As cepas vacinais rotavírus reassortants G1, G2, G3, G4 e G9 foram obtidas de um rearranjo genético entre cepas humanas de rotavírus do grupo A do sorotipo G (D, DS-1, P, ST-3 e AU32) e a cepa bovina UK-Bovine. Neste estudo, a cepa reassortant de rotavírus IB-BRV-4-G4 foi formulada, em duas condições, sem adjuvante (Esquema 1) e com adjuvante (Esquema 2) para imunização de camundongos Balb/c. Três AcMos da classe IgM foram obtidos da fusão utilizando animais do Esquema 1 e com o esquema 2 foram obtidos 3 AcMos, dois da classe IgA e um da subclasse IgG1. Destes últimos, dois AcMo (IgA) reconheceram somente epitopos conformacionais em teste de Dot-blot. Entretanto, o AcMo 3 (IgG1) foi reativo a proteínas virais também por Western Blotting, em uma região de perfil eletroforético coincidente com a glicoproteína VP7 de rotavírus. Os resultados mostram que o esquema II de imunização resultou em AcMo específicos para o sorotipo G4, sendo o AcMo IgG1 um forte candidato a ser utilizado nos testes de potência da vacina pentavalente de rotavírus do Instituto Butantan. / The rotavirus strain (G1, G2, G3, G4 and G9) were obtained of reassortants between human rotavirus of the group A and G serotypes (D, DS-1, P, ST-3 and AU32) and a strain from bovine (UK-Bovine).In this study, IB-BRV-4-G4 reassortant rotavirus strain was used to immunize Balb/c mice. Two schedules of immunization were used in the animals, one with IB-BRV-4-G4 (E1) and other with rotavirus antigen and adjuvant (E2). Three monoclonal antibodies of IgM class were obtained of cells from mice immunized only with rotavirus antigen (E1) and with (E2) was obtained three monoclonal antibodies, two producers the IgA class and one the IgG1 subclass. The IgA clones were reactive to G4 serotype rotavirus antigen only in the Dot-blot test. The mAb IgG1 was also reactive to viral proteins in a region of electrophoresis profile in the Western blotting test that coincided with the glycoprotein VP7 of rotavirus. The results indicate that the IgG1 obtained is specific to G4 serotype of rotavirus. It is candidate to be for use in the potency test of pentavalent vaccine.

As células dendríticas (DCs) expressam receptores que reconhecem padrões moleculares associados a patógenos (PAMPs), sendo capazes de capturar, processar e apresentar antígenos para os linfócitos. O contato com PAMPs induz a maturação das DCs e, consequentemente, a ativação dos linfócitos. Nossa estratégia para desenvolver vacinas é empregar anticorpos monoclonais contra os receptores endocíticos DEC205 e DCIR2, presentes nas subpopulações de DCs, em fusão com o antígeno de interesse. E estimular a maturação delas utilizando os adjuvantes poly I:C, CpG ODN 1826 e flagelina de S. Typhimurium . Imunizamos camundongos selvagens ou knockouts para os receptores TLR 3, 5 e 9 com adjuvantes e anticorpos quiméricos anti-DEC205, anti-DCIR2 ou isotipo controle (Iso) fundidos a proteína MSP119 de Plasmodium vivax. Avaliamos as repostas humoral e celular no grupos imunizados. Os resultados indicam que, seja qual for o adjuvante usado, a maioria dos parâmetros analisados aponta para a superioridade do direcionamento do antígeno para o receptor DEC205. / Dendritic cells (DCs) express receptors that recognize pathogen-associated molecular patterns (PAMPs), and are able to capture, process and present antigens to lymphocytes. In recent years, a immunization strategy that directs antigens to DCs in vivo has been developing. This consists in the use of monoclonal antibodies fused with the antigen of interest. However, effective immune activation occurs mainly when the chimeric antibodies anti-DEC or anti-DCIR2 are administered in the presence of a DC maturation stimulus. We immunized wild type and knockout mice for TLR3, 5 and 9 with adjuvants (poly I:C, CpG ODN 1826 and flagellin from S. typhimurium) and chimeric anti-DEC205, anti-DCIR2 or isotype control fused to the Plasmodium vivax MSP119 protein. After the administration of two doses, we evaluated the humoral and cellular immune responses in the different groups. Our results indicate that, irrespectively of the adjuvant, the majority of parameters analyzed points out to the superiority of antigen targeting to the DEC205 receptor.

A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxina foi construída no vetor pComb3X a partir do RNA total do hibridoma anti-digoxina. Seis clones foram isolados, todos com sequência idêntica na cadeia pesada. A cadeia leve apresentou 2 clones idênticos, um pseudogene e um clone com um aminoácido distinto no CDR2. Quatro clones apresentando variações na sequência do framework1 da cadeia leve foram expressos como fragmentos Fab solúveis. Todos apresentaram ligação à digoxina-BSA por ELISA e Western blotting. A ligação específica do anticorpo também foi confirmada pelo BIAcore, que permitiu ranqueamento entre os clones. / Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is very narrow. To counteract the toxic effect, polyclonal anti-digoxin Fab fragments are commercially available. Our goal was to obtain variants of monoclonal anti-digoxin Fab fragments by phage display technology, which allows the generation of high affinity and specificity antibody fragments. Anti-digoxin Fab fragments combinatorial library was constructed into pComb3X vector from total RNA of anti-digoxin hybridoma. Six clones were isolated and the heavy chain presented the same sequence. For the light chain, 2 clones were identical, one was a pseudogene and other one presented a distinct amino acid in the CDR2. Four clones presenting variations in the framework 1 were induced to express soluble Fab fragments, all positive for anti-digoxin binding in ELISA assays and Western blotting. The specific binding of the antibody was further confirmed by BIAcore, which allowed ranking of the clones.

Angiogênese é a formação de novos vasos sanguíneos a partir de vasos já existentes e é importante em processos fisiológicos, que em adultos é restrita ao reparo tecidual e ao ciclo reprodutivo feminino. Entretanto, doenças, como câncer ou retinopatias, induzem a formação da angiogênese patológica, necessária para a progressão destas patologias. Anticorpos monoclonais constituem uma das classes de biofármacos que mais cresce e com impacto importante nas doenças dependentes de angiogênese. Entre as diversas metodologias para a identificação de anticorpos monoclonais contra alvos terapêuticos, está o phage display. Por causa do fator de crescimento endotelial vascular (VEGF) ser o principal fator responsável pela formação de novos vasos, o principal biofármaco anti-angiogênico disponível na clínica atualmente é um anticorpo monoclonal (bevacizumab) direcionadas contra o VEGF. Embora terapias anti-VEGF sejam eficazes, ainda não são ideais devido a efeitos colaterais indesejados e a resistência medicamentosa. Novas alternativas são necessárias a fim de aperfeiçoar as terapias angiogênicas. O objetivo do nosso estudo é identificar novos alvos moleculares e desenvolver novos agentes terapêuticos para doenças dependentes de angiogênese. Para atingirmos nossa meta escolhemos o sistema de phage display para selecionarmos anticorpos com propriedades angiogênicas. Uma biblioteca de de anticorpos foi desenvolvida em nosso laboratório, dirigida contra a molécula VEGF, em particular uma de suas isoformas. Os animais imunizados desenvolveram anticorpos específicos, detectados por ELISA e Western-blot. A amplificação do pool de genes das cadeias leve e pesada de imunoglobulinas foi realizada para produzir os fragmentos single-chain (ScFv) que foram então clonados no vetor para a construção da biblioteca. A biblioteca de display de anticorpos ScFv será, portanto, analisada em plataformas angiogênicas para isolar anticorpos específicos contra isoformas de VEGF e novos marcadores moleculares de superfície celular expressos por células endoteliais ativadas. / Angiogenesis is the formation of new blood vessels from pre-existing ones and is an important physiological process, which in adults is mostly restricted to wound healing or the female reproductive cycle. However, different illnesses, such as cancer or retinopathies, induce the formation of pathological angiogenesis, necessary for disease progression. Monoclonal antibodies are one of the fastest growing class of biopharmaceuticals with important implications in angiogenesis dependent diseases. Among various methods for the identification of monoclonal antibodies against therapeutic targets is phage display technology. Because the vascular endothelial growth factor (VEGF) is the main molecular factor responsible for the formation of new blood vessels, the major anti-angiogenic drug available in the clinic today is a monoclonal antibody (bevacizumab) directed against VEGF. However, although anti-VEGF therapies are effective, they are not yet ideal due to undesirable side effects and drug resistance. Novel alternatives are necessary to improve on angiogenic therapies. The aim of our study is to identify novel molecular targets and to develop new therapeutic agents for angiogenic dependent diseases. To achieve our goal we have chosen the phage display system in order to select for antibodies with angiogenic properties. An antibody phage library has been developed in our laboratory, directed against VEGF molecule, particularly one of it isoforms. The animals were immunized and developed specific antibodies, detected by ELISA and Western-blot. Amplification of the pool of light and heavy chain Ig genes was performed to produce the single chain (ScFv) fragments for library construction. The ScFv antibody display libraries will be then screened in angiogenic settings to isolate antibodies against specific VEGF isoforms and novel cell surface molecular markers expressed by activated human endothelial cells

Il est admis que l’axe endothéline (endothélines ET-1, -2 et -3 et leurs RCPG ETAR et ETBR), participe à la progression tumorale. Alors qu’ETAR est par exemple surexprimé dans le cancer de l’ovaire, ETBR l’est dans le mélanome. Cette surexpression, ainsi que l’implication d’ETA/BR dans la carcinogenèse, font de ces RCPG une cible tumorale pertinente. En raison de leurs forte spécificité, actions cytotoxiques variées, possibilités de couplage, les anticorps monoclonaux (AcM) sont des outils de choix en diagnostic et thérapie anti-cancéreuse. Cependant, on déplore actuellement l’absence d’AcM ciblant des RCPG sur le marché. Par une technique d’immunisation génique, 4 AcM anti-ETAR et 24 anti-ETBR ont été produits. Les résultats préliminaires obtenus avec les anti-ETAR sont prometteurs puisque ces AcM lient avec une haute affinité ETAR surexprimé dans des cellules CHO, l’un d’eux inhibant fortement la liaison du ligand. Mon travail de thèse s’est cependant concentré sur la caractérisation d’un anti-ETBR. Cet AcM reconnaît de façon spécifique et avec une forte affinité la conformation native d’ETBR surexprimé à la surface de cellules de mélanomes, suggérant l’existence d’une forme tumorale du récepteur. Suite à sa liaison aux cellules UACC-257 (lignée de mélanome), l’AcM se trouve internalisé. Dans ces cellules, malgré son incapacité à inhiber la liaison de l’ET, cet AcM inhibe l’activation de la voie PLC induite par le ligand et est également un fort inhibiteur de la migration due à l’activation de l’axe endothéline. Ces travaux soulignent l’intérêt de cet AcM comme outil diagnostique et thérapeutique dans le cas du mélanome. / It has been admitted that endothelin axis (endothelins ET-1, -2 and -3 and related GPCRs ETAR and ETBR) is involved in tumor progression. For instance, while ETAR is overexpressed in ovarian cancer, ETBR is in melanoma. This overexpression, as well as ETA/BR involvement in carcinogenesis, make these GPCRs a relevant tumor target. Because of their high specificity, various cytotoxic actions, possibilities of coupling, the monoclonal antibodies (mAbs) are useful tools in diagnosis and anti-cancer therapy. However, the absence of mAbs targeting GPCRs on the market is regrettable. Thanks to DNA immunization, 4 anti-ETAR mAbs and 24 anti-ETBR mAbs were produced. Preliminary results obtained with anti-ETAR are promising since these mAbs bind ETAR overexpressed in CHO cells with high affinity, one of them being a potent inhibitor of ligand binding. However, the aim of my PhD research works focused on the characterization of one anti-ETBR. This mAb specifically recognizes with high affinity the native conformation of ETBR overexpressed on the surface of melanoma cells, suggesting the existence of a tumor-specific receptor. Following its binding on UACC-257 cells (melanoma cell line), the mAb is internalized. In these cells, despite its inability to inhibit ET binding, this mAb is able to inhibit the ligand-induced activation of PLC pathway and display a potent inhibition of endothelin axis-induced migration. This work highlights the interest of this mAb as a tool for diagnosis and therapy in melanoma.

La galectine-9 est une lectine animale principalement exprimée dans un contexte inflammatoire et possédant des propriétés à la fois pro-inflammatoires et immunosuppressives. Elle induit la production de cytokines inflammatoires par les cellules du système immunitaire inné tandis qu’elle induit l’apoptose des lymphocytes Th1 CD4+ et favorise l’expansion des lymphocytes Treg. Les propriétés immunomodulatrices de la galectine-9 dépendent en grande partie de son interaction avec le récepteur TIM-3. Cependant, ces deux molécules interagissent chacune avec d’autres protéines et dans plusieurs contextes la responsabilité de l’interaction galectine-9/TIM-3 n’a pas été formellement démontrée. Le développement d’un anticorps neutralisant les effets de la galectine-9 permettrait de préciser son rôle exact dans ces contextes. Par ailleurs, le blocage des voies de signalisations inhibitrices du système immunitaire est aujourd’hui un enjeu majeur en oncologie, comme le démontre le succès récent de la neutralisation du récepteur inhibiteur CTLA-4 pour le traitement des mélanomes. Nous avons produit de nouveaux anticorps monoclonaux dirigés contre la galectine 9 par immunisation de souris avec la partie C-terminale de la protéine. Parmi ces anticorps, 1G3 permet la détection de la galectine-9 sur coupes de tissus humains d’une manière très sensible et spécifique en immunohistochimie. Son utilisation nous a permis de confirmer l’expression constante et intense de la galectine-9 dans les cellules malignes de carcinome nasopharyngé et la forte expression de la galectine-9 dans les cellules de Kupffer présentes dans les tissus hépatiques infectés par le virus de l’hépatite C. Pour la première fois, nous avons mis en évidence une expression de la galectine-9 dans les leukocytes infiltrant les tissus hépatiques infectés par le virus de l’hépatite B. De plus, nous observons une expression de la galectine-9 dans les hépatocytes infectés par ces deux virus, ce qui n’avait pas été démontré jusqu’à présent. Nous avons également caractérisé les capacités fonctionnelles de nos anticorps lors de tests in vitro. L’anticorps 2E12 bloque la fixation de la galectine-9 au récepteur TIM-3 dans un test acellulaire, neutralise l’apoptose induite par la galectine-9 sur cellules de lymphomes T humaines et réduit considérablement l’augmentation de calcium cytosolique induite par la galectine-9 dans les cellules Jurkat. Ces effets de la galectine-9 sont indépendants de TIM-3 dans ce modèle cellulaire. L’anticorps 2E12 constitue un outil puissant pour étudier les fonctions de la galectine-9 à la fois dépendantes et indépendantes de TIM-3. / Galectin-9 is an animal lectin mainly expressed in an inflammatory context which possesses both pro-inflammatory and immunosuppressive properties. It induces inflammatory cytokines production from innate immunity cells whereas it induces apoptosis of Th1 CD4+ lymphocytes and enhance T regulatory lymphocytes expansion. Galectin-9 immunomodulatory properties depends mostly on its interaction with TIM-3 receptor. However, both molecules interact with other proteins, and in several contexts, galectin-9/TIM-3 interaction responsability has not been formally demonstrated. Development of a galectin-9 neutralizing antibody would allow to determine its precise role in those contexts. Moreover, blocking of immune system inhibition pathways is nowaday a major concern in oncology, as the success of CTLA-4 antagonist used for melanoma treatment recently demonstrated. We have produced new monoclonal antibodies against galectin-9 through immunisation of mice with the C-terminus part of the protein. Among those antibodies, 1G3 allows the detection of galectin-9 on human tissue samples in a very sensitive and specific manner in immunohistochemistry. Using 1G3, we could confirm intense and constant expression of galectin-9 in nasopharyngeal carcinoma malignant cells and its strong expression in Kupffer cells infiltrating hepatitis C-infected liver tissue. For the first time, we demonstrated expression of galectin-9 in leukocytes infiltrating hepatitis B-infected liver tissue. Additionally, we could observe galectin-9 expression in infected hepatocytes for both viruses, which had not been demonstrated until now.We also characterized functional properties of our antibodies during in vitro tests. The 2E12 antibody blocks galectin-9 interaction with TIM-3 in an cell-free assay, neutralizes galectin-9-induced apoptosis of human T lymphomas cells and considerably reduces galectin-9-induced increase of intracellular calcium in Jurkat cells. Those effects are independent of TIM-3 in this cell line model. The 2E12 antibody constitutes a powerful too to study both TIM-3-dependent and TIM-3-independent functions of galectin-9.

En France, le cancer colorectal (CCR) est la 2ème cause de décès par cancer. Chez les patients, lorsque la tumeur est localisée, elle est réséquée par chirurgie. Toutefois, 50% des patients sont diagnostiqués à un stade métastatique, ces patients sont alors traités par chimiothérapie (FOLFOX/FOLFIRI), souvent en combinaison avec des thérapies ciblées incluant des anticorps tel que le Cetuximab (anti-EGFR) ou le Bevacizumab (anti-VEGF). Cependant, il reste encore environ 40% des patients qui ne répondent pas au traitement et l’une des causes les mieux établies est l'influence des mutations de la voie RAS en aval du récepteur à l'EGF sur la réponse au Cetuximab. C’est pourquoi l’identification de nouvelles cibles moléculaires accessibles aux anticorps permettrait le développement de nouveaux modèles de thérapie ciblée. Depuis peu, les Claudines ont suscité un intérêt pour le ciblage tumoral comme la claudin-4 dans les cancers de l’endomètre ou de la prostate. Très récemment, les résultats d'une étude clinique ont démontré que la combinaison d'un anticorps anti-Claudin-18.2, l'Acm IMAB362, avec la chimiothérapie prolongeait nettement la survie chez des patients atteints de cancer gastrique avancé.C’est dans cette optique que nous nous sommes focalisés sur les claudines dans le CCR. Tout d’abord en étudiant leur expression dans des échantillons de patients atteints de CCR métastatique. En analysant l’expression des claudines dans les nouveaux sous-types moléculaires, nous avons montré que certaines pouvaient avoir une valeur pronostique. Nous avons également identifié des claudines comme cibles thérapeutiques potentielles dans le CCR, parmi elles la CLDN1. En effet, nous avons montré que la forme membranaire de la CLDN1 était surexprimée dans les tumeurs primaires et métastases du CCR. Nous avons développé un anticorps monoclonal (Acm) ciblant les parties extracellulaires de la CLDN1. Nous avons montré que le ciblage thérapeutique de la CLDN1 par Acm provoquait un ralentissement significatif de la croissance, la migration et l'invasion des cellules tumorales aussi bien in vitro que in vivo. Afin d’améliorer l’efficacité du ciblage de la CLDN1 par Acm, nous avons couplé ce dernier avec une toxine, générant ainsi un Antibody Drug conjugated (ADC). Nous avons montré que le ciblage de la CLDN1 par un ADC diminuait la survie cellulaire in vitro en culture cellulaire 2D, mais également celle des sphéroïdes via un effet cytotoxique. Ce travail a permis d'établir la preuve de concept du ciblage de la CLDN1 aussi bien par Acm que par ADC. Afin de finaliser ce travail, ces résultats doivent être confirmés in vivo. A terme, nous espérons que ce ciblage puisse trouver sa place au sein de l’arsenal thérapeutique du CCR métastatique, en particulier chez les patients résistants aux traitements. / Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery. However, 50% of patients are diagnosed at a metastatic stage, these patients are then treated with chemotherapy (FOLFOX / FOLFIRI), often in combination with targeted therapies including antibodies such as Cetuximab (anti-EGFR) or Bevacizumab (anti -VEGF). Despite these treatment, almost 40% of patients develop resistance. One of the most known resistance mechanisms of resistance is due to the RAS pathway downstream of the EGF receptor in response to Cetuximab. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies. Recently, Claudines have generated interest as targets in cancer, such as claudin-4 in endometrial or prostate cancer. More recently, the results of a clinical study demonstrated that the combination of an anti-Claudin-18.2 antibody, IMAB362 mAb, with chemotherapy significantly prolonged survival in patients with advanced gastric cancer.Therefore, one of our aims was to focus on claudins in CCR. First, by studying their expression in metastatic CRC patient samples. We demonstrated the prognostic value of some claudins, after analyzing their gene expression in the new molecular subtype of CRC. Beside, we identified some claudins as potential therapeutic targets in CCR, among them claudin-1 (CLDN1). Indeed, we showed that the membrane form of CLDN1 is overexpressed in primary tumors and metastases of CRC. Therefore, we developed a monoclonal antibody (mAb) targeting the extracellular parts of CLDN1. We showed that therapeutic targeting of CLDN1 by mAb significantly decreased tumor cell growth, migration and invasion both in vitro and in vivo. In order to improve the efficiency of targeting CLDN1 by mAb, we conjugated it with a toxin, thus generating an Antibody Drug Conjugate (ADC). We showed that CLDN1 targeting by an ADC decreased cell survival in vitro in 2D cell culture, but also spheroids growth via a cytotoxic effect.This work demonstrated the proof of concept of CLDN1 targeting by both mAb and ADC. In order to achieve this work, the next steps remains on testing ADC affect in vivo models. CLDN1 is a good therapeutic target in the CCR. In the long term, we hope that its targeting can find its place within the therapeutic arsenal of metastatic CRC, particularly in treatment of resistant patients.

Since the approval of the first therapeutic monoclonal antibody in 1986, monoclonal antibody has become an important class of drugs within the biopharmaceutical industry, with indications and superior efficacy across multiple therapeutic areas, such as oncology and immunology. Although there has been great advance in this field, there are still challenges that hinder or delay the development and approval of new antibodies. For example, we have seen issues in manufacturing, such as quality, process inconsistency and large manufacturing cost, which can be attributed to production failure, delay in approval and drug shortage. Recently, the development of new technologies, such as Process Analytical Tools (PCT), and the use of statistical tools, such as quality by design (QbD), Design of Experiment (DoE) and Statistical Process Control (SPC), has enabled us to identify critical process parameters and attributes, and monitor manufacturing performance. However, these methods might not be reliable or comprehensive enough to accurately describe the relationship between critical process parameters and attributes, or still lack the ability to forecast manufacturing performance. In this work, by utilizing multiple modeling approaches, we have developed a systematic framework to optimize and control monoclonal antibody manufacturing process. In our first study, we leverage DoE-PCA approach to unambiguously identify critical process parameters to improve process yield and cost of goods, followed by the use of Monte Carlo simulation to validate the impact of parameters on these attributes. In our second study, we use a Bayesian approach to predict product quality for future manufacturing batches, and hence mitigation strategies can be put in place if the data suggest a potential deviation. Finally, we use neural network model to accurately characterize the impurity reduction of each purification step, and ultimately use this model to develop acceptance criteria for the feed based on the predetermined process specifications. Overall, the work in this thesis demonstrates that the framework is powerful and more reliable for process optimization, monitoring and control.

Apesar da forte ativação do sistema imune que ocorre durante a infecção pelo Plasmodium, a memória imunológica à infecção é restrita a pacientes residentes em áreas endêmicas. Dessa forma é importante a geração de métodos capazes de induzir uma resposta imune eficaz e duradoura contra o parasito. Nesse contexto o direcionamento de antígenos para células centrais do sistema imune tem se apresentado como uma alternativa promissora. Produzimos e caracterizamos um anticorpo híbrido específico para a molécula DEC205, um receptor endocítico presente nas células dendríticas, acoplado à proteína MSP-1(19) de P. chabaudi, para fins de imunização e análise da resposta imune celular e humoral. Ensaios de imunização mostraram a indução de resposta humoral em camundongos imunizados com anticorpo híbrido e seu controle isotípico, caracterizada pela produção de IgM. Nossos resultados prévios indicam que o direcionamento de antígenos aliado a outras estratégias de imunizações podem resultar na ativação da resposta imune específica ao parasita. / Despite the strong activation of the immune system that occurs during infection by Plasmodium, the immunological memory to infection is restricted to patients residing in endemic areas. Thus it is important to the generation of methods to induce an effective immune response against the parasite. In this context, the targeting of antigens to the central cells of the immune system has emerged as a promising alternative. We produce and characterize a hybrid antibody molecule specific for DEC205, an endocytic receptor present on dendritic cells, coupled to protein MSP-1(19) of P. chabaudi, for immunization and analysis of cellular and humoral immune response. Immunization tests showed the induction of humoral response in mice immunized with hybrid antibody and isotype control, characterized by production of IgM. Our previous results indicate that targeting antigens combined with other strategies for immunization may result in the activation of specific immune response to the parasite.

Antibodies are one of the most useful molecules with affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human diseases. In recent years, phage-displayed antibody library has been widely adopted to select tailor-made antibodies in a fast, high-throughput mode, as an alternative of traditional hybridoma technology. Although phage display has been introduced for about 20 years, the applications and development of this technology still have a rich space to be explored. / Attempts are made in the present study to extend three applications of the phage displayed antibody library in antibody discovery and engineering. Firstly, a CDR3-randomized phage-displayed scFv library was constructed from genomic DNA of mouse. Following biopanning, anti-peptide of mas oncoprotein scFvs were isolated and identified. These results illustrate the potential use of the genomic phage-displayed library for anti-peptide antibodies selection. Secondly, we described the isolation of anti-idiotypic scFvs against a chimeric anti-CD22 mAb from an immunized phage-displayed scFv library. The isolated anti-Id scFvs were able to capture the immune response of chimeric anti-CD22 mAb with high specificity. This reagent will enhance our understanding of the therapeutic mechanism of anti-CD22 mAb in non-Hodgkin's lymphoma treatment, and may be applied to probe the pharmacokinetics, tissue distribution, and modulation of anti-CD22 mAb in vivo. / In conclusion, we have attempted various approaches to identify specific anti-peptide scFvs, anti-idiotypic scFvs and passive anti-tumor scFvs. These results extend the applications of phage display technology in antibody discovery and engineering. / Our approach enables us to isolate selective and sensitive anti-idiotypic antibodies and could be exploited for other antibodies with clinical and biological applications. Thirdly, we profile a strategy to select and identify markers on tumor cell surface using phage-displayed antibodies from mice bearing xenograft tumor. Our data imply that passive antibodies in cancer patients may be obtained from the immune repertoire of cancer patients. Besides, we found a cell surface antigen was up-regulated more than 3-fold in mas-expressing cells. We further use the targeting antibody to construct a tumor endoprotease-activated immunotoxin. / Zhao, Qi. / Adviser: Wing-Tai Cheung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3499. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 227-250). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.