La vigueur de la réponse immunitaire générée par les cellules dendritiques (DC) a positionné ces cellules comme médiatrices centrales dans l’activation des lymphocytes T. La vulnérabilité des cellules cancéreuses de leucémie myéloïde chronique (LMC) à l’intervention immunitaire résulte apparemment de la capacité des cellules leucémiques de se différencier en DC. Ces DC ont alors la capacité de présenter des peptides provenant des cellules souches leucémiques aux lymphocytes T. Dans ce travail, nous démontrons que la plupart des patients atteints d’une LMC présentent un déficit important en DC au niveau du sang et de la moelle osseuse avant la greffe de cellules souches allogéniques. Les faibles niveaux de DC circulantes résultent en grande partie d’une perte de la diversité au niveau des cellules progénitrices CD34+ leucémiques au niveau de la moelle osseuse. Ces cellules progénitrices CD34+ présentent d’ailleurs une capacité réduite à se différencier en DC in vitro. Nous avons trouvé qu’un décompte faible de DC avant une greffe allogénique était associé à une diminution significative de la survie et une augmentation considérable du risque de développer une des complications mortelles. Puisque la reconstitution des DC suite à la greffe est absente, notre étude appuie aussi la thèse que ce sont les cellules DC pré greffe qui sont primordiales dans l'effet du greffon contre leucémie (GVL). Dans ce contexte, notre étude suggère que le compte des DC avant la greffe allogénique pourrait servir de marqueur pronostique pour identifier les patients LMC à risque de développer certaines complications suite à une greffe allogénique. / The intensity of the immune response that is generated by the dendritic cells (DCs) has established these cells as central mediators in the activation of T lymphocytes. The vulnerability of leukemic cells present in chronic myeloid leukemia is mostly attributable to the ability of the leukemic stems cell to differentiate themselves into potent DCs. The latter are then able to present peptides which come from endogenous origin and from leukemic stem cells to the T lymphocytes. In this thesis, we demonstrate that, before allogeneic stem cell transplant, the majority of patients afflicted by CML have a considerable lack of DCs in their peripheral blood as well as in their bone marrow. The low levels of circulating DCs result to a great extent from a loss of diversity in the CD34+ leukemic progenitor cells located in the bone marrow. Moreover, these CD34+ leukemic progenitor cells display a reduced capacity to differentiate themselves into DCs in vitro. Our findings show that having a low level of DCs prior to an allogeneic transplant is associated with a significant decline in survival rates as well as with increased risks of developing life threatening complications. Since the DCs’ reconstitution following an allogeneic bone marrow transplant is missing, our study supports the thesis that pre graft DCs are essential in the Graft versus leukemia effect (GVL).Therefore, our research suggests that tallying DC before proceeding with an allogeneic transplant could act as a prognostic marker to identify LMC patients who present risks of complications after an allogeneic transplant.

Chimeric antigen receptors (CARs) are artificial molecules composed of an antibody derived antigen recognition domain which is fused with the signal transduction domain derived from the physiological TCR. CAR technology used to transduce patients T-cells and endow them with the specificity to a certain surface antigen, has been a major breakthrough in cancer immunotherapy in the last decade. This strategy has been most successful for treating hematologic malignancies. Various CAR approaches and applications are currently tested mainly in the United States where many clinical trials have been launched. In contrast, in the Czech Republic, there are only a few teams focused on this topic with no clinical trials going on. During my work on this diploma thesis and in close collaboration with MUDr. Pavel Otáhal, PhD., who is working on implementation of CAR technology into the Czech clinics for the treatment of B-cell malignancies, individual functional CARs were prepared and tested. CAR expressing Jurkat T-cell lines were generated using a lentiviral vector transduction system. CAR functionality was determined by two different assays. We have shown that individual CARs are able to recognize the B-cell lineage specific antigens CD19 and CD20 and significantly up-regulate the activation molecule CD69 upon...

Avec quatorze anticorps utilisés en oncologie, les anticorps monoclonaux ont enfin une place de choix dans l'arsenal thérapeutique anticancéreux. Cependant, l'un des modes d'action les plus importants de ces molécules, la cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC), souffre de plusieurs limites en raison de la nécessité d'une interaction optimale avec le FcγRIIIA. L'équipe du Dr. Daniel Baty a conçu de nouveaux formats d'anticorps bispécifiques basés sur l'utilisation de domaine unique d'anticorps de lamas (sdAb) et capables de contourner la plupart de ces limites. Des banques de phages issus de lamas immunisés ont permis de sélectionner deux sdAbs (d'affinités différentes) dirigés contre le FcγRIIIA exprimé par des cellules NK et les macrophages, ainsi qu'un sdAb dirigé contre l'antigène tumoral carcino-embryonnaire (ACE). En utilisant les domaines CH1 et Ck d'IgG1 humaine comme motif d'hétérodimérisation et les sdabs anti-ACE et anti-FcγRIII précédemment sélectionnés, nous avons développé des formats innovants d'anticorps bispécifiques Fab-like nommés bsFabs. Ces molécules sont faciles à produire, très stables et peuvent déclencher la lyse des cellules tumorales par les cellules NK humaines à des concentrations de l'ordre du picomolaire. Elles ne se lient pas au récepteur inhibiteur FcγRIIB, n'entrent pas en compétition avec des IgG sériques pour la liaison aux récepteurs, et leur activité cytotoxique est indépendante de la glycosylation du Fc et du polymorphisme du FcγRIIIA. / With fourteen antibodies used in oncology, monoclonal antibodies finally have a place in the therapeutic arsenal against cancer. However one of the modes of action of the most important of these molecules, cell-mediated cytotoxicity antibody-dependent (ADCC), suffers from several limitations due to the need for optimal interaction with the FcγRIIIA. Daniel Baty's team has developed new formats of bispecific antibodies based on the use of single domain llama antibodies (SdAb) that are capable of circumventing most of the these limitations. Phage libraries from immunized llamas were used to select two sdAbs directed against the FcγRIIIA expressed by NK cells and macrophages (with different affinities for FcγRIII), as well as one SdAb directed against the tumor antigen carcinoembryonic (CEA). Using CH1 and Ck domains of human IgG1 as a motif for heterodimerization and sdabs previously selected, we have developed innovative anti-CEA and anti-FcγRIII formats of bispecific antibodies Fab-like named bsFabs. These molecules are easy to produce, very stable and can trigger tumor cell lysis by human NK cells at picomolar concentrations. They do not bind FcγRIIB inhibitory receptor, do not compete with serum IgG and their cytotoxic activity is independent of FcγRIIIA polymorphism. In addition, they slow tumor growth in mouse model. In terms of pharmacokinetics, although bsFabs have a reasonable tumor retention, one of the limitations of these formats is size, which leads to rapid renal elimination. Such findings led us to consider the creation of new antibody formats to both increase tumor retention and secondly the half-life of antibodies in the body.

L’injection intraveineuse de l’activateur tissulaire du plasminogène (tPA) est le seul traitement aigu de l’ischémie cérébrale autorisé chez l’Homme. Cependant, la thrombolyse présente des limites d’utilisation, comme son étroite fenêtre thérapeutique, un risque hémorragique et une efficacité de recanalisation malgré tout relativement peu élevée. De plus, la littérature suggère fortement que non seulement le tPA endogène, mais aussi exogène (capable de traverser la barrière hémato-encéphalique), a des effets pro-excitotoxiques. Nous avons proposé que cet effet résulte du clivage de la sous unité NR1 du récepteur NMDA. Malgré un effort important de la communauté scientifique pour chercher de nouveaux traitements, tous les espoirs se sont avérés être des échecs. Sur ces bases, ces travaux de thèse ont consisté à : 1) Améliorer les approches précliniques en développant un nouveau modèle d’ischémie cérébrale chez la souris et en incluant dans les études un des principaux facteur de risque des AVC, le vieillissement ; 2) Développer une stratégie d’immunothérapie visant l’interaction tPA/ récepteur NMDA. J’ai ainsi montré qu’il existe une diminution du volume de lésion ischémique corrélée à l’âge et que cette diminution de tPA est due à une diminution d’expression du facteur de transcription D-Site Albumin Binding Protein (DBP). J’ai également développé un modèle innovant d’ischémie thrombo-embolique chez la souris, dans lequel la reperfusion par le tPA est bénéfique, si tant est qu’elle soit réalisée de manière précoce. Sur ce modèle, j’ai apporté par une stratégie d’immunisation active la preuve in vivo du clivage du domaine amino-terminal de la sous-unité NR1 des récepteurs NMDA. Enfin, j’ai produit un anticorps médicament, capable d’empêcher l’interaction du tPA avec la sous-unité NR1 des récepteurs NMDA, dont une injection unique permet de réduire les lésions ischémiques, mais aussi d’augmenter la fenêtre thérapeutique de la thrombolyse, conférant alors une récupération fonctionnelle à long terme. Cette stratégie pourrait donc accroître la proportion de patients traitables après un AVC ischémique aiguë / Reperfusion with tissue plasminogen activator (tPA) is the only approved treatment for ischemic stroke. However, thrombolysis has some limitations, including a narrow therapeutic window, an elevated risk of hemorrhage transformation and a low level of effective recanalization. Moreover, there is a growing body of evidence that both endogenous and exogenous tPA (able to cross the blood-brain barrier) could mediated pro-excitotoxic effects. We have proposed that this noxious effect results from the cleavage of the NR1 subunit of the NMDA receptor. My thesis work consisted in: 1) Improving pre-clinic approaches by developing a new model of thrombo-embolic ischemia in mice and by taking into account a major risk factor for stroke, aging; 2) Developing a strategy of immunotherapy targeting the interaction between tPA and NMDA receptor. I have thus shown that ischemic lesions decrease as a function of age, due to reduced levels of tPA. Moreover, I have identified DBP (D-site albumin Binding Protein), as being the transcription factor responsible for the control of tPA levels as a function of age. I have also developed a new model of thrombo-embolic ischemia in mice, in which tPA-induced thrombolysis is beneficial, provided it is performed soon enough. In this model, I have demonstrated by using a strategy of active immunization the in vivo occurrence of the cleavage of the NMDA receptor NR1 subunit by tPA. Finally, I have produced an antibody able to prevent the interaction between tPA and the NMDA receptor subunit, of which a single injection confers long lasting brain protection and neurological recovery and can also increase the therapeutic window of thrombolysis. This strategy could thus significantly increase the proportion of treatable ischemic stroke patients

La protéine Tat (Trans-activator of transcription) est certainement l’une des cibles présentant le plus d’intérêts dans la lutte contre le VIH-1. En effet, synthétisée précocement, elle joue un rôle central dans le cycle viral et protège les cellules infectées. Secrétée dans le milieu extracellulaire, elle participe à l’immunodéficience en inhibant certaines fonctions ou en induisant l’apoptose des cellules du système immunitaire. Elle est également impliquée directement dans de nombreuses pathologies associées au VIH-1. Dans une première étude, nous avons voulu savoir si la trithérapie était capable d’inhiber la synthèse et la sécrétion de la protéine Tat. Nous avons proposé à des patients infectés, sous trithérapie, ayant une charge virale indétectable, de nous permettre de faire cinq prélèvements sanguins (un tous les trois mois pendant un an) afin de vérifier la présence d’anticorps anti-Tat. Nous avons pu constater que 86% des patients avaient des anticorps anti-Tat mais que ces anticorps pouvaient disparaître ou apparaître chez une majorité de patients, démontrant que la protéine Tat continue d’être sécrétée malgré les antiviraux. Une deuxième étude, sur des sérums de patients, a été effectuée afin de déterminer si la protéine Tat était structurée dans le sang des patients. Il existait une polémique, dans la littérature, sur le fait que la protéine Tat soit naturellement non structurée. Nous avons démontré que la protéine Tat est structurée dans le sérum de patients. De plus, l’activité biologique de la protéine Tat est étroitement liée à l’acquisition de sa conformation. Dans le cadre du développement clinique d’un vaccin anti-Tat dans le laboratoire, nous avons effectué des vaccinations sur des souris afin de déterminer la dose, l’adjuvant, la voie d’administration et le nombre de rappels à effectuer ainsi que la vérification de la tolérance et de la toxicité du vaccin. Des essais cliniques sont en préparation dans le cadre d’un protocole thérapeutique. Le laboratoire développe également une autre approche thérapeutique avec un anticorps monoclonal de souris capable de reconnaitre et de neutraliser les variants Tat représentatifs des cinq principaux sous-types du VIH-1. Cet anticorps qui sera humanisé, servirait à une future immunothérapie en combinaison à la trithérapie pour des patients en phase précoce ou tardive ou encore pour des nouveaux nés dont le système immunitaire est peu fonctionnel. / The protein Tat (Trans-activator of transcription) is definitely one of the most interesting targets in the fight against HIV-1. Synthesized early, it plays a central role in the viral life cycle and protects infected cells. Secreted into the extracellular medium, it participates in the immunodeficiency by inhibiting some functions or inducing apoptosis of immune cells. It is also directly involved in many diseases associated with HIV-1. In a first study, we examined whether HAART was able to inhibit the synthesis and secretion of the Tat protein. We proposed to HIV-1 infected patients under HAART, with undetectable viral load, to allow us to do five blood samplings (one every three months for one year) to verify the presence of antibodies against Tat. We found that 86% of patients had antibodies against Tat but these antibodies could disappear or appear in a majority of patients showing that Tat protein continues to be secreted in spite of antiviral treatment. A second study of patient sera was carried out to determine if Tat was structured in the blood of patients. There was a controversy in the literature about the fact that Tat could be naturally unstructured. We showed that Tat is structured in the serum of patients. In addition, the biological activity of Tat is closely related to the acquisition of its conformation. As part of a clinical development of a Tat vaccine in the laboratory, we carried out vaccinations in mice to determine the dose, the adjuvant, the route of administration, the number of boosts, tolerance and toxicity of the vaccine. Clinical trials are planed with a therapeutic protocol. The laboratory is also developing another therapeutic approach with a mouse monoclonal antibody able to recognize and neutralize Tat variants representative of the five major subtypes of HIV-1. This antibody will be humanized and could be used for future immunotherapy, in combination with HAART for patients in early or late stage of the pathology or to newborn babies who have a weak immune system.

Introdução: A imunoterapia intravesical com o bacilo de Calmette-Guérin (BCG) é o tratamento adjuvante de escolha no câncer superficial de bexiga. Recentemente, os estudos do mecanismo imunoterápico do BCG têm permitido identificar as reações imunológicas e os genes associados ao efeito antitumoral, possibilitando a produção de vacinas recombinantes, possivelmente mais efetivas e com menos efeitos colaterais. Com esses objetivos, associou-se o componente pertussis (S1PT) ao BCG, criando uma variante recombinante (rBCG-S1PT) com capacidade para promover uma resposta imune direcionada ao tipo T helper 1 (Th1), o que poderá elevar a eficácia antitumoral do imunoterápico. Objetivo: Avaliar comparativamente o efeito antitumoral do rBCG-S1PT e do BCG no modelo experimental de carcinoma urotelial de bexiga. Métodos: O estabelecimento do modelo murino ortotópico e singênico de tumor vesical foi realizado através da implantação transuretral das células tumorais de bexiga da linhagem MB49 de camundongo C57BL/6. Experimento I Os animais (modelo experimental) foram divididos em três grupos, os quais receberam 4 aplicações semanais de rBCG-S1PT, BCG, ou soro fisiológico (grupo controle), por via intravesical. Após 7 dias da última aplicação, foram extraídos o baço e a bexiga, com o intuito de inferir o peso tumoral. Em seguida, as bexigas foram submetidas à avaliação do padrão de resposta imunológica e exame anátomo-patológico e imunohistoquímico. Experimento II Realizado como descrito no Experimento I, porém os animais foram acompanhados por 60 dias para análise de sobrevida. Experimento III Este ensaio foi realizado como descrito anteriormente, porém não foi realizada a implantação tumoral, para controle dos achados imunológicos e anátomo-patológicos. Resultados: A taxa média de implantação tumoral foi de aproximadamente 90% dos animais inoculados. Obtivemos redução das médias dos pesos vesicais dos grupos BCG e rBCG-S1PT (p<0,001). Nos dois grupos tratados com os imunoterápicos observou-se aumento significativo da expressão de TNF-, a qual foi mais intensa com o uso do rBCGS1PT (p<0,05). A IL-10 também teve aumento significante de sua expressão no grupo BCG recombinante (p<0,01). Os esplenócitos provenientes dos camundongos que foram tratados com imunoterápicos diminuíram a viabilidade das células tumorais MB49, sendo que este efeito foi mais intenso no grupo rBCG-S1PT. O grupo de animais tratados com rBCG-S1PT apresentou aumento significativo da sobrevida em relação aos outros grupos (Experimento II). As aplicações dos imunoterápicos em animais sem tumor (experimento III) não revelaram diferenças histológicas em relação ao grupo controle e o padrão de resposta imunológica encontrado sugere uma tendência à resposta Th1. Conclusão: Obtivemos sucesso no estabelecimento do modelo murino ortotópico singênico de tumor vesical. O imunoterápico rBCG-S1PT apresentou mais benefícios no tratamento do tumor vesical ortotópico em camundongos em relação ao BCG, como também maior redução da viabilidade das células tumorais in vitro. A cepa rBCG-S1PT apresentou elevação significativamente maior das citocinas da resposta imune Th1 em relação aos demais grupos. Concluimos, então, que os dados apresentados sugerem a possibilidade deste recombinante proporcionar melhor controle clínico do tumor vesical em humanos que a imunoterapia com BCG / Introduction: The intravesical immunotherapy with bacillus Calmette-Guérin (BCG) is the adjuvant treatment of choice in superficial bladder cancer. Recently, studies of the mechanism of BCG have identified the immune reactions favorable and the genes responsible for the antitumor effect, enabling the production of recombinant vaccines, possibly more effective and with fewer side effects. With those goals, the pertussis toxin (S1PT) was combined to BCG, creating a recombinant variant (rBCG-S1PT) with the capacity to promote an immune response targeted to the T helper type 1 (Th1), which may increase the effectiveness of its antitumor effect. Objective: Compare the antitumor effects of rBCG-S1PT and BCG in an experimental model of bladder cancer. Methods: The development of the animal model of bladder cancer was conducted by transurethral instillation of bladder tumor cell line MB49 of the mouse strain C57BL/6, setting the orthotopic and syngeneic murine model. Experiment I - The animal models were divided into three groups, which received 4 weekly intravesical applications of rBCG-S1PT, BCG, or saline (SF - control group). After 7 days of the last instillation, splenectomy was performed for splenocyte culture and the bladders extracted and weighed in order to infer the tumor weight. Then, the bladders were divided into two pieces. The first was used for molecular analysis to assess the pattern of immune response. The second was sent to histopathological analysis. Experiment II - Held as described in Experiment I, but the animals were monitored for 60 days for analysis of survival. Experiment III - This test was carried out as previously described (Experiment I), but with no tumor cells instillation. Results: The rate of tumor implantation was 90% of the animals submitted to tumor inoculation. We obtained reduction of the average weights of bladder in groups BCG and rBCG-S1PT ((p<0,001). In both groups treated with immunotherapy, there was an increase of expression of interleukins TNF-, which was more intense in the group treated with rBCG-S1PT (p<0,05). There was also increased expression of IL-10 in the recombinant BCG (p<0,01). The splenocytes from animals that received immunotherapies had reduced tumor cells viability, more intensely demonstrated in the rBCG-S1PT group. The analysis of survival showed a significant increase in the group of animals treated with rBCGS1PT (Experiment II). The instillation of immunotherapeutic agents in animals without tumor did not demonstrate histological differences when compared to the control group and the immunological response pattern was similar to that of Experiment I (Experiment III). Conclusion: The establishment of the syngeneic orthotopic animal model was successful. The immunotherapy with rBCG-S1PT demonstrated more benefits than BCG in the treatment of bladder cancer in mice, reducing the bladder weight, increasing survival, and reducing tumor cells viability in vitro. The immune response obtained with the rBCG-S1PT expressed higher cytokines related to Th1. All this data may indicate that this recombinant agent may promote better bladder tumor control than BCG imunotherapy

Imunização terapêutica utilizando células dendríticas derivadas de monócitos (MoDCs) pulsadas com antígenos de HIV constitui um meio promissor de potencializar a resposta imune específica anti-HIV em pacientes infectados. Neste contexto, é importante ressaltar que células dendríticas além de estimular a resposta imune específica, podem ser capazes de promover a tolerância periférica em linfócitos T CD4+ e T CD8+ ao induzir deleção, anergia ou através da expansão de células T reguladoras (T regs). Experimentos in vitro foram conduzidos para avaliar a capacidade de MoDCs pulsadas com HIV autólogo inativado em induzir apoptose celular, respostas celulares específicas e a geração de T regs. Os pacientes avaliados neste estudo foram indivíduos infectados pelo HIV, sem uso de tratamento antirretroviral (n = 14) com número de células T CD4+ acima de 350 células/L. MoDCs foram geradas a partir de células mononucleares de sangue periférico e em seguida foram pulsadas com vírus autólogo inativado por Aldrithiol-2, tratadas com estímulo para maturação e então cultivadas com linfócitos autólogos. A apoptose de linfócitos T e MoDCs e a frequência de células efetoras e reguladoras foram avaliadas por citometria de fluxo. Os resultados obtidos mostraram que não houve diferença nos níveis de apoptose de células T CD4+, T CD8+ ou MoDCs entre os grupos pulsadas e não pulsadas com HIV inativado. Foi observado que tanto MoDCs pulsadas quanto aquelas não pulsadas com o vírus autólogo inativado foram capazes de induzir células T CD4+ secretoras de IFN-, enquanto que apenas MoDCs pulsadas levou a um aumento no percentual de células T CD8+ efetoras. Pacientes com contagem de células T CD4+ acima de 500 células/L apresentaram um percentual maior de células T CD4+ secretoras de IFN após estimulo de MoDCs pulsadas. Esta diferença não foi observada em células T CD8 +. T regs também foram induzidas in vitro após cocultivo com MoDCs. Níveis basais mais elevados de T regs foram encontrados em pacientes com carga viral plasmática baixa. Em conjunto, os resultados indicam que MoDCs pulsadas com HIV-1 são capazes de induzir linfócitos T efetores, mas também aumentam a frequência de T regs in vitro. Além disto, pacientes com maior contagem de células T CD4 + foram capazes de responder de forma mais eficiente ao estímulo com MoDCs pulsadas. Viremia persistente na infecção crônica pelo HIV pode estar associada significativamente à perda de T reg / Therapeutic immunization using inactivated autologous HIVpulsed dendritic cells (DCs) is a promising strategy to enhance specific anti-HIV immune responses in infected patients. In this context, it is important to note that DC besides stimulate a specific immune response, may be able to promote tolerance in peripheral CD4 + and CD8 + T cells inducing deletion, anergy or through expansion of regulatory T cells (T reg). In vitro experiments were conducted to evaluate the capacity of autologous HIVstimulated DC to induce apoptosis, effector cellular T cell responses and T reg generation. For these purposes, we used peripheral blood from HAARTnaïve HIVinfected patients (n=14) with CD4+ T cell counts above 350 cell/L for generation of monocytederived DC (MoDC). MoDC were pulsed with aldrithiol-2 (AT-2)-inactivated autologous virus and matured. MoDC were then cocultured with autologous lymphocytes and the apoptosis, production of IFN- and T reg cell frequency were evaluated by flow cytometry. There was no difference in the rate of apoptosis of CD4, CD8 T cells or MoDC between the groups pulsed and not pulsed with inactivated HIV. MoDC pulsed or not with inactivated autologous virus induced IFN- +CD4+ T cells, whereas only pulsed MoDC were able to increase effector CD8+ T cells percentage along the time culture. Patients with CD4+ T cell counts above 500 had an increased percentage of CD4 + T cells secreting IFN- upon DC pulsed with HIV. This difference was not observed in CD8 + T cells. Interestingly, T reg were also induced in vitro after MoDC cocultivation. Higher baseline T reg counts were found in patients with lower plasma viral loads. These results show that MoDC pulsed with HIV-1 are able to induce effector lymphocyte but also elevate the frequency of T reg in vitro. Patients with higher CD4+ T cell counts are able to respond more efficiently to the stimulation with pulsed MoDC, and persistent viremia in chronic HIV infection is associated with significant loss of T reg

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

O câncer colorretal é o terceiro em mortalidade, e apesar das classificações moleculares, entre 30% e 40% dos pacientes apresentam recidiva após quimioterapia, o que aponta a necessidade de novas estratégias terapêuticas. É crescente o estudo de vetores virais para a terapia gênica do câncer, e dados do nosso laboratório mostraram que a entrega combinada dos genes Arf (supressor tumoral e parceiro funcional de p53) e interferon-beta (IFNbeta, citocina imunomodulatória), via vetores adenovirais sob o controle do promotor responsivo à p53 causou indução de morte celular massiva e um efeito imunomodulatório importante. Entretanto, esses resultados foram observados em modelos murinos. O presente trabalho avalia como a combinação da modulação das vias de p53 e IFNbeta impacta células de carcinoma colorretal humano em ensaios in vitro. Para isto, utilizamos nosso adenovírus para transferir p53 (p53) e IFN? como uma estratégia combinada para induzir a morte celular na presença de um modulador imunológico. As linhas celulares HCT116, HCT116p53-/- toleraram uma MOI (multiplicidade de infecção) de 25, enquanto uma MOI de 100 foi suportada por HT29 (mutante p53 R273H). O promotor PG, dependente de p53, como esperado, proporcionou níveis de expressão de GFP reduzidos em HCT116 p53 - / - e HT29, mas a expressão foi aumentada em todas as linhas celulares quando co-transduzida com o vetor codificando p53, mas não com IFNbeta. Em geral, HCT116 e HCT116p53-/- foram mais sensíveis à transferência de p53, enquanto o HT29 foi particularmente afetado pela combinação de p53 + IFNbeta. Esta tendência reflectiu-se na viabilidade celular (curva de crescimento, MTT), formação de colónias e acumulação de células hipodiplóides. Os níveis de morte celular correlacionaram-se com a atividade da caspase 3/7 e coloração com o Anexina V. Os marcadores de morte imunogênico ATP e calreticulina também foram aumentados, especialmente para o HT29 tratado com INFbeta sozinho ou em combinação com p53. Além disso, observamos aumento da expressão de TP63, TP73, bem como alvos transcricionais de p53, como p21 e NOXA, enquanto SESTRIN foi reduzido em ambas as linhagens HCT, mas aumentou em HT29. Adicionalmente, testamos a combinação p53 + IFNbeta em associação com quimioterápicos, revelando cooperação entre a transferência gênica e a doxorrubicina ou 5-FU, mesmo nas menores doses testadas. Ensaios em andamento incluem a avaliacao da ativacao de celulas dentriticas humanas expostas para as celulas tumorais tratadas com os vetores portadores de p53 e/ou IFNbeta. Com este projeto, nossa abordagem de transferência gênica revelará a resposta aos transgenes em modelo humano e também abrirá o caminho para estudar o envolvimento do sistema imune humano / Colorectal cancer is the third leading cause of cancer death and despite new molecular classifications, 30% to 40% of all patients will relapse after chemotherapy, pointing the need for new and innovative therapeutic strategies. The number of studies about viral vectors for gene therapy is growing, and previous data from our laboratory indicates that the combined delivery of Arf (a tumor suppressor gene and functional partner of p53) and interferon beta (IFNbeta, an immunomodulator cytokine), under a p53 responsive promoter, induced massive cell death and an important immunomodulatory effect. However, these results were only observed in murine models. Here we present an RGD-modified adenovirus whose transgene is under the control of a p53 responsive promoter (PG), used to transfer p53 (p53) and IFNbeta as a combined strategy to induce cell death in the presence of an immune modulator. Cell lines HCT116, HCT116 p53 -/- tolerated a MOI (multiplicity of infection) of 25, while a MOI of 100 was supported by HT29 (mutant p53 R273H). The p53-dependent PG promoter, as expected, provided reduced GFP expression levels in HCT116 p53 -/- and HT29, but the expression was increased in all cell lines when co-transduced with the p53 vector, but not with IFNbeta . In general, HCT116 and HCT116p53-/- were more sensitive to p53 gene transfer while HT29 was particularly affected by the combination of p53 + IFNtbeta. This trend was reflected in cell viability (growth curve, MTT), colony formation and accumulation of hypodiploid cells. Levels of cell death correlated with caspase 3/7 activity and staining with Annexin V. Immunogenic markers were also increased, especially for HT29 treated with INFbeta and p53/IFNbeta, as we detected exposure of calreticulin and release of ATP. Also, we observed increased expression of TP63, TP73 as well as p53 transcriptional targets, such as p21 and NOXA; SESTRIN was reduced in both HCTs but increased in HT29. Additionally, we tested the p53+IFNb combination in association with chemotherapeutics, revealing cooperation between gene transfer and doxorubicin or 5-FU even at the lowest doses tested. Ongoing studies include the evaluation of human dendritic cells exposed to tumor cells treated with the vectors for p53/IFNbeta. In conclusion, our combined gene transfer approach has potentiated the killing of colorectal cancer cell lines and may provide an immunomodulatory effect

INTRODUÇÃO: A imunoterapia baseada em células dendríticas derivadas de monócitos (MoDCs) constitui uma estratégia promissora para o tratamento de indivíduos infectados pelo HIV. Devido à sua notória plasticidade, populações heterogêneas de MoDCs podem ser obtidas in vitro, dependendo das condições da cultura. Consequentemente, a capacidade dessas células em secretar citocinas e expressar moléculas que participam do processo de apresentação antigênica (MHC, moléculas de adesão e coestimuladoras) é variável, podendo interferir no perfil e eficácia da resposta imune induzida pela terapia. Em nosso laboratório foi desenvolvido um protocolo clínico de vacinação terapêutica baseada em MoDCs e HIV autólogo inativado para o tratamento de indivíduos cronicamente infectados pelo HIV, não expostos à terapia antirretroviral. Deste modo tornou-se oportuna uma investigação in vitro mais aprofundada sobre a produção viral e as características das MoDCs utilizadas como produto vacinal. OBJETIVOS: Caracterizar o produto vacinal constituído por vírus autólogo e MoDCs de indivíduos infectados pelo HIV utilizados em imunoterapia, com relação a aspectos fenotípicos e funcionais. MÉTODOS: Foram incluídos no estudo 17 indivíduos cronicamente infectados pelo HIV, participantes de um estudo clínico de fase I/II de imunoterapia com MoDCs. Células mononucleares do sangue periférico (PBMCs) foram obtidas a partir de leucaférese e parte do material foi utilizada para isolamento e expansão de HIV em sistema de cultura autólogo ou alogênico. Outra parte das PBMCs foi utilizada como fonte de monócitos para diferenciação em MoDCs imaturas que foram pulsadas ou não com o HIV quimicamente inativado pelo aldrithiol-2 (HIV-AT-2), denominadas respectivamente MoDCs HIV-AT-2 e MoDCs maduras, e posteriormente ativadas com citocinas pró-inflamatórias. MoDCs foram avaliadas fenotípica e funcionalmente quanto à expressão de moléculas de superfície, capacidade fagocítica, potencial migratório, produção de citocinas e habilidade em gerar resposta celular in vitro, avaliada por meio da capacidade em induzir proliferação, produção de citocinas e atividade citotóxica em linfócitos T autólogos. RESULTADOS: O rendimento de partículas virais foi mais elevado quando a expansão do HIV foi realizada em sistema alogênico em comparação ao sistema autólogo. Após estímulo para maturação, tanto MoDCs maduras quanto MoDCs HIV-AT-2 apresentaram aumento na expressão de moléculas de coestimulação, ativação e migração, comparado às MoDCs imaturas. Com relação à caracterização funcional, observamos que MoDCs foram capazes de fagocitar partículas de dextran-FITC, exibiram baixo potencial migratório e baixa produção de citocina polarizante para Th1. Ainda, observamos reduzida atividade citotóxica induzida tanto por MoDCs HIV-AT-2 quanto por MoDCs maduras. Por outro lado, MoDCs HIV-AT-2 promoveram proliferação de linfócitos T autólogos e maior polifuncionalidade em células TCD4+ e TCD8+ em comparação às MoDCs maduras. CONCLUSÃO: A produção de vírus autólogo através de sistema alogênico resulta em maior rendimento viral e potencial imunogênico. O produto vacinal composto por MoDCs HIV-AT-2 é capaz de induzir resposta polifuncional antígeno especifica in vitro / INTRODUCTION: Immunotherapy based on monocyte-derived dendritic cells (MDDCs) is a promising strategy for the treatment of HIV-infected individuals. Due their plasticity, using different combinations of cytokines cocktail in vitro it is possible to obtain a heterogeneous MDDCs population. Consequently the capacity of these cells to secrete cytokines and express molecules that participate in antigen presentation varies (MHC, adhesion and costimulatory molecules) and can interfere in the profile and efficacy of the immune response induced by this therapy. A clinical trial was conducted in our laboratory to evaluate a immunotherapy based on dendritic cells sensitized with autologous inactivated HIV for the treatment of antiretroviral naive chronically HIV-infected individuals. Therefore, it was a good opportunity to study deeply the virus production and expansion in vitro and to characterize MDDCs used as a vaccine. OBJECTIVE. To characterize MDDCs in context of their phenotype and function as well as investigating viral production and expansion in autologous and allogenic systems. METHODS: 17 patients underwent apheresis before vaccination and their peripheral blood mononuclear cells (PBMCs) were used for autologous virus production and expansion of the virus was carried out in both autologous and allogenic systems. Monocytes were differentiated into immature MDDCs that were pulsed/or not with autologous chemically (aldrithiol-2) inactivated HIV particles (HIV-AT-2). These pulsed (HIV-AT-2 MDDCs) and non-pulsed (mature MDDCs) cells were then activated by proinflammatory cytokines. Phenotypic (cell surface marker) and functional analysis (phagocytosis, transmigration and cytokines production) of MDDCs and their priming and stimulation of lymphocyte (proliferation, polyfunctionality and cytotoxicity) was performed using flow cytometry. RESULTS. Viral yield was higher when expanded in allogenic compared to autologous system. After stimulation with proinflammatory cytokines, both HIV-AT-2 MDDCs and mature MDDCs presented increased costimulation expression, activation and migratory molecules compared to immature MDDCs. Regarding to functional characterization, we observed that MDDCs were able to phagocytize FITC-Dextran and exhibitted a low migratory potential and low production of Th1 polarizing response cytokines. Moreover we observed reduced cytotoxic activity induced by HIV-AT-2 MDDCs and mature MDDCs. On the other hand we also observed that HIV-AT-2 MDDCs were capable of inducing proliferation and polyfunctionality of autologous CD4+ and CD8+ T-lymphocytes compared to mature MDDCs. CONCLUSION. Allogenic system was found to be more efficient in increased viral yield in relation to autologous system. Besides, virus expanded in allogenic system showed a more immunogenic profile. Vaccine product (HIV-AT-2 MDDCs) was able to induce antigen specific polyfunctional response