Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)

Through the years, cancer therapies have progressed rapidly, pouring out novel treatments such as gene therapy, small molecule therapies and immunotherapy. One such immunotherapy, adoptive cell transfer (ACT), augmented through the addition of a chimeric antigen receptor (CAR), has proven success in treatment of hematological malignancies. Additionally, oncolytic viruses (OV) and OV-based (OVV) therapies, have shown promising results in both clinical and pre-clinical studies. In most instances, when applied as a monotherapy, the aforementioned treatment methods are incapable of inducing complete tumour remission. The Wan lab has developed an approach combining ACT with OVV therapies that dramatically increase therapeutic benefit resulting in complete regression of well-established solid tumours. Despite promising results, certain tumours can still escape this combination therapy through antigen loss resulting in antigen negative relapse (ANR). To further augment the therapy, the addition of a secondary receptor (CAR) provides the ACT multiple avenues of attack to prevent ANR. In this dissertation, we define culture conditions that promote strong expression of the CAR alongside confirmation of function in an in vitro setting. Following, it is demonstrated that OVV boosted dual-targeting T cells carry strong T cell activity by measure of cytokine release in vivo. Despite promising T cell activity data, dual-specific T cells are unable to improve tumour control and survival once relapse occurs. The failure to control relapse remains unclear however evidence points towards lack of T cell persistence, poor CAR function in vivo and a lack of endogenous T cell response leading to compounding effects that prevent dual-targeting T cells from preventing ANR. Although dual specific therapies have shown poor efficacy in preventing ANR, further study must be completed to identify areas of improvement – such as persistence, as the potential for success in using dual-targeting T cells coupled with OVVs still lies untapped. / Thesis / Master of Science (MSc)

No description available.

No description available.

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.

L’allogreffe de cellules souches hématopoïétiques (CSH) est un traitement efficace des Hémopathies malignes. Cependant, les complications des allogreffes parmi lesquelles les infections virales sont associées parfois à une morbidité et une mortalité importantes. Ces infections surviennent en l’absence de reconstitution immunitaire. Un monitoring régulier de la charge virale des principaux agents infectieux impliqués est réalisé mais amène parfois à la mise en oeuvre abusive de traitements anti-viraux qui ne sont pas dénués de toxicité.Dans ce travail, nous proposons d’associer à ce monitoring un suivi régulier de la reconstitution immunitaire spécifique afin de cibler parmi les patients présentant une réactivation ceux qui nécessitent un traitement curatif de ceux qui pourront maîtriser l’infection par leur système immunitaire. Nous illustrons ce propos avec le virus d’Epstein Barr (EBV) et avons en cours une étude sur l’Adénovirus (ADV).Dans certains cas parfaitement ciblés, les traitements anti-viraux s’avèrent inefficaces. C’est pourquoi dans ce travail, nous présentons la mise au point d’une technique de grade clinique de production de lymphocytes T cytotoxiques anti-ADV (CTL anti-ADV) en condition GMP (Good Manufacturing Practice), grâce au système CliniMACS et au Cytokine Capture System de Miltenyi, afin de proposer une immunothérapie adoptive.Nous décrivons par la suite trois expériences cliniques de traitement compassionnel d’une infection ADV post-allogreffe de CSH. Enfin, nous présentons les résultats préliminaires de la production de CTL bispécifique anti-ADV et CMV / Hematopoietic stem cells Transplantation (HSCT) is a well recognized strategy for treatment of haematological malignancies. However, HSCT complications among which the viral infections a reassociated with high morbidity and mortality. These infections arise in the absence of immune reconstitution. Monitoring of viral reactivations after allogeneic HSCT is necessary, to identify patients at risk of viral infections, but not sufficient, as patients may be abusively treated. In this work we propose to combine viral DNA load assessment with specific immune monitoring to target patients who need to be treated. We report a retrospective study investigating EBV infection and EBV-specific immune recovery using the functional IFN Elispot assay in 40 allogeneic HSCT patients. We initiated a similar study with ADV which is pending. However, although patients are correctly targeted, anti-viral treatment is sometimes not effective. We present a study on the development of a complete clinical grade generation of Human anti-Adenovirus cytotoxic T cells in GMP (Good Manufacturing Practice) conditions, thanks to the system CliniMACS and the Cytokine Capture System, to propose an adoptive immunotherapy to the recipient.We describe afterwards three clinical experiments of treatment of an ADV infection after HSCT.Finally, we present the preliminary results of the anti-ADV and -CMV bi-specific CTL production.

L’allogreffe de cellules souches hématopoïétiques (CSH) est un des seuls traitements curatifs des hémopathies bénignes ou malignes et des déficits immunitaires primitifs. Cependant, les infections notamment virales ainsi que la réaction du greffon contre l’hôte comptent parmi les complications les plus fréquentes des allogreffes associées à une morbidité et une mortalité élevées. Les infections virales surviennent souvent en l’absence de reconstitution immunitaire spécifique dans un contexte d’immunosuppression liée à la GVHD elle-même ou à la prophylaxie ou au traitement de la GVHD. Les traitements médicamenteux anti-viraux préconisés présentent une efficacité inconstante dans ce contexte d’immunodéficience et ne sont pas dénués de toxicité. L’alternative thérapeutique prometteuse est l’immunothérapie adoptive cellulaire notamment celle qui consiste en l’injection de lymphocytes T spécifiques anti-viraux isolés par technique immunomagnétique (VSTs). Cependant, ces lymphocytes T peuvent être la cible des traitements immunosuppresseurs administrés pour la GVHD mais également par eux-mêmes être potentiellement la cause de la survenue ou de la réactivation d’une GVHD. Nous avons montré dans ce travail que l’efficacité des VSTs, qui repose sur leur expansion in vivo lors de la rencontre avec le virus circulant, est principalement permise par les sous-populations lymphocytaires les plus immatures, même si elles ne sont présentes qu’en faible proportion. Nous défendons dans ce travail le fait que l’efficacité des VST ainsi que leur persistance repose prioritairement sur la présence des sous-populations lymphocytaires T les plus immatures et ce quel que soit le degré de compatibilité HLA entre les VSTs et le receveur. De plus, leur sensibilité modérée aux corticoïdes, que nous avons étudiée in vitro, ne justifie pas la modulation de l’immunosuppression lors de l’injection des ADV-VSTs, comme observé in vivo dans le protocole clinique multicentrique de phase I/II que nous avons mené entre 2012 et 2015. En effet, ce protocole clinique ne rapporte aucune GVHD de novo après injection d’ADV-VSTs ; en revanche, la modulation de l’immunosuppression peut potentiellement être incriminée dans la réactivation de GVHD dans les semaines suivant l’injection des ADV-VSTs. La réalisation d’un essai comparatif de phase II permettra de prouver très clairement le rôle des VSTs dans la réactivation de GVHD. / Hematopoietic stem cell transplantation (HSCT) is one of the only curative treatments for benign or malignant hematological diseases and primary immune deficiencies. However, viral infections and graft-versus-host disease (GVHD) are among the most frequent complications after HSCT associated with high morbidity and mortality. Viral infections often occur in the absence of specific immune reconstitution in the context of immunosuppression related to GVHD itself or to the prophylaxis or treatment of GVHD. The recommended anti-viral drug treatments have an inconsistent efficacy in this context of immunodeficiency and are not devoid of toxicity. The promising therapeutic alternative is adoptive immunotherapy, in particular the infusion of specific anti-viral T lymphocytes isolated by immunomagnetic technique (VSTs). However, these T lymphocytes may be targeted by immunosuppressive treatments administered for GVHD, but also may be the cause of the onset or reactivation of GVHD. We have shown in this work that the efficacy of VSTs, which is based on their in vivo expansion when they encounter the circulating virus, is mainly allowed by the most immature lymphocyte subpopulations, even in a small proportion. We argue in this work that the efficacy of VSTs and their persistence is mainly based on the presence of the most immature T lymphocyte subpopulations and this regardless of the degree of HLA compatibility between the VSTs and the recipient. Moreover, their moderate sensitivity to corticosteroids, which we have studied in vitro, does not justify the modulation of immunosuppression at the time of infusion of ADV-VSTs, as observed in vivo in the multicenter phase I / II clinical trial we conducted between 2012 and 2015. Indeed, this clinical trial does not report any de novo GVHD after ADV-VSTs infusion. On the other hand, modulation of immunosuppression may potentially be incriminated in the reactivation of GVHD within weeks of ADV-VST infusion. A Phase II comparative trial will bring the evidence of efficacy and will clearly determine the role of VSTs in the reactivation of GVHD

Adoptiver Transfer EBV-spezifischer, polyklonaler T-Zelllinien findet Anwendung bei Prophylaxe und Therapie EBV-assoziierter Erkrankungen. Der Ansatz hat den Nachteil der aufwändigen Herstellung der T-Zelllinien, welche aufgrund der Stimulation mit EBV transformierten B-Zelllinien oft nicht die gewünschten EBV Antigene, sondern immundominante EBV Antigene erkennen. Eine Alternative zum polyklonalen T Zelltransfer stellt die Übertragung EBV-spezifischer T-Zellrezeptoren (TCRs) dar. Dadurch können subdominante EBV Antigene angegangen werden, die von Tumorzellen tatsächlich exprimiert werden. In den hier beschriebenen Arbeiten, verwendeten wir peptidbeladene dendritische Zellen (DCs), um selektiv CD4+ T-Zellen gegen ein Epitop aus dem EBV Protein EBNA2 anzureichern. Es gibt Hinweise darauf, dass DCs sich besonders zur Stimulation von T-Zellen eignen, die subdominante EBV Antigene erkennen. Die TCR Gene eines solchen CD4+ T-Zellklons, sowie zweier CD8+ Klone, wurden in Vektoren kloniert, mit denen die EBV Spezifität der Klone auf andere T-Zellen übertragen werden sollte. Wie bereits zuvor in anderen Laboren beobachtet, waren auch unsere TCR modifizierten T-Zellen zunächst nicht in der Lage, EBV infizierte Zielzellen effektiv zu attackieren. Erst durch Modifikation der Vektorstrategie (2A Peptidlinker als Ersatz für das IRES Element (internal ribosomal entry site)) sowie der TCR Gene (Codon-Optimierung) konnte eine deutlich verbesserte Expression und Funktion der modifizierten T-Zellen erreicht werden. Außerdem hing die Effektivität der modifizierten T-Zellen essentiell von der als Zielzelle verwendeten LCL ab. Die hier beschriebenen Arbeiten zeigen die erfolgreiche Übertragung von TCRs gegen EBV Antigene auf T-Zellen. Die so modifizierten T-Zellen erlangten anti-EBV Aktivität und sprechen daher für die prinzipielle Anwendbarkeit TCR-modifizierter T-Zellen zur Behandlung EBV-assoziierter Erkrankungen. / Adoptive transfer of polyclonal Epstein-Barr-virus (EBV)-specific T cell lines has been used as prophylaxis and therapy in patients with EBV-associated malignancies. This approach, however, is limited by the difficult expansion of polyclonal T cells directed mainly against dominant EBV antigens presented on EBV-transformed B cell lines (LCLs). Isolating EBV-specific T cell receptors (TCRs) for transduction of T cells is an alternative strategy to confer T cell immunity against EBV antigens including subdominant EBV antigens. In this study, we have used peptide-pulsed DCs to selectively expand EBV-specific CD4+ T cell clones against an EBNA2-derived epitope. Data suggested that peptide-pulsed DCs are particularly effective in stimulating T cells specific for subdominant EBV antigens. TCR genes from one of these clones as well as from two CD8+ T cell clones were identified by RACE PCR. TCR alpha and beta chains where then cloned into retroviral vectors for transduction of T cells to equip them with anti-EBV specificity. The TCR-modified T cells where then tested for their function towards LCLs to assess the chances for the use of EBV-redirected T cells in adoptive immunotherapy of EBV-associated disease. Like in previous reports, our EBV-specific TCRs at first did not confer effective activity against LCLs. Instead, we had to apply modifications to the TCR vectors to improve expression and function of the introduced TCRs. Codon optimization as well as replacement of the IRES site by a 2A peptide linker was required to significantly increase expression and function of transduced TCRs. Also, we found that the effectiveness of TCR transduced T cells is dependent on the target cell chosen. Our data show successful transfer of functionally active EBV-specific TCRs into T cells to render them effective against LCLs, representing the basis for the development of TCR-transgenic T cells for adoptive T cell transfer in EBV-associated disease.

Die spezifische adoptive Immuntherapie ist ein hoffnungsvoller Ansatz zur Behandlung von Tumoren. Die aufwendige individuelle Bereitstellung primärer Effektorlymphozyten könnte durch den Einsatz etablierter tumorantigenspezifischer Effektorzellinien vermieden werden. In dieser Arbeit wurde untersucht, ob sich ein Tumortargeting der humanen Natürlichen Killer-(NK)-Zellinie YT durch den Gentransfer chimärer Immunglobulin-T-Zellrezeptoren (cIgTCRs) erreichen läßt. Die cIgTCR-Konstrukte wurden aus single-chain-Fv-Fragmenten (scFv), dem IgG1-Fc-Teil und der CD3-Zeta-Signalkette erzeugt. Die scFv-Fragmente wurden aus den humanisierten Antikörpern BW431/26 und HuM195, die spezifisch für das karzinoembryonale Antigen (CEA) bzw. CD33 sind, konstruiert und zeigten als scFv-hFc-Fusionsproteine eine spezifische Bindung an Tumorzellen. Die YT-Zellen wurden mit den cIgTCR-Genkonstrukten über Elektroporation transfiziert und über immunologische Verfahren angereichert. In-vitro-Studien ergaben eine spezifische Lyse von CEA+ Kolonkarzinomzellinien durch die scBW431/26-hFcZeta+ YT-Zellen. Die Zytotoxizität korrelierte mit der Expression des cIgTCR-Antigens auf den Tumorzellen und wurde durch zirkulierendes CEA nicht gehemmt. Die scHuM195-hFcZeta+ YT-Zellen zeigten eine spezifische Lyse der CD33+ myeloischen Leukämiezellinie KG1. Die Bestrahlung wurde zur Wachstumsbegrenzung der YT-Zellen eingesetzt. Die spezifische Zytotoxizität der scBW431/26-hFcZeta+ YT-Zellen gegenüber CEA+ Tumorzellen war einen Tag nach Bestrahlung unverändert. Die Koinjektion von CEA+ Tumorzellen mit bestrahlten scBW431/26-hFcZeta+ YT-Zellen führte zu einer signifikanten Hemmung des Tumorwachstums in NOD/SCID-Mäusen. Die cIgTCR+ YT-Zellen zeigten in vitro eine geringe Sensibilität gegenüber allogenen Blutlymphozyten. Die Ergebnisse zeigen, daß die Zytotoxizität der NK-Zellinie YT tumorantigenspezifisch durch cIgTCR-Gentransfer erweitert wird und ein Potential zur Behandlung minimaler Tumorerkrankungen besteht. / The specific adoptive immunotherapy is a promising strategy for cancer treatment. The utilization of established tumor antigen specific effector cell lines could bypass the expendable individual preparation and often limited specificity of primary effector lymphocytes. This study investigated the tumor targeting of the human Natural Killer (NK) cell line YT by gene transfer of chimeric immunoglobulin T cell receptors (cIgTCRs). The cIgTCR constructs were generated of single chain antibody fragments (scFv), the IgG1 Fc part and the CD3 Zeta chain. The scFv fragments were constructed of the humanized antibodies BW431/26 and HuM195 with specificity for the carcinoembryonic antigen (CEA) and CD33, respectively, and showed as scFv-Fc fusion proteins a specific binding to tumor cells. YT cells were transfected with the cIgTCR gene constructs by electroporation and enriched by immunological cell separation. In vitro studies revealed a specific lysis of CEA+ colon carcinoma cell lines by scBW431/26-hFcZeta+ YT cells. The cytotoxicity correlated with the expression of the cIgTCR antigen on the tumor cells and was not inhibited in the presence of soluble CEA. The scHuM195-hFcZeta+ YT cells mediated a specific lysis of the CD33+ myeloic leukemia cell line KG1. The irradiation was used to limit the growth of the YT cell line. The specific cytotoxicity of the scBW431/26-hFcZeta+ YT cells against CEA+ tumor cells was unaltered one day after irradiation. The coinjection of CEA+ tumor cells and irradiated scBW431/26-hFcZeta+ YT cells led to a significant growth inhibition in NOD/SCID mice. The cIgTCR+ YT cells showed a low susceptibility to the cytotoxicity of allogeneic blood lymphocytes in vitro. The results demonstrated that the cytotoxicity of the human NK cell line YT can be specifically extended to tumor antigens by cIgTCR gene transfer. The employment of receptor gene modified YT cells could be a useful tool for the adoptive immunotherapy of minimal tumor diseases.

For the past 60 years, American citizens have turned to international adoption as a way to build their families. Unfortunately, international adoptees often spend the first months or years of their young lives in conditions of poverty and/or institutionalized care. Additionally, current U.S. immigration laws dictate that the children receive only a cursory health screening before arrival. As a result, many of the children adopted by Americans arrive to the United States with significant physical, emotional, behavioral, and developmental health problems. Twelve mothers who had adopted children internationally were interviewed for this study. Their stories of obtaining health care for their newly adopted children were shared in descriptive narratives. The themes that emerged from the data analysis were seeing healthy children despite the challenges, struggling to help the children, needing help, missing lost pieces, being different, and wanting more from providers. The overall essence derived from the mothers' experiences was that seeking health care for their newly adopted children was one of hope to achieve wellness for their children and normalcy for their families within the context of loss and a desire for more support. These findings have significant implications for the health care providers who care for them. / by Natalie L. Murphy. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.