abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates. / Dissertation/Thesis / Masters Thesis Industrial Engineering 2020

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23084号 / 医博第4711号 / 新制||医||1049(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 木村 剛, 教授 森本 尚樹, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les maladies ischémiques du myocarde sont actuellement la principale cause de mortalité dans les pays développés. Malgré la diminution des délais de reperfusion et l’amélioration de la prise en charge médicale, la mortalité de l’infarctus du myocarde reste stable. Le développement de nouvelles solutions thérapeutiques reste donc nécessaire. Parmi ces nouveaux traitements, les dérivés cellulaires d'origine cardiaque (obtenues après mise en culture de fragments de biopsies) semblent prometteurs. Ces cellules sont obtenues à partir de biopsies myocardiques qui mises en culture vont spontanément s’organiser en cluster multicellulaires tridimensionnels appelés cardiosphères. La modification des conditions de culture permet ensuite d’obtenir une population de cellules uniques appelés cellules dérivés des cardiosphères (CDCs). Ces CDCs sont capables de diminuer la taille de l’infarctus dans l’infarctus constitué (études précliniques et cliniques) ainsi que dans l’infarctus aigu (ischémie reperfusion). Cependant plusieurs études réalisées chez le petit animal suggèrent que 1. Les cardiosphères pourraient avoir un potentiel thérapeutique supérieur à celui des CDCs et 2. Les exosomes sécrétés par les CDCs semblent être les médiateurs de leur effet, et l’injection des exosomes seuls pourraient permettre d’obtenir le même effet. Néanmoins, aucune étude n’a jusqu’à présent évalué les cardiosphères et les exosomes dans un modèle pre-clinique de gros animal. L’objectif de cette thèse sera donc de comparer les effets des cardiosphères, des CDCs et des exosomes sécrétés par les CDCs dans des modèles porcins d’ischémie reperfusion et d’infarctus constitué.Méthodes : Dans un premier temps, la technique de délivrance du traitement, la dose optimale et la sécurité seront évaluées ; dans un 2e temps des études randomisées contre placebo seront réalisées pour évaluer l’efficacité. Le modèle animal utilisé sera un modèle porcin d’un infarctus du myocarde reperfusé réalisé par occlusion de l’artère interventriculaire antérieure (après la première diagonale) par un ballon d’angioplastie. Pour les études d’ischémie reperfusion, les animaux seront traités 30 minutes après la reperfusion et suivi pendant 48 heures. Pour les études dans l’infarctus constitué, les animaux seront traités 4 semaines après l’infarctus puis suivi 4 semaines supplémentaires. L’efficacité sera évaluée par histologie (étendue du no-reflow et taille de l’infarctus) pour l’ischémie reperfusion, et par IRM (taille de la cicatrice et fonction ventriculaire gauche) pour l’infarctus constitué.Résultats attendus : nous espérons confirmer les résultats obtenus chez le petit animal à savoir démontrer une efficacité de cardiosphères supérieure à celle des CDCs, et une efficacité des exosomes au moins égale à celles des CDCs mais avec un profil de sécurité (notamment immunologique) supérieur. / Background: Ischemic heart disease is the first cause of death in western countries. Despite early reperfusion and improvement of medical care, myocardial infarction (MI) mortality remains constant. Therefore new treatments are desirable. Among those new treatments, heart derived cells (obtained from cardiac explants) are promising. Those cells are grown from cardiac explants which, in culture, will spontaneously self-organized in three-dimensional multicellular cluster named cardiospheres. When replated in adherent surface, those cardiospheres will yield to a population of single cells named cardiosphere-derived cells (CDCs). Allogenic CDCs have been shown to decrease infarct size both in convalescent MI (pre-clinical and clinical studies) and in ischemia-reperfusion. However several small animal studies suggest that 1. Cardiospheres may be more potent than CDCs and 2. CDC-secreted exosomes are likely to be the mediator of CDC effect and their injection may recapitulate the effect of CDCs. Nevertheless, no study has assessed the efficacy of cardiospheres and CDC-derived exosomes in a relevant pre-clinical large-animal model. We aim to compare the effect of cardiospheres, CDCs and CDC-derived exosomes in pig models of convalescent MI (adverse remodeling) and acute MI (ischemia reperfusion).Methods: First, delivery, optimal dose and safety will be optimized. Then, randomized placebo-controlled study will be performed to assess efficacy. For all studies, MI will be performed by balloon occlusion of the left anterior descending artery after the 1st diagonal. For the acute studies, pigs will be treated 30 minutes after reperfusion and followed for 48 hours. For the chronic studies, pigs will be treated 4 weeks after MI and followed for 4 additional weeks. Efficacy will be evaluated by histology (no-reflow and infarct size) for the acute studies and by MRI (scar size and left ventricular function) for the chronic studies.Expected results: we expect to confirm the results obtained in small animal models. Efficacy of cardiospheres may be better than CDCs, and exosomes should be at least as effective as CDCs but with a more favorable safety profile (especially immunological).

Eine Vielzahl von Studien konnte den nützlichen Effekt einer Knochenmarkzelltherapie nach Schlaganfall an Ratten nachweisen. Diese Ergebnisse ließen sich jedoch in klinischen Studien nicht reproduzieren. Eine mögliche Ursache können die Unterschiede zwischen den eingesetzten Versuchstieren und Zellspendern sowie den Patienten im klinischen Bereich sein. Während die eingesetzten Tiere und Spender meist jung und gesund waren, ist der typische Schlaganfallpatient in der Regel älter und leidet an einer Vielzahl von Begleiterkrankungen. Ziel dieser Studie war es, die Auswirkungen von erhöhtem Alter und Begleiterkrankungen auf die Effektivität einer Knochenmarkzelltherapie des Schlaganfalles zu untersuchen. Zu diesem Zwecke wurde die Arteria cerebri media von 18 Monate alten spontan-hypertensiven Ratten operativ verschlossen. Nach 24 Stunden wurden entweder Zellen eines jungen oder eines alten Spender transplantiert. Tiere, die eine äquivalente Menge an PBS erhielten, dienten als Kontrolle. Im Anschluss wurden das neurofunktionelle Defizit und das Infarktvolumen über einen Zeitraum von 63 Tagen mittels verschiedener Verhaltenstest und magnetresonanztomographischer Bildgebung analysiert. Nach Induktion des Schlaganfalls kam es zu einer Abnahme der funktionellen Leistung in allen Verhaltensversuchen, die sich jedoch bis zu Tag 27 wieder erholte. Im MRT war im gleichen Zeitraum eine Reduktion des Infarktvolumens zu beobachten. In der zweiten Versuchshälfte kam es anschließend zu keinen weiteren Veränderungen, weder funktional noch morphologisch. Ein Einfluss der Therapie auf die Regeneration oder die Größe des Infarkts, war zu keinem Zeitpunkt feststellbar. Anhand dieser Ergebnisse muss in Frage gestellt werden, ob gealterte und komorbide Tiere durch einer Zelltherapie nach Schlaganfall profitieren können und ob sich Zellen älterer Spender als Therapeutikum für einer Knochenmarkzelltherapie des Schlaganfalls eignen.

Most Adoptive Cell Therapies (ACT), including CAR T cell therapies, suffer failure because of the severe side effects due to loss-of-control of the therapeutic cells once they are inside the patient’s body, suggesting that novel strategies must be developed for a better in vivo control of these engineered cells. In the meantime, CAR T cell therapies targeting solid tumors have not experienced the remarkable success achieved with hematopoietic cancers, mainly due to continuous tumor antigen exposure and a suppressive tumor microenvironment. Here we designed a private passageway fusion receptor, which is composed of a ligand binding domain and a glycosylphosphatidylinositol (GPI) anchoring domain, to be expressed and localized to the surface of CAR T cells independently to the classical CAR T construct. These ligand binding domains preserve high binding affinity towards their cognate ligands and are only expressed on the CAR T cells that have been transduced. Therefore, cytotoxic drugs or immunosuppressants linked to the corresponding targeting ligands are shown to be specifically delivered to these fusion receptor positive CAR T cells for lowering the activity of the over-activated CAR T cells. On the other hand, we discovered that a potent TLR7 agonist is able to enhance the lysis effect of the exhausted CAR T cells in a co-culture model. Serial releasable and non-releasable targeted TLR7 agonists were prepared and tested. Based on these data, we suggest that our secret passageway fusion receptor platform provides a better control of the activity of CAR T cells using the corresponding targeting ligand-payload conjugates in a dose dependent manner and function as a doorway for the delivery of instructions to CAR T cells for versatile purposes.

Due to their immunomodulatory and regenerative potential, mesenchymal stem cells (MSCs) represent a promising therapeutic tool for cell-based therapy, organ transplantation or tissue engineering. To improve clinical applicability of MSCs, new methods to increase their delivery and efficacy have been tested in the latest years but the mechanism of observed alterations has not yet been described. In the present project we focused on studying the effect of several factors that can significantly affect the therapeutic success of MSC-based treatment. Initially, we analysed the therapeutic effect of MSCs applied locally on nanofiber scaffold with incorporated cyclosporine A (CsA) in a mouse model of allogeneic skin transplantation. Our results indicate that application of MSCs in the presence of CsA direct M1/M2 macrophage polarization towards regulatory phenotype. This phenotype switching is accompanied by decreased production of nitric oxide (NO) and interferon  (IFN-) and increase production of interleukin 10 (IL-10), and may result in suppression of the local inflammatory reaction. The next goal of proposed study was to analyse the effect of the treatment based on MSCs combined with immunosuppressive drugs with different mechanism of action on the balance among distinct T cell subpopulations. We...

T cells engineered with chimeric antigen receptors (CAR-T) have had breakthrough successes in cancer immunotherapy with FDA approvals. Still, the high cost of personalized CAR-T cell therapy hinders the accessibility from the public as a therapeutic option, and the lack of uniquely defined cancer-specific antigens brings the risk of life-threatening on-target, off-tumor toxicity. Changing the immune cell therapy platform to Natural Killer cells (NK cells) can be an option to develop allogenic off-the-shelf cell therapy. To reduce the on-target, off-tumor toxicity, developing an inhibitory CAR (iCAR) for cell therapy is necessary for precise logic computation and implementation of iCAR can broaden the spectrum of treatable cancers. This dissertation optimized the primary NK cell expansion platform with autologous PBMC and set robust virus transduction using pseudotyped retrovirus on the primary NK cells to use the NK cells as the cell therapy platform. I engineered iCAR candidates to suppress in response to a “safety antigen” on healthy cells, first in T cells and validated in NK cells, showing its efficacy against various versions of activating CAR (aCAR) drove killing of targets expressing activating antigen, while an inhibitory CAR (iCAR) suppressed cytotoxicity against targets expressing a safety antigen. Therapeutic immune cells equipped with the right pair of aCAR and iCAR platform will improve safety for currently validated cancer antigens and enable new therapies for previously unaddressed indications. / 2024-05-23T00:00:00Z

Der Schlaganfall stellt die häufigste Ursache für dauerhafte körperliche Behinderungen im Erwachsenenalter dar. Derzeit steht aufgrund des engen therapeutischen Zeitfensters lediglich für 10% aller Schlaganfallpatienten die kausale Thrombolysetherapie zur Eröffnung des verschlossenen Blutgefäßes zur Verfügung. Eine vielversprechende Therapieoption stellen humane Nabelschnurblutzellen dar, deren Effektivität bisher ausschließlich bei jungen und meist gesunden Tieren nachgewiesen wurde. Die vorliegende Arbeit untersuchte die Wirksamkeit intravenös applizierter, kryokonservierter mononukleärer Zellen des humanen Nabelschnurblutes auf die Entwicklung eines experimentellen Schlaganfalles in jungen und alten hypertensiven Ratten. Die sensomotorische Regeneration der Tiere wurde anhand der BeamWalk- und mNSS-Tests untersucht. Im Rahmen dieser Arbeit wurde außerdem der LadderRung Walking-Test etabliert. Parallel durchgeführte MRT-Untersuchungen dienten zur Analyse des Infarktvolumens. Nach Ablauf des 64-tägigen Beobachtungszeitraumes wurde die Proliferationsaktivität von Astrozyten im Randbereich des Infarktes überprüft. Die Auswertungen sensomotorischer Fähigkeiten, der Infarktvolumina und der Zellproliferation ergaben keine signifikanten Unterschiede zwischen Therapie- und Kontrollgruppe beider Altersstufen. Junge und alte Ratten entwickelten vergleichbare Infarktvolumina und wiesen ähnliche motorische Fähigkeiten auf. Ein erhöhtes Alter der Versuchstiere war darüber hinaus mit methodischen Problemen behaftet, was die sensomotorische Funktionalitätsprüfung alter Tiere eingeschränkte und eine histopathologische Auswertung ausschloss.

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20569号 / 医科博第79号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 江藤 浩之, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM