Дисертації з теми "CD20 antibodies"

Immunoglobulin E (IgE) is fundamental to the allergic response and the functions of IgE are mediated by its Fc region binding to two receptors, FcεRI and CD23 (FcεRII). The interaction of IgE with other proteins have complicated our investigations of the unique role each receptor plays. To solve this, a small-scale library of IgE-Fc proteins was designed with two key positions, one at each receptor-binding site mutated. The unpredictable allosteric nature of IgE prevents rational engineering approaches, thus the design of a membrane-bound IgE-Fc-GFP-tagged protein allowed for the generation of a membrane-surface display library of stable cell lines. A FACS selection assay identified IgE-Fc proteins with weakened binding to a single IgE-receptor, which serves as a proof-of-principle for this concept. Additional studies into human CD23 and the differences between it and murine CD23 revealed additional levels of regulation for IgE-binding not seen in other species and this is due to its unique properties. Human CD23 is an unusual antibody receptor, being a calcium dependent (C-type) lectin that has lost its carbohydrate binding capability. Ca2+ binds to and increases CD23’s affinity for IgE, and one of two Ca2+ binding sites usually present in C-type lectins is absent in human but present in murine CD23. To understand if the loss of the second Ca2+ binding site has led to a regulatory gain/loss of function in human CD23, a panel of CD23 mutant proteins with increasingly ‘mouse-like’ sequences was generated. The insertion of the second Ca2+ binding site was verified by HSQC-NMR whilst molecular dynamic simulations provided a means of understanding the flexibility of the proteins. It revealed that binding of two Ca2+ ions tethers the soluble CD23 loops into position in the most mouse-like mutant protein, limiting possible conformations for IgE binding. Complementary Biacore experiments indicated that higher calcium binding affinity may have come at a cost of weakened IgE binding, as data in the presence and absence of Ca2+ showed decreased binding affinities of the proteins for human IgE. This regulatory difference between murine and human soluble CD23 could inform the development of CD23/IgE inhibitor therapeutics for the treatment of allergy.

Le rituximab est un anticorps monoclonal (AcMo) IgG1 chimérique qui cible le CD20, une protéine présente à la surface de la plupart des lymphocytes B. Il est indiqué dans la polyarthrite rhumatoïde (PR) et est utilisé dans les vascularites associées aux anticorps (ANCA) anti-cytoplasme des neutrophiles (AAV). Il existe une grande variabilité interindividuelle de la pharmacocinétique (PK) du rituximab et de sa relation concentration-réponse. Le rituximab se lie au CD20, et les complexes rituximab-CD20 formés sont éliminés par le système immunitaire. L’élimination médiée par la cible n'a jamais été décrite dans les maladies inflammatoires auto-immunes. Ce travail de thèse visait à étudier la PK et la relation concentration-réponse du rituximab par modélisation PK et pharmacocinétique-pharmacodynamique (PK-PD) de population. Ces travaux ont été réalisés grâce à : - 52 patients atteints de PR, suivis dans le Service de rhumatologie du CHRU de Tours, où les concentrations de rituximab, la numération CD4+ et le score d'activité de la maladie sur 28 articulations (DAS28) ont été mesurés. - 92 patients de l'essai RAVE, où les concentrations de rituximab, d'anticorps anti-protéinase 3 (PR3-ANCA) et d’anti-myéloperoxydase (MPO-ANCA) ont été mesurées. Chez les patients atteints de PR, la PK du rituximab a été décrite à l'aide d'un modèle à bicompartimental. Aucune élimination médiée par la cible n'a été détectée. Cela peut être dû à (i) une faible quantité d'antigène-cible par rapport à l’oncologie, et/ou (ii) à des données de PK peu denses. Chez les patients atteints d’AAV, une élimination non-linéaire médiée par la cible a été détectée au début du suivi. Un modèle TMDD simplifié a été utilisé, où le rituximab se fixait de façon irréversible sur le CD20, modélisé en tant que variable «latente». La variable latente peut être en partie interprétée comme la fraction de CD20 (circulante ou exprimée sur la membrane des cellules B) et disponible pour la liaison au rituximab. L'élimination médiée par la cible, négligeable, à la fin du suivi dans les AAV, peut être due aux numérations de cellules B faibles à la fin du suivi. La relation quantitative entre la déplétion des cellules B du sang, des cellules immunitaires, et la réponse clinique reste floue, aussi bien dans la PR que dans les AAV. De plus, cette relation, très variable selon les patients, n’a jamais été quantifiée à l’aide de la modélisation PK-PD. - Dans la PR, les relations entre les concentrations de rituximab et les numérations CD19+, entre les numérations CD19+ et CD4+, et entre les numérations CD4+ et le DAS28 ont respectivement été décrites à l'aide de modèles de durée de vie cellulaire « cell lifespan », de réponse indirecte et directs (Emax). Cette étude a montré que (i) l’amplitude de la déplétion CD19+ n’était pas associée à une déplétion CD4+ ou à une diminution du DAS28, et (ii) que la diminution du DAS28 était doublée en présence de déplétion des CD4+ comparé à son absence, et (iii) des concentrations plus élevées de rituximab étaient liées à une meilleure réponse clinique. - Dans les AAV, la relation entre les concentrations de rituximab et les niveaux d'ANCA a été décrite à l'aide d'un modèle de transit semi-mécanistique avec rétrocontrôle négatif. Ce modèle a montré que (i) la diminution des niveaux d'ANCA induite par le rituximab était profonde mais retardée et plus soutenue chez les patients avec MPO-ANCA que chez ceux avec PR3-ANCA, et (ii) des concentrations plus élevées de rituximab étaient associées à une diminution plus importante des niveaux d'ANCA. Des administrations répétées de rituximab seraient susceptibles d’améliorer la réponse clinique au rituximab et diminuer le risque de rechute. En conclusion, ce travail a été le premier à montrer une PK non linéaire du rituximab dans une maladie auto-immune (AAV), et à quantifier la relation complexe entre la déplétion des cellules B induite par le rituximab, la diminution des CD4+, et son efficacité clinique
Rituximab is a chimeric IgG1 monoclonal antibody (mAb) that targets CD20, a protein on the surface of most B lymphocytes. It is approved as a second line treatment in rheumatoid arthritis (RA) patients, and is used in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). There is a large interindividual variability in rituximab pharmacokinetics (PK), and its concentration–response relationship. Rituximab binds to CD20 with high affinity and specificity and form mAb-target complexes which are eliminated by immune system. This elimination is usually described using target-mediated drug disposition (TMDD) models. For rituximab, TMDD was never reported in autoimmune inflammatory diseases, as RA or AAV. This PhD work aimed at studying the variability of PK and cconcentration-response relationship of rituximab using population PK and pharmacokinetic-pharmacodynamic (PK-PD) modeling. This work was done using data from : - 52 RA patients of the rheumatology department of Tours University Hospital, where rituximab concentrations, CD4+ counts (biomarker) and disease activity score in 28 joints (DAS28, clinical response) were measured. - 92 AAV patients included in the RAVE trial (Rituximab for AAV), where rituximab concentrations, antibodies to proteinase 3 (PR3-ANCA), and antibodies to myeloperoxidase (MPO-ANCA) (biomarkers) were measured. In RA patients, rituximab PK was described using a two-compartment model with linear (endogenous) elimination. No target-elimination of rituximab or influence of B-cell count was detected. This may be due to (i) a low amount of target antigen compared to B-cell neoplasia, and/or (ii) insufficiently dense PK data. In AAV patients, nonlinear target-mediated elimination was detected at the beginning of the follow-up. A simplified TMDD model was used, where target was modeled as a latent variable, and irreversible binding to rituximab on its target was assumed. Latent variable may be partly interpreted as the fraction of CD20 (circulating or present at the B-cell membrane) available for rituximab binding. The negligible target-mediated elimination towards the end of the follow-up in RA and AAV may be due to the sustained B-cell depletion after rituximab treatment. Rituximab-induced B cell depletion was shown to alter count and function of other immune cells. However, quantitative relationship between depletion of blood B cells, immune cells, and clinical response in both RA and AAV remain unclear. In addition, this relationship, highly variable among patients, was never quantified using PK-PD modeling. In RA patients, the relationship between rituximab concentrations and CD19+ counts, between CD19+ and CD4+ counts, and between CD4+ counts and DAS28 (clinical activity score in RA) was described using cell lifespan, indirect response and direct Emax models, respectively. This study showed that (i) the amplitude of CD19+ count depletion was not associated with CD4+ decrease or DAS28, and (ii) CD4+ decrease leads to two-fold DAS28 decrease compared to no CD4+ decrease, and (iii) higher rituximab concentrations were related to a better clinical improvement. In AAV patients, the relationship between rituximab concentrations and ANCA levels was described using a semi-mechanistic transit model with negative feedback. This model showed that (i) rituximab-induced decrease of ANCA levels was deep but delayed, and more sustained in patients with MPO-ANCA than in those with PR3-ANCA, and (ii) higher rituximab concentrations were associated with deeper decrease of ANCA levels. Moreover, repeated rituximab courses may improve clinical response to rituximab, and increase the time to disease relapse. Overall, this work was the first to report nonlinear PK of rituximab in autoimmune disease (AAV), and to quantify the complex relationship between rituximab-induced B-cell depletion, CD4+ decrease, and the clinical response

CD23 is the low affinity IgE receptor. It is a type II integral transmembrane glycoprotein that can be shed from the cell surface forming soluble products of approximately 37, 33, 29, 25 and 16kDa. It appears that membrane and soluble CD23 have opposing regulatory functions and that inhibition of CD23 shedding may have potential to alleviate both allergic and inflammatory diseases. This has focused attention on the endogenous protease(s) responsible for CD23 shedding, leading to the demonstration that both the 37 and 33kDA sCD23 fragments are cleaved from the cell surface by a metalloprotease. In order to characterise the cleavage events within CD23, anti-neoepitope antibodies specific for the newly created amino and carboxy termini of the two predominant cleavage sties within CD23 (producing the 37 and 25kDa soluble CD23 products) were raised. Characterisation of these antibodies demonstrated that the proteolytic cleavage events responsible for creating the 37 and 25kDa sCD23 fragments are independent of each other. Furthermore, two different proteases were shown to be responsible for cleaving these two fragments. The work described in this thesis confirms previous reports that 37kDA sCD23 is cleaved by a metalloprotease, however cleavage of the 25kDa fragment was not inhibited by metalloprotease inhibitors. The production of the two different sized sCD23 molecules by different proteases has important implications for targeting the proteolytic cleavage events to alleviate symptoms of allergic and inflammatory diseases. This emphasises the importance of defining the biological functions of mCD23 and each of the sCD23 molecules.

Como a imunoproteção na Paracoccidioidomicose (PCM) é principalmente mediada por células T, investigamos o impacto da deficiência de CD28, molécula co-estimulatória de linfócitos T, na gravidade da infecção primária e secundária pelo Paracoccidioides brasiliensis. Quando comparados a camundongos C57Bl/6 normais (WT), camundongos CD28-deficientes (CD28KO) apresentaram infecção mais grave associada à produção diminuída de anticorpos e citocinas. Além disso, a pré-imunização de animais deficientes e normais resultou em imunoproteção equivalente. Inesperadamente, a sobrevida de animais CD28KO foi significativamente maior que a dos WT, apesar da sua elevada carga fúngica tecidual. Em conclusão, nosso trabalho mostrou que a deficiência de CD28 resulta em PCM mais grave, porém não letal, associada a resposta imune deficiente. Além disso, verificamos que carga fúngica elevada, na ausência da imunidade adaptativa efetora, não ocasiona diminuição de sobrevida, revelando que a resposta imune na PCM pode tanto proteger como ser deletéria aos hospedeiros.
As immunoprotection in Paracoccidioidomycosis (PCM) is mainly mediated by T cells, and CD28 is a costimulatory molecule for T lymphocytes, we investigated the influence of CD28 deficiency in primary and secondary PCM. Compared with normal C57BL/6 mice, CD28-deficient (CD28KO) mice developed a more severe infection associated with impaired antibody and cytokine production. In addition, CD28KO and WT mice previously immunized by the s.c. route developed equivalent immunoprotection when challenged by the pulmonary route. Interestingly, CD28KO mice presented increased mean survival time despite their elevated fungal loads in the lungs. In conclusion, our work showed, for the first time, that CD28-deficiency results in more severe, but not overwhelming, PCM. Furthermore, in the absence of effector adaptative immunity, elevated fungal loads do not cause lethal infections, revealing the protective and deleterious effects of immune responses to Paracoccidioides brasiliensis infected hosts.

Cancer, historically considered a genetic disease, is currently acknowledged to affect the whole body. Our immune system is one key player that can elicit a response against malignant cells but can also promote tumorigenesis. Tumors avoid immune recognition by creating a suppressive microenvironment and inducing tolerance. T-cells are regarded a major effector cell type in tumor immunotherapy. An important ”switch” needed for T-cell activation involves so-called costimulatory and coinhibitory receptors. In this thesis, experimental tumor models were used to investigate the potential of immunomodulatory antibodies to stimulate immune cells and subsequently eliminate tumors. First, systemic antibody blockade of two negative checkpoint regulators (CTLA-4 and PD-1) present on T-cells was evaluated in combination with local CpG therapy or standard BCG treatment. Indeed, this combinatorial therapy with CpG augmented anti-tumor effects with increased levels of tumor-directed T-cells and reduced tumor-infiltrating Tregs. Secondly, as these immunomodulatory antibodies elicit severe side effects in patients, a local low-dose delivery regimen was explored as an alternative to systemic bolus treatment. Our results demonstrated that an approximately seven times lower dose of aCTLA-4, compared to systemic delivery, could eradicate both primary and distant tumors. CD40-expressing APCs are another potential target in antibody-mediated cancer therapy. CD40-stimulated dendritic cells (DCs) have the capability to activate tumor-directed T-cells to kill tumor cells. We next sought to investigate agonistic CD40 antibody efficacy and in vivo biodistribution when delivered locally compared to the equivalent systemic dose. Anti-tumor effects were dependent on CD8+ T-cells, host CD40 expression and the presence of tumor antigen at the injection site. CD40 antibodies were cleared from the circulation and accumulated in lymphoid organs, where, upon repeated aCD40 dosing, target APC populations increased in numbers and upregulated their surface CD40 expression. Lastly, CD40 agonist antibodies were mixed with nanoparticles to enhance their stimulatory properties. B-cells demonstrated increased proliferative capacity and DCs became more activated when exposed to the cocktail. Further, this combination reduced serum levels of pro-inflammatory cytokines compared to plain antibodies.       The results herein advocate further exploratory studies of the delivery of monoclonal antibodies at the tumor site in order to improve anti-tumor effects and reduce toxicity.

As células da linhagem macrofágica são fundamentais na infecção por T. cruzi. Além do reconhecimento por PRRs, a interação com anticorpos e/ou complemento facilita a internalização. Avaliamos o papel in vivo e in vitro de IgM e IgG específicas na saída de parasitas Sylvio X10/4 do sangue, um clone miotrópico de T. cruzi que causa infecção sem parasitemia patente. Devido à sua saída espontânea, estudamos a remoção no sexto dia de infecção, momento em que a parasitemia subpatente aumenta. Camundongos foram infectados e tratados com soro normal (NMS), soro crônico (B6) e soro de animais CD28KO crônicos (que produzem somente IgM). O grupo tratado com soro de CD28KO apresentou uma remoção significativa, porém menos eficiente do que com soro crônico (B6). Tais resultados foram reproduzidos em estudo in vitro na invasão de macrófagos (derivados de medula óssea ou do peritônio) com os distintos tratamentos. Concluímos que os macrófagos são fundamentais na remoção dos parasitas e os anticorpos, não somente IgG, mas também os da classe IgM reforçam este processo.
The cells of the macrophage lineage are essential for the infection by T. cruzi. Besides the recognition by PRRs, interaction with antibodies and/or complement facilitates internalization. We evaluated the role in vivo and in vitro of specific IgM and IgG in the blood output of parasites Sylvio X10/4, clone myotropic of T. cruzi which causes infection without patent parasitemia. Due to its spontaneous exit, we studied the removal on the sixth day of infection, when the parasitemia subpatent increases. Mice were infected and treated with normal mouse serum (NMS), chronic serum (B6) and serum from CD28KO chronic mice (which produce only IgM). The group treated with CD28KO serum showed a significant removal, but less efficiently than with chronic serum (B6). These results were reproduced in vitro study on the invasion of macrophages (derived from bone marrow or peritoneum) with these different treatments. We conclude that macrophages are essential in the removal of parasites and antibodies, not only IgG, but also IgM enhance this process.

Cytokine-induced killer (CIK) cells are a heterogeneous population of lymphocytes obtained in vitro within 21 days from mononuclear cells under the influence of cytokines. CIK cells show potent MHC-unrestricted cytotoxicity against a variety of tumor cells, in particular hematological malignancies, and minimal tendency to induce graft-versus-host disease. The expanded bulk CIK culture consists of over 90% CD3+ cells, of which the majority coexpress CD56 and the remaining cells are CD56-. CD3+CD56+ “true” CIK cells are terminally differentiated non dividing lymphocytes which could deliver potent MHC-unrestricted cytotoxicity for the immediate destruction of tumor cells. The other less cytotoxic CD3+CD56- cell subset represents a progenitor reservoir that could proliferate and differentiate into CD3+CD56+ CIK cells. CD3+CD56+ CIK cells express activating NK receptors including NKG2D, DNAM-1 and low levels of NKp30. Cell signalling not only through TCR/CD3, but also through NKG2D, DNAM-1 and NKp30, leads to CIK cell activation resulting in granule exocytosis and cytotoxicity. Antibody blocking experiments revealed that NKG2D, DNAM-1 and NKp30 are actually involved in tumor cell recognition and killing. Anti-CMV specific CIK cells could be expanded in standard CIK conditions and mediate both specific, MHC-restricted recognition of a CMV-pulsed autologous target and NK-like non specific cytolytic activity against leukemic cell targets. Antibody blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, two frequently associated complications in transplanted patients. B-cell non-Hodgkin lymphoma is only partially susceptible to CIK-mediated lysis. The addition of anti-CD20 monoclonal antibodies GA101 or rituximab increased cytotoxicity mediated by CIK cell cultures by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum inhibited NK-cell activation induced by rituximab, but not activation induced by GA101. Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 mcg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. The combined use of CIK cells with anti-CD20 mAbs could represent a novel immunotherapy protocol for the treatment of B lymphoma patients with resistant disease.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Nowadays, cancer is one of the most feared diseases, affecting each day more and more people worldwide. The importance of new cancer treatment researches is very clear since the ones that has been used are not very effective and may lead to drug resistance, implying in a constant dose increasing which can lead to toxicity issues. Collateral effects and the instability generated in the patient’s organism are also reasons why the necessity of discovering new cancer treatments is imminent. A treatment alternative that has aroused interest is the use of monoclonal antibodies as immunotherapics, since they act by stimulating the patient’s immune system neutralizing the tumor cells in a very efficient and specific way. This kind of antibody can be produced by culturing hybrid animal cells, better known as hybridoma, under strictly controlled conditions so they can be studied and used in human beings. For this reason, the major goal of this project was the production of murine monoclonal antibodies using hybridoma cell culture in order to stablish an efficient culture methodology for hybridomas PC-61 or DTA1 producers of monoclonal antibodies anti-CD25 and anti-GITR, respectively, with high quality and enough amounts using Fetal Bovine Serum (FBS) free medium to, in the future, carry out animal model studies of their potential as therapeutic agents for cancer treatment. Both hybridomas were cultivated on a small scale with RPMI medium and addition of SFB, for comparative purposes and only one was selcted for the second step. The sequential adaptation methodology test, consisted in a gradual percent’s reduction of medium with serum at the same time that increase the percentage of commercial medium without serum, and was selected the medium without SFB in which the hybridoma was better adapted. After was carried out on a laboratory scale in a system type spinner flask (500 ml) with the commercial medium selected in the previous step, in controlled conditions of temperature (37 ° C) and pH (7.2). Based on analyzes of cell culture results, amino acid consumption and monoclonal antibodies quantification , SFM commercial medium SFB-free provided better results for culturing the PC-61 hybridoma, allowing the pilot scale culture reached even higher cell densities than in the standard medium with addition of FBS.
O câncer é uma das doenças mais temidas da atualidade, e atinge cada vez mais pessoas em todo o mundo. A importância de pesquisas sobre novos tratamentos na luta contra o câncer é clara e consensual, uma vez que os que vem sendo utilizados, não são muito eficientes, causam resistência à medicação utilizada o que implica na utilização de doses crescentes que por sua vez podem gerar problemas de toxicidade. Os efeitos colaterais e a instabilidade gerada no organismo do paciente, também são fatores da necessidade de pesquisar novos caminhos para o tratamento do câncer. Uma das alternativas de tratamento que tem despertado interesse é a utilização de anticorpos monoclonais (mAbs) como imunoterápicos, os quais agem estimulando o próprio sistema imune do paciente neutralizando a ação das células tumorais de forma eficiente e específica. A produção de tais anticorpos pode ser feita mediante o cultivo de células animais híbridas, mais conhecidas como hibridomas, sobre condições estritamente controladas para que possam ser estudados e utilizados em humanos. Por essa razão definiu-se como objetivo desse trabalho a produção anticorpos monoclonais murinos por meio de cultivo de hibridomas com a finalidade de estabelecer uma metodologia eficiente de cultivo dos hibridomas PC-61 ou DTA1 secretores dos mAbs anti-CD25 e antiGITR respectivamente, com qualidade e em quantidades suficientes utilizando meios livres de soro fetal bovino (SFB), para a seguir efetuar estudos em modelo animal de seu potencial como agentes terapêuticos no tratamento de câncer. Os dois hibridomas foram cultivados em pequena escala utilizando meio RPMI-1640 e adição de SFB, para fins comparativos e somente um foi selecionado para a próxima etapa. O teste da metodologia de adaptação sequencial, onde houve a redução gradativa da porcentagem de meio RPMI-1640 com 10% de SFB e o aumento da porcentagem de meio comercial sem SFB, e foi selecionado o meio livre de SFB em que o hibridoma melhor se adaptou. Posteriormente foi realizado o cultivo do hibridoma em escala laboratorial em sistema de frasco agitado biorreator do tipo Spinner (500 mL) com o meio livre de SFB selecionado na etapa anterior, sob condições bem controladas de temperatura (37ºC), pH (7,2). Com base nas análises dos resultados dos cultivos celulares, metabolismo de aminoácidos e quantificação de mAbs, o meio comercial SFM livre de SFB proporcionou melhor resultados para o cultivo do hibridoma PC-61, permitindo que o cultivo em escala laboratorial atingisse densidades celulares ainda maiores que no meio padrão com adição de SFB. Como consequência desse vasto crescimento celular em quantidades abundantes de mAbs foram conseguidas para iniciar num futuro próximo ensaios em modelos animais.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016
Lymphoma is the third most common neoplasia in the world. Within lymphomas, non-Hodgkin lymphoma (NHL) is the most common affecting mainly B cells. For more than three decades, chemotherapy and radiotherapy have been the only treatments available, but since the discovery of Rituximab, a new era of lymphoma therapy was inaugurated, once it was the first monoclonal antibody approved against the CD20 receptor. The CD20 receptor is an antigen expressed on the B-cells surface, and it is present nearly in all of its maturational development, being absent only in the stages of the pro-B lymphocyte and plasma cells. Furthermore, the CD20 receptor is also present on >90% of the B-cell NHL. These characteristics make this receptor an ideal target for immunotherapy. Besides the success of Rituximab, various mechanisms of resistance have been developed by the tumoral cells against this antibody, such as the decrease of the CD20 expression on B-cells, immunogenicity, structural changes affecting the binding region of the CD20 antibody or alterations in the cell membrane. New anti-CD20 antibodies have been developed to overcome the disadvantages of Rituximab, such as Ofatumumab and Obinutuzumab, presenting a different immunogenicity and binding to the different epitopes on the target with a different affinity. Apart from the progress in the lymphoma therapy, a significant population of patients still succumbs to this disease, as tumoral cells are always changing, making the search for better antibodies a never-ending process. Thus, the aim of this project consisted in the development and characterization of a new antibody against the CD20 receptor. To achieve this goal, the potential of rabbit derived single-domain antibodies (sdAbs) as therapeutic molecules were explored. For that, one rabbit was immunized with the CD20 receptor and an immune VH and VL sdAb library was generated. Then, a subtractive cell phage display screening was used for antibody selection. This approach allowed a specific selection of one sdAb in the VL format against the CD20 receptor in cells. In summary, the strategy explored in the present project resulted in an antibody that, according to its characteristics, could be a promising candidate in the treatment of NHL and other B-cell malignancies.
Os linfomas são a terceira neoplasia mais comum no mundo, sendo os linfomas não-Hodgkin os mais frequentes. Dentro dos linfomas não-Hodgkin, cerca de 85-90% são de células B. Os linfomas desenvolvem-se maioritariamente a partir dos nódulos linfáticos, no entanto podem formar-se a partir de qualquer outro tecido. Durante mais de três décadas, os únicos tratamentos disponíveis para esta doença eram a quimioterapia e a radioterapia. No entanto, com a descoberta do Rituximab, um anticorpo específico para o recetor CD20 presente nos linfomas de células B, começou uma nova era no campo das imunoterapias. O anticorpo Rituximab, uma vez que foi o primeiro a ser descoberto, faz parte da primeira geração de anticorpos anti-CD20. Este é um anticorpo quimérico, uma vez que é constituído por um IgG1 glicosilado composto por uma região constante kappa humana e regiões variáveis leves e pesadas murinas. Este anticorpo atua essencialmente através de três principais mecanismos de ação: citotoxicidade mediada por anticorpos (ADCC); citotoxicidade dependente do complemento (CDC) e indução da apoptose. O recetor CD20 é uma molécula expressa na superfície das células B e está presente na maioria das fases de maturação destas células, com exceção da fase de linfócitos pró-B e nas células plasmáticas. Para além disso, está presente em mais de 90% dos linfomas não-Hodgkin de células B. No entanto, apesar da sua função ainda não ser totalmente conhecida, pensa-se que possa estar envolvido no fluxo de cálcio. Este recetor é uma fosfoproteína não glicosilada com 4 domínios membranares: os domínios N e C terminal que são intracitoplasmáticos e 2 loops adicionais que são extracelulares. Devido a estas suas características, o recetor CD20 é considerado um bom alvo para imunoterapias, nomeadamente terapias para linfomas não-Hodgkin de células B. Apesar do sucesso do Rituximab, têm vindo a ser descritos vários mecanismos de resistência das células tumorais contra este anticorpo, nomeadamente a diminuição da expressão de CD20, mudanças estruturais que afetam o local de ligação do anticorpo à molécula ou alterações na membrana da célula, o que diminuiu a sua eficácia. Este aspeto fez com que fosse necessária a contínua procura por novos anticorpos para colmatar as lacunas do Rituximab. Assim, ao longo dos anos, foram descobertos novos anticorpos específicos para CD20, como o Ofatumumab e o Obinutuzumab, que têm uma diferente imunogenicidade e reconhecem diferentes epítopos na molécula CD20 aos quais se ligam com diferentes afinidades, atuando assim com diferentes mecanismos de ação. Ainda assim, este tipo de doença continua a afetar imensas pessoas, muitas vezes não existindo um tratamento eficaz devido às múltiplas resistências das células tumorais. Tendo em conta este contexto e uma vez que os anticorpos disponíveis no mercado não conseguem resolver estes problemas, a procura por novos anticorpos mais vantajosos continua. Atendendo a esta necessidade, o objetivo deste projeto é o desenvolvimento e caracterização de um anticorpo de pequeno domínio específico para CD20. Os anticorpos de pequenos domínios têm várias vantagens em relação aos IgGs completos, principalmente devido ao seu reduzido tamanho, que faz com que estes anticorpos tenham melhor acesso ao alvo na superfície da célula, para além disso podem ainda ser facilmente expressos em bactérias como proteína. Têm ainda a vantagem, como moléculas terapêuticas, de serem mais estáveis em circulação que os anticorpos completos. Estas características permitem uma administração de maiores quantidades por grama de produto, o que leva a um aumento significativo da potência por dose e na redução do custo de produção. Para o desenvolvimento do projeto, inicialmente foi imunizado um coelho com o péptido CD20 e com células HEK 293T transfetadas com o recetor CD20. Antes de cada imunização, o soro era recolhido para ser avaliado através de ELISA. Ao dia 89, quando foi atingido um elevado título de anticorpos, procedeu-se à recolha do soro final e à eutanásia do coelho. Os ensaios de ELISA permitiram confirmar que o soro extraído após as imunizações estava a reconhecer não só as células Raji, que contêm à sua superfície CD20, mas também o péptido CD20, por outro lado o soro recolhido antes das imunizações não reconhecia nem as células Raji, nem o péptido. Após a eutanásia, foram recolhidos o baço e a medula que são os órgãos onde se encontra um maior número de células plasmáticas que produzem anticorpos. A partir destes órgãos, procedeu-se à extração de ARN usando o reagente Tri. A partir do ARN extraído foi possível sintetizar a primeira cadeia de ADN. O cADN sintetizado foi utilizado para a construção de bibliotecas imunes de pequenos domínios de anticorpos específicos para o recetor CD20, através da amplificação por PCR das famílias de cadeias variáveis leves e pesadas. Esses produtos resultantes da amplificação foram clonados no vetor pComb3x, que é necessário na técnica de phage display, resultando numa biblioteca imune com uma grande diversidade, ideal para este tipo de seleção. Em seguida, as bibliotecas resultantes foram selecionadas através da tecnologia de phage display que permite a seleção de anticorpos específicos para CD20 em células que contenham esse recetor. Esta tecnologia baseia-se na engenharia genética de bacteriófagos e em várias rondas de seleção contra o antigénio e propagação dos fagos. Resumidamente, os fagos vão conter à sua superfície o fenótipo correspondente ao genótipo encapsulado no seu interior que contem a sequência correspondente aos fragmentos de ADN obtidos através da construção das bibliotecas de anticorpos. Seguem-se várias rondas de seleção onde vão sendo eliminados os anticorpos-fagos que não se ligam ou que possuem uma fraca ligação às células que contêm o antigénio, neste caso o recetor CD20, através de várias lavagens. Estas rondas vão sendo repetidas até obtermos uma população de anticorpos específica para o alvo. Para este phage display, foram utilizados 3 tipos de células: células Raji, que está descrito que expressam o CD20, pois são células de linfoma B; células HEK 293T que não expressam CD20 e por isso foram utilizadas para eliminar os anticorpos não específicos para este recetor; células Jurkat, que tal como as anteriores não possuem CD20, pois tratam-se de células T de leucemia aguda e que por isso foram também utilizadas para eliminar anticorpos não específicos. Esta seleção resultou num conjunto de anticorpos-fagos específicos para o recetor CD20, resultado que foi confirmado através de Western Blot. Após a seleção por phage display, foi necessário fazer uma seleção em larga escala para avaliar os anticorpos que melhor eram expressos e que melhor se ligavam ao alvo. Para esta avaliação da expressão foi necessário clonar o ADN resultante dos fagos selecionados por phage display, num outro vetor que permitisse a expressão de proteína, neste caso o vetor pT7-PL. Esta avaliação da ligação e expressão foi feita através de ensaios de ELISA com extrato proteico de células Raji e células Raji. Para além disso, após várias seleções os melhores 10 clones foram sequenciados para avaliar a homologia entre si e verificar os CDRs. Através do alinhamento da sequência de aminoácidos obtida a partir da sequenciação, verificou-se que 9 dos clones eram iguais, restando assim apenas 2 clones. A especificidade dos dois clones selecionados para o recetor CD20 foi avaliada através de Western Blot, sendo que um deles (anticorpo no formato VL) era específico para o recetor pretendido. Estas metodologias levaram à seleção de um anticorpo de pequeno domínio específico para CD20, que devido às suas características únicas e diferentes dos que já existem no mercado poderá ser um promissor candidato para ser usado como agente terapêutico para linfomas de células B que expressem o recetor CD20.

My study is focused on understanding the mechanisms underlying the modulation of NK cell responsiveness and plasticity induced by tumor targeting therapeutic anti-CD20 monoclonal antibodies (mAbs) nowadays routinely used in the treatment of B-cell malignancies and autoimmune disorders. Anti-CD20 mAbs are grouped into type I and II subtypes. Type I mAbs induce CD20 redistribution into lipid rafts and display a remarkable ability to activate complement-dependent cytotoxicity (CDC). On the other hand, type II mAbs, which are not able to localize CD20 complexes into lipid rafts and induce weak or no CDC, evoke more homotypic adhesion and direct killing of target cells. Both type I and II mAbs demonstrate efficient phagocytosis and antibody-dependent cytotoxicity (ADCC). Natural Killer (NK) cell-mediated ADCC, based on the recognition of IgG-opsonized targets by the low affinity Fc receptor for IgG FcgammaRIIIA/CD16, represents one of the main mechanisms by which anti-CD20 mAbs mediate their anti-tumor effects. Besides ADCC, CD16 ligation also results in the production of cytokines such as IFN-gamma that plays a key role in the shaping of adaptive immune responses. Rituximab is a chimeric type I anti-CD20 mAb of 1st generation and is considered the reference molecule for the comparison with new generation anti-CD20 mAbs, designed to optimize clinical efficacy. Among them, obinutuzumab is a humanized Fc-glycoengineered type II anti-CD20 mAb of 3rd generation designed to increase the affinity for CD16 receptor and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, I demonstrated that CD16 affinity ligation conditions may dictate both the amplitude of NK responsiveness (cytotoxicity and IFN-gamma production) as well as the ability to shift the NK functional program. Indeed, I observed that the interaction of NK cells with obinutuzumab-opsonized targets results in enhanced cytotoxicity and IFN-gamma production as compared with the parental non-glycoengineered mAb or the reference molecule rituximab, independently from the CD16-158V/F allotype. The affinity ligation conditions also strictly correlate with the ability to induce CD16 surface down-modulation and lysosomal targeting of receptor-coupled signaling elements. Indeed, a preferential degradation of FcepsilonRIgamma chain and Syk tyrosine kinase was observed upon obinutuzumab stimulation independently from the CD16-158V/F allotype. Notably, although the down-regulation of FcepsilonRIgamma/Syk module hesitates in the impairment of cytotoxic function induced by CD16, NKp46 and NKp30 activating receptors, obinutuzumab-experienced NK cells exhibit an increased ability to produce IFN-gamma in response to cytokines and target stimulation as well as to obinutuzumab-mediated CD16 re-stimulation. Relying on the observation that obinutuzumab-experienced NK cells, under molecular and functional profile, resemble the distinctive features of the long-lived and highly functional “memory” NK cells, a population recently identified in HCMV seropositive individuals, I assessed the capability of anti-CD20 mAbs to affect the expansion as well as the phenotypic and functional properties of the “memory” NK subset. My data show that the majority of the analysed healthy donors is HCMV seropositive and exhibits a detectable population of “memory” NK cells (CD3- CD56+ FcepsilonRIgamma- CD16+) accounting for 3 to 50% of peripheral blood NK cells. I observed that “memory” NK cells selectively undergo 2- to 12-fold expansion upon co-culturing with anti-CD20-opsonized targets; on the opposite, the proliferation of “conventional” NK cells (CD3- CD56+ FcepsilonRIgamma+ CD16+) is not affected by CD16 stimulation. I also noted that anti-CD20 mAb in vitro expanded “memory” NK cells show the molecular and functional hallmarks of their freshly isolated counterpart, including the increased expression of NKG2C receptor, the reduced expression of NKp46 receptor associated to an enhanced functional activity in response to CD16 re-stimulation, particularly in terms of IFN-gamma production.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
In the past few years, great discoveries and improvements have been done in drug development field and a market that was previously populated just by chemical drugs is now growing to include the so called biopharmaceuticals. As biotechnologically-derived drugs rely on the fundamental understanding of the related disease, it can be predicted that they will play a major – if not dominant – role in the drug development arena of the next decades. Following the success of recombinant proteins, therapeutic Monoclonal Antibodies (mAbs) represent the second wave of innovation created by the biotechnology industry. Overcoming the problems initially raised by these biopharmaceuticals, the recent generations of mAbs have managed to reduce some of the immunogenicity problems observed with murine mAbs. Other concerns remain, however, and the adverse events arising during the long-life experience will continue to be an important factor to monitor. The target specificity of mAbs associated to the fact that they are large protein molecules make the emergence of off-target or metabolite–related toxicities less probable, being immunogenicity and target-mediated effects the most relevant determinants of toxicity. This project focus was in the correlation and comparison of mAbs’ adverse events with their specific mechanism of action. The data here analysed comprises mainly European data (EMA) but also some United States data [1]. Due to the large diversity of mechanisms for the marketed mAbs, the analysis has been restricted to three pharmacological classes of mAbs: anti-TNFα, anti-VGEF and anti-CD20 mAbs. Adverse events were compared within each mAb class and then compared through all mAbs classes. There were antibody-related adverse events reported transversally for all mAbs but there were also some class-related adverse events which were only reported in specific classes, with specific mechanisms of action. For class-related adverse events it was observed that additional key factors – like administration routes, mAbs structural configurations and profile of patients receiving those mAbs – may also impact the adverse events and cannot be excluded in safety profile characterisation. It was confirmed that the adverse events reported for all mAbs analysed were strictly related to mAbs’ mechanism of action. Hence, characterisation of each mAb specifities together with a precise understanding of the mAbs mechanism of action is crucial for mAbs safety profile characterisation.

碩士
輔仁大學
生命科學系碩士班
96
The immune system is a collection of mechanisms within an organism that protects against disease by identifying and killing pathogens and tumor cells. However, overactive effector immune responses to autologous or allogeneic antigens can result in immune-mediated diseases such as hypersensitivity and autoimmune diseases. One of the therapeutic objectives in autoimmune diseases is blocking of activation, proliferation and cytokine production in T lymphocytes. There are immunosuppressive drugs used for autoimmune diseases, but they almost accompany with serious side effects. Therefore, we attempted to identify other immunomodulators from natural products that could be as an adjuvant treatment for autoimmune diseases. The previous data indicated that the natural products kaempferol 3-O-a-L-[2,4-di-(E)-p-coumaroyl] rhamnopyranoside (C39H32O14; MW=724; K3), arctigenin (C21H24O6; MW=372; AC), and (±)-3,9-dihydroeucomin (C17H16O5; MW=300; DC) isolated from Cinnamomum kotoense, Arctium lappa and Agave sisalana, respectively, inhibited human peripheral blood mononuclear cells (PBMC) proliferation stimulated by phytohemagglutinin (PHA). In the present study, human T lymphocytes were purified from PBMC and used as target cells. The regulation of K3, AC, and DC in human T lymphocytes immune responses induced by anti-CD3 and anti-CD28 antibodies (anti-CD3/CD28 Abs) was studied. The results indicated that (1) Primary human T lymphocytes has been isolated from PBMC by nylon wool method and their purities are about 80%. (2) Both IL-2 and IFN-r mRNA expression and protein production could be detected in T lymphocytes induced by anti-CD3/CD28 Abs at 2 hr and 24 hr postactivtion, respectively. The cell proliferation in T lymphocytes could be detected at 72 hr postactivation. (4) Both IL-2 and IFN-r gene expression in T lymphocytes activated anti-CD3/CD28 Abs were regulated by ERK, P38, NF-kB, and NF-AT signaling pathways. (5) All K3, AC and DC reduced IL-2 and IFN-rmRNA transcripts and protein production and cell proliferation in T cells induced by anti-CD3 and anti-CD28 antibodies without cytotoxicity. (6) Both K3 and AC did not affect ERK and P38 activation in T lymphocytes stimulated with anti-CD3/CD28 Abs. DC decreased P38 phosphorylation in activated T lymphocytes. (7) The preliminary data from luciferase reporter assay showed that K3 and AC decreased NF-AT activation. (8) The results from electrophoresis mobility shift assay (EMSA) demonstrated that K3 impaired NF-AT and NF-kB DNA binding activities. We suggested that K3 suppressed IL-2 and IFN-r gene expression in T lymphocytes induced by anti-CD3/CD28 Abs related to reduction of NF-AT and NF-B activity. Furthermore, the detailed action mechanisms behind the differential effects of K3, AC, and DC on human T lymphocytes will be elucidated.