und rekrutieren gezielt zytotoxische Effektorzellen zu Tumorzellen. Zur Krebsimmuntherapie sind BiTE-Antikörper (bispecific T cell engager) bereits gut untersucht. Diese Antikörper sind gegen ein tumorassoziiertes Antigen sowie gegen CD3ε, das konstante Molekül des T Zell-Rezeptor-Komplexes, gerichtet. Diese Arbeit beschreibt zum einen die Entwicklung eines bispezifischen Antikörpers, der CD3ε und den mit Rhabdomyosarkom assoziierten fetalen Acetylcholinrezeptor erkennt. Zum anderen präsentiert sie ein neues, zweiteiliges trispezifisches Antikörperformat, das zur Behandlung von Leukämie und anderen bösartigen Erkrankungen des blutbildenden Systems im Zusammenhang mit hämatopoetischer Stammzelltransplantation (HSZT) genutzt werden könnte. Für eine HSZT wird ein HLA-identischer Spender bevorzugt. Dieser steht jedoch nur sehr selten zur Verfügung. In Fällen mit nur einer Unstimmigkeit in den HLA-Merkmalen zwischen Patient und Spender könnte diese HLA-Unstimmigkeit nun zur gezielten Krebsbehandlung ausgenutzt werden. In dieser Arbeit wurde ein trispezifisches HLA-A2 × CD45 × CD3 Antikörperkonstrukt speziell für solche Fälle entwickelt, in denen der Patient HLA-A2-positiv ist, der Spender jedoch nicht. Dies trifft in Deutschland auf ungefähr die Hälfte aller Fälle zu, da HLA-A2 hier als häufigstes HLA-Molekül vorkommt. Mit der Kombination aus HLA-A2 und dem Pan-Leukozytenmarker CD45 (leucocyte-common antigen) als Ziel, wird eine hochspezifische, von zwei Antigenen abhängige, zielgerichtete Tumoransteuerung (tumour targeting) möglich. Genauer gesagt wurden zwei Einzelketten-Antikörperkonstrukte entwickelt: i) ein HLA A2-spezifisches single-chain variable fragment (scFv) und ii) ein CD45-spezifisches scFv, jeweils verbunden mit der VL- bzw. der VH-Domäne eines CD3ε-spezifischen Antikörpers. Es stellte sich heraus, dass nach gleichzeitiger Bindung der beiden Konstrukte an dieselbe HLA-A2- und CD45-exprimierende Zelle sich die beiden einzelnen, ungepaarten variablen Domänen eines CD3ε-spezifischen Antikörpers zu einem funktionellen scFv zusammenfügen. Dieses Zusammenfügen sollte in einer therapeutischen Situation ausschließlich auf den Blutkrebszellen des Empfängers geschehen, was zur T-Zell-vermittelten Zerstörung der Krebszellen führen würde. Auf diese Weise könnte ein Rückfall der Erkrankung vermieden und eventuell sogar auf die Standardtherapie (Bestrahlung und Chemotherapie) verzichtet werden. Für die beiden beschriebenen Ansätze wurden die Antikörperkonstrukte periplasmatisch in E. coli exprimiert, über einen His-Tag aufgereinigt und biochemisch charakterisiert. Ihre Bindung an die jeweiligen Zielantigene wurde mittels Durchflusszytometrie nachgewiesen. Die stimulatorischen Eigenschaften der Antikörper wurden durch eine Messung der IL-2-Freisetzung nach Inkubation zusammen mit T-Zellen und antigenexprimierenden Zielzellen untersucht. Sowohl der gegen Rhabdomyosarkom gerichtete BiTE-Antikörper, als auch der zusammengefügte trispezifische Antikörper gegen Blutkrebs vermittelten konzentrationsabhängig eine T Zellaktivierung bei nanomolaren Konzentrationen. Für den trispezifischen Antikörper erwies sich dieser Effekt tatsächlich als abhängig von zwei Antigenen, da er durch eine vorausgehende Inkubation mit entweder einem HLA-A2- oder einem CD45-spezifischen scFv-Fragment geblockt werden konnte und nicht auf Zellen auftrat, die nur ein Antigen (CD45+) oder keins von beiden (HLA-A2- CD45-) tragen. Darüber hinaus rekrutierten die Antikörper beider Ansätze T-Zellen zur Zerstörung von Tumorzellen in vitro

博士
國立臺灣大學
醫學工程學研究所
103
Cancer is a terrible disease characterized by abnormal cell growth and metastasis, which is the potential to invade or spread to other parts of the body. Although many clinical therapies are used to kill the cancer cells, these therapies usually cause a variety of side effects and show low survival in some particular type of cancer. Cancer immunotherapy is the use of one person’s immune system to treat or destroy cancer. Over the years, the researchers believe that successful stimulation of the patient’s own immune system are able to eliminate cancer and are able to significantly improve the survival rate of cancer patients without producing serious side effects. Therefore, how to induce effective immune responses to treat various types of cancer has been an extremely important issue. Alpha-Galactosylceramide is a potent vaccine adjuvant in protecting against tumors. In this study, α-GalCer was incorporated into lipid-bilayer of liposome and exposed galactose molecules on the outer surface of liposome. Ovalbumin (OVA) was then encapsulated into α-GalCer incorporated liposomes. We evaluate whether α-GalCer incorporated liposomes could act as an effective DC-targeted mucosal vaccine that could facilitate antigen delivery familiar to galactosylated liposomes we have published. The immunoregulatory effects of α-GalCer incorporated liposomes were also evaluated. The particle sizes of each liposome formulation were controlled to are approximately 1000-1100 nm and OVA encapsulation efficiencies are approximately 30-40%. We demonstrated that α-GalCer incorporated liposomes effectively facilitated antigen uptake by mouse bone-marrow derived dendritic cells (BMDCs) in vitro, led to higher expression of maturation markers, including CD80, CD86 and MHC II, and induced higher production of pro-inflammatory cytokines. In vivo uptake of α-GalCer incorporated liposomes by DCs in nasopharynx-associated lymphoid tissue (NALT) showed the similar results. C57BL/6 mice immunized intranasally with OVA-encapsulated α-GalCer incorporated liposomes produced high levels of OVA-specific IgG antibodies in their serum and secretory-IgA (s-IgA) in nasal wash fluid. Spleen cells from mice receiving α-GalCer incorporated liposomes were re-stimulated with OVA and showed significantly augmented levels of IFN-γ and IL-4, and increase number of IFN-γ producing CD8+ cells. In addition, intranasal administration of α-GalCer incorporated liposomes resulted in complete protection against EG7 tumor challenge in C57BL/6 mice. Taken together, these results indicate that nasal administration of α-GalCer incorporated liposomes mediates the development of an effective immunity against tumors and might be useful for further clinical anti-tumoral applications.

In recent years, immunotherapy approaches for the treatment of cancer have been intensively investigated and substantial benefit observed in clinical trials has led to the marketing authorization of various immunotherapeutic drugs including monoclonal antibodies and cytokine-based therapies. Even though, several studies demonstrated effectivity of cancer immunotherapy, improvement is still required to address issues such as efficacy and safety. Development of suitable delivery systems might contribute to the improvement of immunotherapeutic approaches for cancer, whereby one strategy involves the application of viral vectors. For this purpose, viruses can be used in two ways: Either as replication-deficient viral vectors that solely deliver the therapeutic gene of interest or as replication-competent oncolytic viruses that directly kill tumor cells and are additionally engineered to encode immunomodulatory transgenes. The latter strategy was addressed in the first part of the thesis which aimed at combining the oncolytic activity of semireplication-competent vesicular stomatitis virus (srVSV) with the viral-mediated expression of immunotherapeutic transgenes in order to induce a long-lasting antitumor immune response. The second part of the thesis intended to generate and characterize replication-deficient, receptor-targeted adeno-associated viral (AAV) vectors for the tumor-specific delivery of immune checkpoint inhibitors. The srVSV system is based on two trans-complementing, propagation-deficient VSV vectors, VSVΔG and VSVΔL. Both vectors were armed with the immunostimulatory transgenes granulocyte-macrophage colony-stimulating factor (GM-CSF), FMS-like tyrosine kinase 3 ligand (Flt3L), B7 and the tumor-associated antigens Her2/neu as well as CTLA4-Her2/neu to enhance srVSV-mediated antitumor immune responses. After insertion of the immunotherapeutic transgenes into the VSVΔG as well as VSVΔL vector genomes, all recombinant vectors were successfully generated de novo and analyses confirmed srVSV-mediated transgene expression in vitro. As VSV is exquisitely sensitive to type I interferon (IFN)-induced antiviral responses, different murine tumor cell lines were analyzed for their IFN sensitivity to identify a suitable VSV-permissive syngeneic tumor mouse model for the preclinical studies. The murine colon cancer cell line MC38 was identified as a potentially appropriate tumor model since MC38 cells were productively infectable even when pretreated with high doses of IFNα. First preclinical data showed the capability of transgene-armed as well as control srVSV to induce an antitumor immune response in the MC38 tumor model eventually leading to cure in some animals. However, the in vivo data showed only a marginally improved therapeutic efficacy of transgene-armed versus control srVSV. Accordingly, more preclinical studies are required to clarify whether arming of srVSV with immunomodulatory payload indeed improves srVSV therapy efficacy. Monoclonal antibodies directed against immune checkpoints, such as programmed cell death protein-1 (PD-1) and its ligand PD-L1, have shown promising results for the treatment of certain cancer types eventually leading to the marketing authorization of several products. However, drawbacks of this therapy include lack of efficacy in some patients and treatment-related toxicity. Probably the systemic administration of these antibodies does not only promote the activation of immune responses in the tumor microenvironment but also in healthy tissue. Therefore, this project aimed at the specific delivery of immune checkpoint inhibitors precisely to sites of tumor growth. To this end, Her2/neu-targeted AAV (Her2-AAV) vectors were equipped with the coding sequence of PD-1- or PD-L1-specific inhibitors. In vitro analyses showed that the inhibitors were readily detectable in the supernatant of AAV-transduced tumor cells. Furthermore, AAV-encoded αPD-1 as well as αPD-L1 specifically bound their target antigens. In vivo imaging analyses revealed that Her2-AAV successfully targeted tumor cells upon systemic injection into immunocompetent BALB/c mice bearing subcutaneously growing Her2/neu-positive RENCA tumors, while non-targeted AAV2 transduced mainly liver. Finally, in vivo delivery of AAV-encoded αPD-1 was assessed in the aforementioned mouse model. Mice injected with AAV2 showed the highest αPD-1 levels in the liver. In contrast, Her2-AAV successfully redirected the immune checkpoint inhibitor from liver to Her2/neu-positive tumor tissue upon systemic injection. In conclusion, this study is a proof of concept that tumor-targeted AAV vectors can be used for the delivery of immune checkpoint inhibitors to the site of tumor growth. It will pave the way for further investigations addressing toxicity and efficacy of vector-mediated compared to systemic immune checkpoint modulation.

The objective of this project is to develop novel treatments, using expression-targeted gene therapy and minicell technology, to replace current methodologies used in the clinic for the treatment of carcinomas, especially transitional cell carcinoma of the bladder. The expression-targeted gene therapy procedure involves cancer-specific DNA elements (promoters) to drive the expression of engineered suicide genes to induce apoptosis in cancer cells. Minicells, a kind of bacterial derivative , prevent tumor recurrence and growth through targeted toxicity and an induced immune response that is similar to that induced by Bacille Calmette-Guerin (BCG), but without the risk of infection due to lack of chromosomes. The osteopontin promoter (popn) was selected via currently accepted methods by comparing endogenous gene expression between normal and cancerous cells. The opn gene is expressed in far greater amounts in cancer cells, so it was reasoned that the opn promoter would be more active in cancer cells as well. Reporter constructs using popn were transfected into both cancerous and normal cell types, with maximum Popn-driven reporter intensity in the cancer cells showing up as strong (102.7%) compared to Pcmv-driven positive controls. Popn-driven reporter intensity was reduced by ~90% in the non-cancer cells. Further enhancements to targeting and expression were obtained through the incorporation of single-nucleotide polymorphisms (SNPs) in the promoter sequence. Further investigations to confirm a correlation between endogenous opn mRNA levels and Popn-driven reporter expression produced a surprising lack of correlation (R2=0.24). However, taking into account opn mRNA splicing variants, a strong negative correlation was determined between mRNA levels of the variant opn-a and Popn-driven transgene activity (R2=0.95). Three novel cancer-specific promoter pran, pbrms1 and pmcm5 were identified through a new screening logic. The activities of those promoters were verified to be much higher in the tested cancer cell lines than the current gold standard used to target gene expression to cancer cells: the promoter of human telomerase reverse transcriptase (phTERT). A constitutively active, apoptosis-inducing analog of caspase 3, referred to as Reverse Caspase3 (RevCasp3), was engineered via gene recombination and cloned into expression-targeted plasmid constructs. These constructs showed excellent activity in inducing apoptosis within the cancer cells tested. Moreover, Pran-RevCasp3 constructs were shown to have significant, cancer-specific killing action within both human and murine cell in vitro. The therapeutic effects of minicell constructs known as VAX-IP were tested within our orthotopic, murine model of transitional cell carcinoma of the bladder. In trials focused on the prevention of tumor growth and tumor implantation, bell-shaped curves were produced by data reflecting the relation between drug dose and tumor burden. The median and average bladder weights, used as a surrogate for tumor burden, decreased with increasing doses of VAX-IP minicells administered via intravesical, transurethral delivery. Activity was lost at high doses of VAX-IP minicells. Compared with the sham-treated group, 1x108 VAX-IP minicells, delivered at 24 hours post-surgery with repeated administrations every 7 days for a total of four treatments, yielded a significant survival advantage to the treated animals (P=0.03).
acase@tulane.edu

Differently from lung adenocarcinoma, effective targeted therapies for lung squamous-cell cancer (SQLC) are still missing, although a series of molecular pathways are constantly altered. In this regard, the prognostic and/or predictive impact of potential drivers needs to be elucidated, in order to create a global portrait of SQLC patients. Nowadays, one of the main emerging research strategies in cancer is centered on the study of the genome of exceptional responder and prognostic outlier patients. Adopting this idea, we retrospectively analysed a multicenter series of 573 surgically resected SQLC patients and we built one of the first risk classification model for SQLC, which was afterwards validated in a larger cohort of more than 1000 patients. This model, based on a combination of simple and easily available clinicopathological parameters, was able to effectively stratify resected SQLC patients in risk classes with a good prognostic accuracy. Once identified the best and worst prognostic performers, we investigated their molecular portrait, principally by next-generation sequencing (NGS), and their expression profile to identify recurrent molecular alterations and explore their association with prognosis. Sixty and forty-seven patients were evaluated as training and validation cohort, respectively. Copy number variation (CNV) was the most frequent genomic event. Molecular alterations were distributed regardless of prognosis, except from DDR2 mutations in good prognosis group and SMAD4 loss in poor prognosis group. The potentially druggable PI3KCA/mTOR axis represented the most frequently altered pathway (42%), with PI3KCA mutations and RICTOR high gain reported only in poor prognosis group. Upon transcriptome analysis, RICTOR high gain patients presented an increased pathway activity. The final aim of the overall project was to evaluate the molecular profile of outliers resected SQLC taking the advantage to adopt modern technologies in order to identify those molecular aberrations potentially able to predict the probability of disease recurrence and the efficacy of agents selectively targeting these candidate pathways. This integrated multi-step analysis performed in almost one hundred resected SQLC patients identified altered pathways with a biological impact in SQLC oncogenesis, revealing RICTOR high gain as a candidate therapeutical target for selective inhibition in SQLC.

Les immunothérapies de type immune checkpoint blockade (ICB) ont révolutionné les approches thérapeutiques en oncologie médicale et ont largement contribué au fait que l’immunothérapie est maintenant considérée comme le quatrième pilier des traitements anticancer, aux côtés d’approches traditionnelles telles que la chirurgie, la radiothérapie et la chimiothérapie. Malgré les résultats encourageants des études cliniques évaluant ce type d’immunothérapie, la majorité des patients décèderont des suites de leur maladie. Conséquemment, le domaine de recherche visant à comprendre les mécanismes de résistance aux immunothérapies est en expansion constante. Plusieurs stratégies visant à améliorer les issues cliniques ont été proposées, parmi lesquelles figurent: 1) la recherche de nouvelles cibles thérapeutiques dans le microenvironnement immun tumoral et; 2) les études de combinaisons thérapeutiques où une immunothérapie est jumelée à d’autres types de modalités thérapeutiques potentiellement synergiques. Chacune des études présentées dans cette thèse de recherche s’apparente à l’une ou l’autre de ces stratégies. Dans le cadre de notre première étude, nous démontrons que la protéine WISP1 représente une cible prometteuse à l’intérieur du microenvironnement de plusieurs types de tumeurs solides étant donné son association avec différentes variables pronostiques et proinflammatoires, ainsi qu’avec un programme épigénétique complexe, la transition épithélialemésenchymateuse (Epithelial-Mesenchymal Transition; EMT). De plus, nous démontrons que les niveaux d’expression de WISP1 sont significativement plus élevés au sein des tumeurs démontrant une résistance primaire aux immunothérapies de type ICB, particulièrement lorsque qu’une signature reliée à l’EMT peut être retrouvée de façon concomitante. Pour notre deuxième étude, nous avons utilisé des modèles murins in vivo de cancer pulmonaire non à petites cellules KRAS-mutés afin de tester différentes combinaisons thérapeutiques jumelant une thérapie dite ciblée (i.e., un inhibiteur de MEK) a différentes immunothérapies de type ICB. Nos résultats démontrent que l’ajout d’une immunothérapie anti-CTLA-4 à l’inhibiteur de MEK AZD6244 (selumetinib) et une immunothérapie anti-PD-L1 augmente significativement la survie, et que ces bénéfices sont associés à une diminution de marqueurs reliés à l’EMT. Il existe donc un lien commun entre ces deux études qui repose sur l’importance de l’EMT comme facteur favorisant la résistance thérapeutique aux immunothérapies. De plus, nous démontrons pour la première fois que les bénéfices associés à la triple combinaison thérapeutique susmentionnée peuvent être corrélés à une diminution d’expression de marqueurs liés à l’EMT. Par conséquent, nos résultats sont discutés en tant que base potentielle pour de futures études visant à réduire la résistance thérapeutique reliée à l’EMT. Nous discutons également de la valeur translationnelle de nos résultats à travers le développement d’une étude clinique.
Immune checkpoint blockade (ICB) has revolutionized therapeutic approaches in the field of medical oncology and has largely contributed to the fact that immunotherapy is now being regarded as the fourth pillar of cancer treatment alongside surgery, radiotherapy and chemotherapy. Despite encouraging results from clinical trials using ICB, most patients ultimately relapse or succumb to their disease. Therefore, the field of immunotherapeutic resistance research is rapidly expanding. Many strategies to improve ICB responses have been undertaken, including: 1) the search for novel, actionable targets in the immune tumor microenvironment (TME) and; 2) therapeutic combination studies where an ICB backbone is combined with different, synergistic treatment modalities. Each of the studies presented in this research thesis embraces one of these strategies. In our first study, we show that WISP1 represents a promising TME target in multiple solid tumor types by demonstrating its association with prognostic and pro-inflammatory variables, as well as to a complex epigenetic program termed Epithelial-Mesenchymal Transition (EMT). Furthermore, we show that increased WISP1 expression is associated to primary resistance to ICB, particularly when EMT-related signatures are found concomitantly. In our second study, we used in vivo mouse models of KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) to test different therapeutic combinations of targeted therapies (i.e., MEK inhibitor) and ICB. We found that the addition of anti-CTLA-4 ICB to MEK inhibitor AZD6244 (selumetinib) and anti-PD-L1 ICB increases survival, and that these benefits are associated with the downregulation of EMT-related markers. Therefore, there exists a common link between these studies, which relies on the significance of EMT as a detrimental factor within the TME and its association with ICB resistance. Moreover, we show for the first time that the benefits of ICB combination therapy can be associated to the downregulation of EMT markers in vivo. Consequently, we discuss how our results may constitute the basis for future work aiming at reducing EMT-mediated therapeutic resistance, as well as the translational relevance of our pre-clinical results through the development of a clinical trial.