L’objectif de cette thèse est de définir des biomarqueurs relatifs à l’immunothérapie allergénique (ITA). Il peut s’agir de biomarqueurs prédictifs d’une réponse au traitement qui vont permettre aux cliniciens d’adapter les schémas thérapeutiques ou de biomarqueurs d’efficacité facilitant le suivi clinique des patients au cours du traitement. La stratégie de recherche est basée sur une hypothèse qui consiste à dire que les cellules dendritiques (DCs) sont impliquées dans le succès de l’immunothérapie. En particulier, nous supposons que le traitement induit une baisse des DCs effectrices et une augmentation des DCs tolérogènes.Dans une première partie, un criblage de molécules biologiques et pharmacologiques a été entrepris sur les DCs dérivées des monocytes afin de générer in vitro des DCs effectrices de type DC1 etDC17 et des DCs régulatrices. Quatre molécules ont ainsi été identifiées pour leurs propriétés polarisantes. En particulier, les protéases d’Aspergillus oryzae se sont révélées être des inducteurs forts de tolérance. Le phénotype des DCs régulatrices obtenu a été étudié en détail ainsi que la polarisation et la fonctionnalité des lymphocytes T générés après cocultures.Dans une deuxième partie, deux approches de protéomique quantitative (la 2D-DIGE et la LCMS/MS sans marquage) ont été utilisées pour comparer les protéomes des DCs régulatrices et desDCs effectrices. Le différentiel d’expression des protéines les plus pertinentes a été validé au niveau transcriptionnel et protéique dans différents modèles. Le suivi des marqueurs dans des cellules du sang de patients traités ou non par ITA lors d’une étude clinique randomisée, contrôlée, en double aveugle, a permis de définir deux nouveaux biomarqueurs d’efficacité précoce de l’immunothérapie. Ces marqueurs pourront être suivis lors des traitements de désensibilisation pour distinguer les patients répondeurs des non-répondeurs. Par ailleurs, le suivi de ces biomarqueurs pourrait être essentiel dans d’autres pathologies comme les maladies auto-immunes ou encore la transplantation. / The aim of this thesis is to define biomarkers of allergen-specific immunotherapy (SIT).These biomarkers can be predictive of a clinical response or could be efficacy biomarkersable to discriminate responders versus non responder patients. The research strategy is based on the following hypothesis: if immunotherapy works, effector DCs are decreased where as regulatory DCs are increased locally or in the peripheral blood.First, we screened several biological or pharmacological agents to identify effector orregulatory DCs polarization agents. Four distinct molecules lead to the generation of eitherDC1, DC17 or regulatory DCs. In particular, proteases from Aspergillus Oryzae were clearinducer of tolerogenic DCs. The phenotype of those cells and the CD4+ T cell polarization induced after coculture were characterized extensively.In a second part, two proteomic approaches were used to compare the whole cell proteome of generated DCs. Most pertinent markers of polarization were validated in several cellular models. Markers were also followed in a randomized, double blind, placebo controlled clinical trial testing the efficacy of grass pollen tablets. Two markers were up regulated in patients who responded to the treatment pointing to a potential role of these proteins as early efficacy biomarkers. These markers are of crucial interest in the follow up of patients after SIT and could also be used in other diseases like autoimmune diseases or transplantation.

Cancer is the second most common cause of death in the United States behind heart disease. While current treatments such as surgery, chemotherapy, and radiation therapy are effective and widely used, medicine is moving towards more targeted and personalized therapies. Immunotherapy is one such treatment that utilizes the patient’s own immune system to target and eliminate tumor cells. It allows for the patient’s adaptive immune system to bypass the self-tolerance mechanisms used by the cancerous cells and be activated against the cancer. Two such self-tolerant mechanisms that are co-opted by tumor cells are the interactions between CTLA-4 and T lymphocytes and the interactions between PD-1 and PD-L1. Blocking these interactions allows for the recruitment of CTLs to the site of the tumor and subsequent attack. CTLA-4 and PD-1 are inhibitory costimulators that play a role in the suppression of the adaptive immune system. The interaction of these receptors with their respective ligands leads to self-tolerance, and is a common mechanism used as a protective measure against autoimmune reactions. Monoclonal antibodies against these two receptors and ligand have been tested in clinical trials and have shown efficacy against ovarian cancers, non-small cell lung carcinomas, colon cancers, and melanomas. By targeting the inhibitory signals, these monoclonal antibodies expose cancer cells as being “non-self” thus prompting the immune system to attack. Now, studies are focusing on combination therapies, which combine chemotherapeutics or other monoclonal antibodies with PD-1 and CTLA-4 inhibitors to enhance the effectiveness of the drug. However, drawbacks and side effects to the therapy range from fatigue and nausea to development of autoimmune diseases. It brings forward that future studies will need a panel of predictive biomarkers to identify the best candidates for the immunotherapy. While there are many obstacles, such as a lower than expected efficacy of the immunotherapy, the progress made has important implications in the development of personalized medicine.

Cells have long been known to sense and respond to mechanical stimuli in their environment. In the adoptive immune system particularly, cells are highly specialized and responsible for detecting and eliminating pathogens from the body. T cell mechanosensing is a relatively new field that explores how force transmission in cell-cell interaction elicits both inter- and intra-cell signaling. Owing to recent advances in genetic manipulation of T cells, it has emerged as new tool in immunotherapy. We recently demonstrated human T cell activation in response to mechanical rigidity of surfaces presenting activating antibodies CD3 and CD28. The work in this dissertation highlights new progress in the basic science of T cell mechanosensing, and the utilization of this knowledge toward the development of a more specialized expansion platform for adoptive immunotherapies. Human T cells are known to trigger more readily on softer PDMS substrates, where Young's Modulus is less than 100 kPa as compared to surfaces of 2 MPa. While the range of effective rigidities has been established, it is important to explore local differences in substrates that may also contribute to these findings. We have isolated the rigidity-dependence of cell-cell interactions apart from material properties to optimize design for a clinical cell expansion platform. Though PDMS is a well understood biomaterial and has found extensive use in cellular engineering, a PA gel substrate model allows for rigidity to be tuned more closely across this specific range of rigidities and provides control over ligand density and orientation. These rigidity-based trends will be instrumental in adapting models of mechanobiology to describe T cell activation via the immune synapse. In what is generally accepted as the clinical gold-standard for T cell expansion, rigid (GPa) antibody-coated polystyrene beads provide an increase in the ratio of stimulating surface area-per-volume, over standard culture dishes. Herein we describe the development of a soft-material fiber-based system with particular focus on maintaining mechanical properties of PDMS to exploit rigidity-based expansion trends, investigated through atomic force microscopy. This system is designed to ease risks associated with bead-cell separation while preserving a large area-to-volume ratio. Exposing T cells to electrospun mesh of varying rigidities, fiber diameters, and mesh densities over short (3 day) and long (15 day) time periods have allowed for this system's optimization. By capitalizing on the mechanisms by which rigidity mediates cell activation, clinical cell expansion can be improved to provide greater expansion in a single growth period, direct the phenotypic makeup of expanded populations, and treat more patients faster. This technology may even reach some cell populations that are not responsive to current treatments. The aims of this work are focused to identify key material properties that drive the expansion of T cells and optimize them in the design of a rigidity-based cell expansion platform.

Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV.

Glioblastoma multiforme is the most common and lethal primary brain tumour in adults. Its aggressive and invasive phenotype makes it resistant to current standards of care, with a patient median survival following treatment of only 14 months. Potent and safe therapeutics are necessary to improve patient prognosis. Globally, efforts are being made in immunotherapies to combat such deleterious tumours. Preliminary work in the Stojdl lab has developed a novel oncolytic virus platform for brain cancer therapy that is non-toxic and exhibits potent anti-tumour efficacy. This platform is based on the rhabdovirus Farmington, identified for its potent oncolytic properties and engineering malleability. Herein, we begin to show our capability to discover and vaccinate against immunogenic neoepitopes derived from a mouse cancer mutanome. Engineering Farmington virus to express neoepitopes, allows for robust tumour specific immune proliferation following a prime vaccination. Overcoming problems of targeting self-antigen and antigen loss variants, a multi-neoepitope vaccine, presented here, is one of many alternative approaches to help combat cancer resistance. Despite achieving robust anti-tumour immunity by vaccination, selectivity of the tumour microenvironment remains an enormous challenge. Cumulative efforts in immunotherapy research will help drive novel therapeutics, like Farmington, into clinic and, ultimately, improve patient’s prognosis and quality of life.

Recent advances in the field of cancer immunotherapy have enabled this therapeutic approach to enter the mainstream of modern cancer treatment. In particular, adoptive T cell therapy (ACT) is a potentially powerful immunotherapy approach that relies on the administration of tumor-specific T cells into the patient. There are several strategies to obtain tumor-reactive cytotoxic T lymphocytes (CTLs), which have already been shown to induce remarkable responses in the clinical setting. However, there are concerns and limitations regarding the conventional approaches to obtain tumor-reactive T cells, such as accuracy of the procedure and reproducibility. Therefore, we aimed to develop two approaches to improve the precision and efficacy of tumor-reactive T cells therapy. These two techniques could constitute effective, safe and broadly applicable alternatives to the conventional methods for obtaining tumor-specific CTLs. The first approach of this study is the so called “Doublet Technology”. Here, we demonstrate that peptide-human leukocyte antigen-T cell receptor (pHLA-TCR) interactions that involve immune reactive peptides are stable and strong. Therefore, the CTLs that are bound by their TCR to tumor cells can be selected and isolated through FACS-based cell sorting taking advantage of this stable interaction between the CTLs and the target cells. The CTLs from acute myeloid leukemia (AML) patients obtained with this technique show cytolytic activity against blast cells suggesting a potential clinical use of these CTLs. “Doublet Technology” offers a personalized therapy in which there is no need for a priori knowledge of the exact tumor antigen. The second approach of this study is the Chimeric Antigen Receptor (CAR) Technology. We design several CARs targeting the B-Cell Maturation Antigen (BCMA). BCMA CAR T cells show antigen-specific cytolytic activity, production of cytokines including IFN-γ and IL-2, as well as productive proliferation. Although we confirm the presence of soluble BCMA in serum of multiple myeloma (MM) patients, we demonstrate that the presence of soluble protein does not abrogate the efficacy of BCMA CAR T cells suggesting that BCMA CAR T cells can be used in the clinical setting to treat MM patients. The high antigen specificity of CAR T cells allows efficient tumor cell eradication and makes CAR Technology attractive for broadly applicable therapies. / Durch jüngste Fortschritte auf dem Gebiet der Krebsimmuntherapie konnte dieser therapeutische Ansatz in der Mitte moderner Krebsbehandlungen ankommen. Insbesondere die adoptive T-Zelltherapie (ACT), die auf der Verabreichung tumorspezifischer T-Zellen an den Patienten beruht, stellt einen potentiell schlagkräftigen immuntherapeutischen Ansatz dar. Es existieren bereits verschiedene Strategien um tumorreaktive zytotoxische T-Lymphozyten (CTL) herzustellen, von denen bereits gezeigt wurde, dass sie klinisch bemerkenswerte Antworten hervorrufen. Dennoch gibt es Bedenken und Grenzen bezüglich dieser konventionellen Ansätze zur Herstellung tumorreaktiver T-Zellen, wie zum Beispiel die Genauigkeit und Reproduzierbarkeit des Verfahrens. Daher arbeiteten wir an der Entwicklung zweier Ansätze um die Präzision und Effizienz der tumorreaktiven T-Zelltherapie zu verbessern. Diese beiden Techniken könnten effektive, sichere und breit anwendbare Alternativen zu den konventionellen Methoden der tumorspezifischen CTL-Gewinnung darstellen. Der erste Ansatz dieser Studie wird als „Doublet Technology“ bezeichnet. Hierbei zeigen wir, dass die Interaktionen zwischen Peptid/MHC-Komplex und T-Zellrezeptor (pHLA-TCR), die immunreaktive Peptide involvieren, stabil und solide sind. Außerdem zeigen wir, dass CTLs, die über ihren TCR an Tumorzellen gebunden sind, selektioniert und durch FACS-basierte Zellsortierung isoliert werden können. Hierbei wird die Stabilität der Interaktion von CTLs und Zielzellen genutzt. Die CTLs von Patienten mit Akuter Myeloischer Leukämie (AML), die auf diese Weise gewonnen werden, zeigen zytolytische Aktivität gegenüber Blasten, was auf einen potentiellen klinischen Nutzen dieser CTLs hinweisen könnte. Die „Doublet Technology“ bietet eine personalisierte Therapie, die kein vorheriges Wissen über ein exaktes Tumorantigen erfordert. Der zweite Ansatz dieser Studie ist die Chimere Antigenrezeptor (CAR) Technologie. Wir entwickeln verschiedene CARs gegen das B-Zellmaturationsantigen (BCMA). BCMA-CAR T-Zellen zeigen antigenspezifische zytolytische Aktivität, Produktion der Zytokine IFN-γ und IL-2 sowie produktive Proliferation. Obwohl wir bestätigen, dass lösliches BCMA im Serum von Multiplen Myelompatienten zu finden ist, zeigen wir auch, dass dieses lösliche Protein nicht die Effizienz von BCMA-CAR T-Zellen beeinträchtigt und somit BCMA-CAR T-Zellen zur Behandlung von Multiplen Myelompatienten klinisch genutzt werden können. Die hohe Antigenspezifität der CAR-T-Zellen erlaubt eine effiziente Vernichtung von Tumorzellen und macht die CAR-Technologie attraktiv für breit einsetzbare Therapien.

Signal Transducer and Activator of Transcription 3 (STAT3), a common oncogenic mediator, is constitutively activated in many types of human cancers and plays a critical role in tumor growth and cancer immune evasion. The focus of this dissertation is the delivery of STAT3 inhibitor cucurbitacins to tumors using polymeric nano-carriers for the inhibition of tumor growth and modulation of tumor-induced immunosuppression. The anticancer and immunomodulatory activity of STAT3 inhibitor JSI-124 (cucurbitacin I) was studied in mice carrying B16 tumor. The results showed that JSI-124 + CpG or 7-acyl lipid A combination therapy modulated immunosuppression in tumor environment and generated superior anti-tumor effects compared to monotherapy. In further studies, a sensitive and reproducible liquid chromatography-mass spectroscopy (LC-MS) method was developed and validated for quantitative analysis of STAT3 inhibitor cucurbitacins in vitro and in biological samples. Moreover, nano-delivery systems based on poly(ethylene oxide)-block-poly(-caprolactone) (PEO-b-PCL) micelles and its analogues containing physically encapsulated cucurbitacin and poly(D,L -lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing chemically conjugated JSI-124 for the delivery of STAT3 inhibitor to tumor and dendritic cells (DCs) were developed and characterized. Polymeric micelles of different PCL based core structure were able to significantly increase the water solubility of STAT3 inhibitor cucurbitacins, and slow the rate of drug release by a diffusion dependent mechanism. The chemical structure of the micellar core was found to control the release rate of cucurbitacin from the micelles. PLGA NPs containing conjugated JSI-124, on the other hand, demonstrated a degradation dependent drug release profile over a 1-month period. Both nanoparticulate formulations exhibited potent anticancer and STAT3 inhibitory activity against B16 cancer. Moreover PLGA-JSI-124 NPs suppressed STAT3 activation in immunosuppressed p-STAT3highDCs and significantly improved their function in stimulating T cell proliferation in vitro. These findings show that JSI-124 esters of PLGA NPs can potentially provide a useful platform for JSI-124 delivery to tumor and its targeted delivery to DCs. The results of this research not only proved the principle of STAT3 inhibition in tumors as an efficient intervention for enhancing the therapeutic efficacy of TLR ligand-based cancer immunotherapy, but led to development of nano-delivery systems with potential application in cancer chemo-and immunotherapy. / Pharmaceutical Sciences

Currently, in most situations, the autologous immune system is unable to eradicate the residual leukemic burden persisting after chemo-radiotherapy, but a balance can be established between leukemic and immune cells leading to a clinical remission for several months or years. If this balance is broken, a clinical relapse can occur. The high incidence of relapses in human cancers demonstrates the frequent inefficacy of the immune system to control these residual cells. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has been proven to be the most effective way to reinforce the immune reaction against leukemia, graft-versus-leukemia (GVL) effect and, so, achieve a definitive eradication of the residual disease in a significant proportion of patients. Indeed, the whole concept of HSCT evolved from an organ transplant concept (to replace a defective ill organ with a new healthy one) to the concept of creating an extraordinary immunotherapeutic platform in which the donor immune system contributes to the eradication of the residual leukemic cells. Thus, the past and present issues remain those of finding the best immunomodulatory modalities to achieve a full engraftment, a powerful GVL effect and no or moderate graft-versus-host disease (GVHD). Different ways to reach this goal, such as post transplant cytokine modulation, specific or global cellular depletion of the graft and post transplant global or specific donor immune cell add-backs, are still extensively studied. Nevertheless, the persistent high relapse rate (RR) observed in leukemia patients after HSCT remains the most important cause of death before transplant-related toxicities. Moreover, since only about 40 to 70% (depending on the ethnic context) of patients with high-risk hematological malignancies, eligible for allogeneic HSCT, have a fully HLA-matched sibling or matched unrelated donor (MUD), a great deal of effort has been invested to make the use of an alternative haploidentical sibling donor feasible. The advantage of this procedure is the immediate availability of a donor for almost all patients. The aim of the work described in this thesis has been to implement a strategy to transplant a patient using a HLA haploidentical donor. The strategy is to try to improve DFS that could be applied both in the autologous or allogeneic context: first, by using nonspecific immune manipulation post transplant and then, by developing specific strategies directed against leukemia antigens. Particularly in the allogeneic situation, the aim was to increase the GVL effect without inducing or aggravating the deleterious GVHD. The first part of this thesis described our own clinical results, consisting of three consecutive phase I/II studies, in which we tried to determine the feasibility of giving prophylactic donor lymphocyte infusions (DLI) post transplant and the effect of replacing granulocyte colony-stimulating factor (G-CSF), typically used to speed up neutrophil recovery, with granulocyte macrophage colony-stimulating factor (GM-CSF), which is known for its immunomodulatory properties. The slow immune reconstitution in haploidentical transplant is chiefly responsible for the high incidence of early lethal viral and fungal infections, and most probably for early relapses; therefore, we sought to accelerate and strengthen the post transplant immune reconstitution without increasing the GVHD rate. Thus, we have studied the impact of post transplant growth factor administration and of unselected DLI in haploidentical transplant. We have also implemented, in our center, anti-cytomegalovirus (CMV) specific T cell generation and infusion to improve anti-CMV immune reconstitution. Since then, our results have been pooled in a multi-center analysis performed by the European Bone Marrow Transplantation group (EBMT) allowing us to compare our results with those of the entire group. We have also participated in the design of an ongoing study aimed at selectively depleting the graft from alloreactive T cells, and improving post transplant T cell add-backs. In our attempts to generate and expand ex vivo lymphocytes (directed against pathogens (CMV) and leukemia-associated antigens, Wilms' tumor gene 1 (WT1) and to use them in vivo, we found inconsistent results (in the case of WT1) using classical clinical grade dendritic cells (DC) generated and matured in bags, as was the case for the majority of the teams worldwide. This led us to question the full functionality of these DC and we undertook a thorough comparative analysis of DC generated and differentiated in bags and in plates (typical for most pre-clinical studies). This analysis showed us that one cannot transpose pre-clinical studies (using culture plates) directly to clinical protocols (generally using clinical grade culture bags) and that DC generated in bags are functionally deficient. We learned that, if we want to use a DC vaccine to improve the GVL effect in haploidentical transplant, we will have to be careful about the technique by which they are generated. To improve immunotherapeutic approaches, the understanding of the mechanisms underlying tumor tolerance and how to manipulate them is critical in the development of new effective immunotherapeutic clinical trials. This is why we currently focus on how to obtain effective in vivo anti-leukemia immune reactions using an ex-vivo manipulated product to trigger the immunotherapeutic response. More specifically, we are analyzing the impact of regulatory T cell (Tregs) depletion and function for an adequate anti-leukemic immune response. This pre-clinical work aims at improving the outcome of leukemia patients who have relapsed and been put back into second remission and at decreasing the RR after HSCT, especially in the field of haploidentical transplantation. In conclusion, haploidentical transplantation has become a valuable tool. The results are at least similar to those obtained using MUD when performed in the same group of patients. Specific immunomodulation post transplant can affect events such as GVHD and GVL, but clinically we are still at the level of nonspecific manipulations. It is our hope that ongoing pre-clinical work will enable us to perform specific anti-pathogen and anti-leukemia immune manipulation that will favorably influence the patient outcome. / Dans la majorité des situations, le système immunitaire autologue est incapable d’éradiquer les cellules leucémiques résiduelles qui échappent à la radiothérapie et à la chimiothérapie, cependant un équilibre peut s’établir entre les cellules leucémiques et immunitaires aboutissant à une rémission pouvant durer plusieurs mois ou années. Si cet équilibre se rompt, une rechute clinique peut se déclarer. Dans ce contexte, il est prouvé que la greffe allogénique de cellules souches hématopoïétiques est le moyen le plus efficace de renforcer les réactions immunitaires contre la leucémie par la réaction du greffon contre la leucémie et ainsi d’obtenir une éradication définitive de la maladie résiduelle chez un nombre significatif de patients. En effet, le concept global de l’allogreffe de cellules souches hématopoïétiques a évolué du concept de transplantation d’organe (remplacement d’un organe malade par un nouvel organe sain) vers celui de créer une extraordinaire plateforme d’immunothérapie à travers laquelle le système immunitaire du donneur contribue à l’éradication des cellules leucémiques persistantes. Donc, la problématique reste celle de trouver les meilleures modalités d’immunomodulation pour achever une prise du greffon, un effet anti-leucémique puissant du greffon, et l’absence ou un minimum d’effet du greffon contre l’hôte. Différentes stratégies existent pour atteindre cet objectif, comme l’utilisation de cytokines pour moduler la reconstitution immunitaire, des déplétions cellulaires globales ou spécifiques du greffon et l’infusion de cellules immunes «globales» ou spécifiques du donneur après greffe. Ces stratégies sont encore largement à l’étude. Néanmoins, la persistance d’un taux de rechute élevé observé chez les patients leucémiques, après allogreffe reste la cause principale de décès, avant celle liée à la toxicité de la greffe. De plus, étant donné que seulement environ 40 à 70% (dépendant de l’origine ethnique) des patients avec une hémopathie à haut risque, éligibles pour une greffe allogénique, ont un donneur familial ou non familial complètement HLA compatible, des efforts importants ont été développés pour rendre faisable l’utilisation de donneurs familiaux alternatifs, haploidentiques. L’avantage de cette approche est l’accès immédiat à un donneur pour quasiment tous les patients. Le but du travail décrit dans cette thèse a été l’implémentation d’une stratégie d’allogreffe utilisant un donneur haploidentique. Le travail vise également à développer de façon plus large des stratégies qui peuvent améliorer le taux de survie sans rechute, non seulement dans le contexte des greffes haploidentiques, mais également dans le cadre des greffes allogéniques en général, ainsi que dans les situations autologues : premièrement, par la manipulation immunitaire non spécifique après greffe et ensuite par le développement de stratégies spécifiques dirigées contre des antigènes leucémiques. En particulier dans la situation allogénique, le but a été d’augmenter l’effet du greffon contre la leucémie sans induire ou aggraver l’effet délétère du greffon contre l’hôte. La première partie de la thèse décrit les résultats cliniques de notre propre protocole de greffe haploidentique, qui a consisté en trois études consécutives de phase I/II. Dans ces études, nous avons voulu déterminer la faisabilité de réaliser des infusions prophylactiques de lymphocytes du donneur après transplantation, et l’impact du remplacement du « granulocyte colony-stimulating factor » (G-CSF), largement utilisé pour permettre une récupération en polynucléaires neutrophiles plus rapide, par du « granulocyte-macrophage colony-stimulating factor » (GM-CSF), lequel est connu pour ses propriétés immunomodulatrices différentes. La reconstitution immunitaire très lente après greffe haploidentique est majoritairement responsable de l’incidence élevée de décès par infections virales et fungiques précoces, et très probablement des rechutes précoces. C’est pourquoi nous avons cherché à accélérer et à renforcer la reconstitution immunitaire post-greffe sans augmenter la fréquence de réaction du greffon contre l’hôte. Nous avons donc étudié l’impact de l’administration de facteurs de croissance et l’infusion de lymphocytes non sélectionnés du donneur en post greffe haploidentique. Nous avons également implémenté dans notre centre, la génération et l’infusion de lymphocytes T spécifiques anti-cytomégalovirus (CMV) afin d’améliorer la reconstitution immunitaire anti-CMV. D’autre part, nos résultats ont été regroupés dans une étude multicentrique menée par le groupe européen de transplantation de moelle osseuse (EBMT), ce qui nous a permis de comparer nos résultats avec ceux de l’entièreté du groupe. Nous avons parallèlement participé à la conception d’une étude actuellement en cours ayant pour but d’améliorer la reconstitution immunitaire après greffe par la déplétion sélective du greffon en lymphocytes T alloréactifs et par l’infusion après greffe de lymphocytes T du donneur également sélectivement déplétés en lymphocytes T alloréactifs. Afin d’optimaliser l’effet anti-leucémique du système immunitaire, nous avons débuté un protocole de vaccination par cellules dendritiques (DCs). Ces cellules dendritiques étaient chargées en lysat de blastes leucémiques dans le cas de patients présentant au diagnostic une leucémie aigue surexprimant l’oncogène 1 de la tumeur de Wilms (WT1). Néanmoins dans nos travaux de génération et d’expansion ex-vivo de lymphocytes T spécifiques de l’antigène WT1, utilisant les DCs de grade clinique, générées et maturées en poches, nous avons rencontré des résultats inconsistants, comme c’était le cas dans la majorité des protocoles cliniques internationaux de vaccination. Nous nous sommes alors posé la question de la fonctionnalité globale de ces cellules et nous avons entrepris une analyse comparative poussée des DCs générées et différenciées en poches ou en plaques. Les DCs générées en plaques sont celles utilisées dans la plupart des travaux précliniques. Cette analyse nous a montré que l’on ne pouvait pas directement transposer les résultats précliniques basés sur des DCs générées en plaques dans des protocoles cliniques basés sur des DCs générées en poches, car ces dernières présentent des déficits fonctionnels importants. Nous avons appris que si l’on voulait utiliser un vaccin à base de cellules dendritiques pour améliorer l’effet du greffon contre la leucémie dans les greffes allogéniques, nous devions être très attentifs quant au protocole utilisé pour la génération de ces vaccins cellulaires. Pour améliorer les approches immunothérapeutiques, la connaissance des mécanismes qui établissent la tolérance tumorale et des façons de manipuler ceux-ci, est critique dans le développement de nouveaux protocoles efficaces. C’est pourquoi nous nous concentrons actuellement sur les conditions nécessaires à l’obtention in vivo d’une réaction immune anti-leucémique efficace lors de l’utilisation d’un produit cellulaire manipulé ex vivo. Plus spécifiquement, nous analysons l’impact de la déplétion en lymphocytes T régulateurs (Tregs) sur la réponse anti-leucémique. Ce travail préclinique a pour but d’améliorer le devenir de patients leucémiques qui ont rechutés et ont été mis en seconde rémission, ainsi que de diminuer le taux de rechute après allogreffe, spécifiquement après greffe haploidentique. En conclusion, la transplantation haploidentique est actuellement un outil précieux pour de nombreux patients. Les résultats sont au minimum similaires à ceux qui sont obtenus par les greffes non-familiales HLA identiques lorsqu’elles sont pratiquées dans les mêmes groupes de patients. L’immunomodulation spécifique après greffe peut affecter des événements comme la réaction du greffon contre l’hôte et la réaction du greffon contre la leucémie, mais en pratique clinique nous en sommes encore au niveau de la manipulation aspécifique. Nous espérons que les travaux précliniques actuels vont nous permettre d’appliquer des stratégies spécifiques et d’obtenir une manipulation immune anti-leucémique qui aura une influence favorable significative sur le devenir des patients.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

For the treatment of cancer, immunotherapy has some inherent advantages over other treatment modalities: disseminated disease can be eradicated due to the systemic nature of immunity, the immune system is effective against a wide range of targets, long-term memory can offer added protection against disease relapse, immunotherapy should be relatively non-toxic, and it can be synergistically combined with other treatment platforms such as radiation and chemotherapy. Type 1 immune responses are thought to be superior for the treatment of cancer and, as the quintessential Th1 polarizing cytokine, interleukin-12 (IL-12) holds much promise; however, optimal therapeutic protocols have yet to be developed and clinical results have fallen short of this promise. The in vivo IL-12 experiments described here highlight a characteristic of cellular therapy that has not previously been appreciated. That is, the effect of cell-mediated cytokine delivery on the immediate microenvironment and how that affects the immune response initiated. This observation has implications for the clinical application of IL-12 therapy but may also prove to be an important consideration when studying other immunostimulants. I have herein developed a novel in vitro assay system that I have used to dissect the cellular responses to IL-12 and to identify the signals that are required for activation of a cluster of differentiation 4 (CD4)+ effector population that affects leukaemia cell clearance both in vitro and in vivo. This work, and the future studies proposed, will expand our understanding of the potential of IL-12 immunotherapy and enhance our ability to manipulate therapeutic conditions to favour the desired response. Moreover, the in vitro assay system offers a method for further characterization of CD4+ effector cells and the development of protocols to initiate their potent anticancer activity.