Thèses sur le sujet « Anti-CD20 monoclonal antibodie »

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.

Linfomas são doenças originárias do sistema linfático, descritos por Thomas Hodgkin em 1932. São tradicionalmente classificados em dois grupos básicos: doenças de Hodgkin e linfomas do tipo não hodgkin (LNH). Inicialmente, pacientes com LNH foram tratados com radioterapia apenas ou combinada com imunoterapia usando-se anticorpo monoclonal anti-CD20 (ex., Rituximab-Mabthera, Roche). Porém, radioimunoterapia é uma nova modalidade de tratamento para pacientes portadores de LNH, na qual radiação citotóxica proveniente de radioisótopos terapêuticos é depositada nos tumores via anticorpos monoclonais (Acms). Este estudo concentrou-se nas condições de marcação do Acm anti-CD20 (Rituximab-Mabthera, Roche), com radioiodo (131I), pelo método direto, usando-se Cloramina-T como agente oxidante. Foram estudados parâmetros de marcação tais como: método de purificação, influência de tempo de incubação, influência de massa de agente oxidante, estabilidade in vitro e in vivo, imunoreatividade do anticorpo e distribuição biológica do anticorpo marcado em camundongos Swiss sadios. Produto de alta pureza radioquímica foi obtido, sem diferença notável entre os métodos aplicados. Não foi observada nenhuma influência direta do tempo de incubação na pureza radioquímica do anticorpo marcado. Um pequeno decréscimo na pureza radioquímica foi observado quando variou-se a massa do Acm sem variar a atividade do radioiodo. Após purificação, o anticorpo marcado apresentou pureza radioquímica de aproximadamente 100 %. Observou-se um produto de alta pureza radioquímica quando o anticorpo foi marcado na condição padrão. O anticorpo anti-CD20 apresentou variações de pureza radioquímica quando sua estabilidade foi testada em cinco condições estabilizadoras diferentes. Entretanto, a condição em que ácido gentísico foi combinado com congelamento demonstrou-se apropriada e capaz de minimizar os efeitos de autoradiólise do anticorpo marcado com alta atividade terapêutica de iodo-131. O anticorpo marcado apresentou imunoreatividade abaixo da relatada na literatura. A distribuição biológica em camundongos Swiss sadios revelou captação elevada no pulmão, fígado, e intestino delgado. O clareamento sanguíneo do anticorpo marcado foi relativamente rápido. Contudo, dados experimentais revelaram que anticorpos monoclonais anti-CD20 podem ser seguramente marcados com alta atividade de iodo-131 usando o método de Cloramina-T. O uso de cromatografia em camada delgada (ITLC-SG) na avaliação de pureza radioquímica mostrou-se apropriado, eficiente, prático, além de simples e rápido. O método de purificação utilizado demonstrou-se eficiente para a separação do anticorpo marcado do iodo livre. A abordagem inédita apresentada neste estudo, no sentido de viabilizar transporte e comercialização do produto marcado, referiu-se ao estudo de estabilidade do Acm marcado. A distribuição biológica do anticorpo demonstrou-se compatível com a distribuição do anticorpo íntegro indicando ótima estabilidade relativa in vivo. Os resultados deste estudo confirmam a potencialidade do anticorpo para a radioimunoterapia de linfomas do tipo não-Hodgkin.
Lymphomas are malignancies of the lymphatic system, described by Thomas Hodgkin in 1932. Traditionally, lymphomas are classified in two basic groups: Hodgkin disease and non-Hodgkin lymphoma (NHL). Patients with NHL were earlier treated with radiotherapy alone or in combination with immunotherapy using monoclonal antibody anti-CD20 (ex., Rituximab-Mabthera, Roche). However, Radioimmunotherapy is a new modality of treatment for patients with NHL, in which cytotoxic radiation from therapeutic radioisotopes is delivered to tumors through monoclonal antibodies. This study focused on labeling conditions of monoclonal antibody anti-CD20 (Rituximab-Mabthera, Roche) with iodine-131, by direct radioiodination method using Chloramine-T as oxidizing agent. Labeling parameters investigated were: Radiochemical purity (RP), method of purification, incubation time, antibody mass, oxidative agent mass, stability in vitro, stability in vivo, immunoreactivity and biological distribution performed in normal Swiss mouse. Product of high radiochemical purity was obtained with no notable difference between the methods applied. No clear evidence of direct influence of incubation time on radiochemical purity of the labeled antibody was observed. Whereas, a clear evidence of direct influence of activity on radiochemical purity of the labeled antibody was observed when antibody mass was varied. After purification, the labeled product presented radiochemical purity of approximately 100 %. Product of superior radiochemical yield was observed when standard condition of labeling was used. The labeled product presented variation in radiochemical purity using five different stabilizer conditions. The condition in which gentisic acid was combined with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with high therapeutic activity of iodine-131. The labeled product presented low immunoreactivity when compared to the literature. Biological distribution in normal Swiss mouse demonstrated high uptake of the labeled antibody in lungs, liver, and small intestine. The progressive loss of activity in blood indicates fast blood clearance of the labeled antibody that is eliminated through the kidney, in urine. The experimental data proved that mAb anti-CD20 can be securely labeled with high therapeutic activity of iodine-131 using Chloramine-T method. Radiochemical purity determined by chromatographic plates (ITLC-SG) proved to be appropriate, efficient, practical and simple. The purification method demonstrated to be appropriate and efficient for separating the labeled antibody from free iodine. The results of stability of the labeled antibody presented in this study suggest that the product can be transported and commercialized using the condition in which gentisic acid was combined with freeze. In vivo distribution of the labeled antibody shows to be compatible with integral antibody distribution, indicating good in vivo stability. Results obtained in this study confirmed the potential of the labeled product anti-CD20-131I for radioimmunotherapy of non-Hodgkin lymphoma (NHL).

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Les anticorps monoclonaux (AcM) anti-CD20 sont essentiels pour le traitement du lymphome non hodgkinien et de la leucémie lymphoïde chronique (LLC). Les AcM agissent soit en activant directement la signalisation apoptotique dans les cellules cibles, soit via le système immunitaire. Dans une étude préclinique, nous avons montré que le traitement avec AcM anti-CD20, rituximab et GA101, induit l'expression de la protéine early growth response 1 (EGR-1) (Dalle et al., 2011). EGR-1 est un facteur de transcription régulé par le calcium (Ca2+) et CD20 est impliqué dans la régulation du flux calcique transmembranaire. Nous avons donc étudié le rôle d'EGR-1 et du flux Ca2+ dans l'activité cytotoxique des AcM anti-CD20. Nous avons montré qu'EGR-1 est rapidement induit suite à l'exposition au rituximab et à GA101. La baisse de l'expression d'EGR-1 par shRNA a supprimé l'effet cytotoxique du GA101 à la fois in vitro et in vivo, indiquant qu'EGR-1 est requis pour la mort cellulaire médiée par CD20. De plus, la surexpression d'EGR-1 augmente la sensibilité au GA101 in vitro et in vivo. En outre, nos résultats indiquent que les AcM anti-CD20 induisent un flux Ca2+. Le blocage du flux Ca2+ par inhibiteurs de canaux calciques (ICC) a aboli l'induction d'EGR-1 ainsi que l'efficacité du GA101 in vivo et ex vivo dans des échantillons de LLC. Plus important, nos données indiquent que les patients recevant des ICC ont une moins bonne réponse au traitement par les AcM anti-CD20. En conclusion, nous avons identifié EGR-1 comme potentiel biomarqueur pour prédire la réponse à la thérapie anti-CD20 et démontré que les ICC ont un impact négatif sur l'efficacité des AcM anti-CD20 chez les patients
Anti-CD20 monoclonal antibodies (mAbs) are an essential component of the treatment of patients with non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). They mediate their antitumor effects by activating the immune system or by direct apoptotic signaling in target cells. In a previous preclinical study, we showed that treatment with anti-CD20 mAbs, rituximab and GA101, resulted in upregulated expression of early growth factor 1 (EGR-1) (Dalle et al. 2011). EGR-1 is a calcium (Ca2+) regulated transcription factor and CD20 is hypothesized to regulate transmembrane Ca2+ flux. Therefore, we aimed to assess the role of EGR-1 and Ca2+ flux in the cytotoxic activity of anti-CD20 mAbs. We have shown that EGR-1 expression is rapidly upregulated in CD20+ cells following rituximab and GA101 exposure. Decreasing EGR-1 expression by shRNA abolishes the direct cytotoxic effect of GA101 both in vitro and in vivo, indicating that EGR-1 is required for CD20-mediated cell death. Additionally, the overexpression of EGR-1 enhances the cytotoxic activity of GA101 both in vitro and in vivo. Furthermore, our results indicate that anti-CD20 mAbs induce calcium influx. Blocking the Ca2+ flux with calcium channel blockers (CCB) abolishes EGR-1 induction and impaires the GA101 efficacy in vivo and ex vivo in CLL blood samples. More importantly, our data indicate that patients receiving CCBs and anti-CD20 therapy have worst progression free survival and overall survival. In conclusion we have identified EGR-1 as a potential biomarker to predict response to anti-CD20 therapy. We demonstrated that co-treatement with CCBs negatively impacts the outcome of patients receiving anti-CD20 mAbs

Research into cancer therapeutics has utilized the antigen specificity of antibodies to provide targeted cancer treatments. One notable therapeutic is rituximab, a chimeric human IgG1 antibody that has specificity to the CD20 protein on the surface of B cells. This antibody is currently used in the treatment of leukaemia and lymphoma. However, due to reasons not fully understood patients often relapse and become resistant to therapy. Therefore an increasingly important area of research is to uncover the key mechanisms of action of anti-CD20 antibodies. It has been demonstrated that antibody Fc: FcγR interactions are critical foreffective anti-CD20 antibody dependent cellular cytotoxicity (ADCC). During this project the role of Fc: FcγR interactions and their ability to mediate anti-CD20 mediated B cell depletion was explored. We assessed two different types of anti-CD20 antibody: type I, rituximab has been shown to have high levels of antibody:antigen internalisation following CD20 engagement. This process has been termed antigenic modulation. In contrast, type II, obinutuzumab do not display a high level of antigenic modulation. We assessed the ability of rituximab and obinutuzumab to deplete normal and malignant B cells. Additionally, macrophages and NK cells have both been implicated as key FcγRexpressing effector cells. Therefore, we assessed rituximab and obinutuzumab using in vitro macrophage or NK cell mediated ADCC assays and looked at the impact antigenic modulation had on effector cell activity. In vivo analysis revealed that obinutuzumab give more sustained B cell depletion compared to rituximab. Furthermore, we demonstrate that this sustained depletion is due to a lack of antigenic modulation preventing NK cell and macrophage mediated ADCC. In contrast rituximab undergoes significant internalisation preventing both NK cell and macrophage mediated ADCC. In addition, it has been well documented that the tumour microenvironment will affect the phenotype of the surrounding stroma, particularly macrophages. Established tumours are typically associated with low levels of apoptosis. Therefore, we investigated the impact dysregulated apoptosis had on the ability of macrophages to orchestrate ADCC. Using apoptosis resistant Vav Bcl-2 mice, as well as other apoptosis defective strains, we demonstrate that there is a decrease in the activatory to inhibitory FcγR ratio on effector cell subsets. This was largely due to an increase in inhibitory FcγRIIb. Subsequent analysisrevealed that an increase in FcγRIIb may be due to the autoimmune-likephenotype of the Vav Bcl-2 mouse. This knowledge will be beneficial to future consideration of antibody selection and provides further avenues of research into antibody and FcγR interactions.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia of the Western world, and unfortunately despite advances in chemotherapeutic agents CLL remains incurable to date. CLL is a highly heterogeneous disease. CLL patients exhibiting progressive disease often become refractory to standard first line therapies, underpinning the need for novel drugs. An emerging field is targeted monoclonal antibody (MAb) development. Rituximab (RTX) a chimeric anti-CD20 MAb is registered as a first line therapy in combination with fludarabine and cyclophosphamide. A next generation type I, human IgG1 anti-CD20 MAb, ofatumumab (OFA), binds to a novel epitope of CD20 resulting in higher potency of complement dependent cytotoxicity (CDC) induction. OFA has recently been given FDA approval for treatment in combination with chlorambucil for previously untreated CLL patients when fludarabine based regimes are not appropriate. OFA is also licensed as a single agent for the treatment of CLL patients refractory to both fludarbine and anti-CD52 MAb alemtuzumab. This research was conducted to establish what factors within CLL biology may influence RTX or OFA efficacy, in order to highlight the improved activity of OFA in comparison to RTX for the treatment of CLL. The ability of OFA to elicit a CDC response is an important part of its efficacy. Within this study we have demonstrated that OFA activity is limited in the treatment of CLL by several different factors including; complement levels, CD20 expression, microenvironmental stimulation and CLL genetic mutations. Our study identified that a large proportion, 37.5%, of CLL patients harbour complement deficiencies within their sera, this then led to a significant amount of complement exhaustion with successive OFA treatments, abrogating CDC induction. The detection of excessive complement exhaustion potentially has a clinical impact on the administration of anti-CD20 MAbs. CD20 expression levels also impact upon CDC induction by OFA, CD20 expression levels display a positive linear correlation with the percentage of CDC induction. Importantly OFA is superior in the induction of CDC in CLL patients with low CD20 expression levels in comparison to RTX. It is well established that CLL patients have lower CD20 expression compared to other B cell malignancies, which is an important factor for anti-CD20 MAb efficacy as CDC induction is critically dependent on the expression levels of the target antigen on the surface of the cell. Recurrent mutations within NOTCH1 (NOTCH1MUT) have recently been identified as a poor prognostic marker within CLL. Emerging data suggests this mutation confers resistance against anti-CD20 MAbs, possibly through a down-regulation of CD20. We demonstrate that NOTCH1MUT CLL cells do display reduced expression levels of CD20, however this alone did not appear to contribute to all the reduced susceptibility towards anti-CD20 MAb activity. Preliminary gene expression data in combination with Ca2+ flux analysis suggest that NOTCH1MUT CLL cells have deregulated Ca2+ signalling which highlights the role of CD20 as a store-operated Ca2+ channel, and provides another mechanism for their resistance. Microenvironmental stimulation also impacts upon CLL cell response to RTX and OFA. Mimicking CLL cell interaction with their microenvironmental niche, caused an increase in CDC induction within different CLL cytogenetic subsets, independently to changes in CD20 expression. This is important as the interaction of CLL cells with other components of the microenvironment cause an up-regulation of anti-apoptotic proteins aiding CLL survival and promoting drug resistance. In conclusion this study highlights the increased efficacy of OFA as a chemotherapeutic agent in the treatment of CLL in comparison to RTX. Collectively these results also demonstrate that a change within the dosing schedule of OFA and complement supplementation using an exogenous source such as fresh frozen plasma could diminish some of the limitations in CDC induction, and potentially lead to enhanced clinical efficacy.

CD20 est une cible thérapeutique validée pour l’immunothérapie des néoplasmes lymphoïdesdes cellules B, incluant la Leucémie Lymphoïde Chronique (LLC). Nous avons comparé les effets de rituximab et de GA101 (nouvel anticorps anti-CD20) contre les cellules LLC fraiches in vitro. Le marquage avec Annexine V a démontré une induction de l’apoptose après l’exposition au rituximab et GA101.Contrairement au rituximab, GA101 induisait une réduction du potentiel transmembranaire mitochondrial, uneffet qui peut être partiellement inhibé par la cyclosporine A et qui est partiellement caspase-dépendant. GA101induisait aussi la production des espèces d’oxygènes réactives. L’analyse du niveau d’expression des protéinespro- et anti-apoptotiques après exposition aux anticorps a démontré une forte hétérogénéité entre les échantillons.Bax subissait une activation de conformation et une translocation mitochondriale suite à l’exposition aux anticorps d’une manière caspase-indépendante. GA101, mais pas rituximab, induisait le clivage des caspase-8, -9et -3. En transfectant les cellules LLC avec un siRNA ciblant Bcl-xL utilisant la sonoporation, nous avons trouvéque la réduction du niveau d’expression de Bcl-xL est associée à une augmentation de la sensibilité aux anticorps. Nos résultats suggèrent que les voies de signalisation apoptotiques diffèrent entre rituximab et GA101avec une implication de la voie mitochondriale avec le GA101. L’inhibition de Bcl-xL peut constituer une façon pour sensibiliser les cellules LLC aux effets apoptotiques des anticorps anti-CD20
CD20 is a validated target for the immunotherapy of B lymphoid neoplasms, including ChronicLymphocytic Leukemia (CLL). We compared the activities of rituximab and GA101 (novel anti-CD20 antibody)on fresh human CLL cells in vitro. AnnexinV staining demonstrated induction of apoptosis after exposure torituximab or GA101. Unlike rituximab, GA101 induced a reduction of the mitochondrial transmembranepotential, an effect which could be partially inhibited by cyclosporin A and which was partially caspasedependent.GA101 was also found to induce the production of Reactive Oxygen Species. Analysis of pro- andanti-apoptotic protein content after exposure to antibodies demonstrated a strong degree of heterogeneity between samples. Bax underwent conformational activation and mitochondrial translocation upon exposure toantibodies in a caspase-independent manner. GA101 but not rituximab induced cleavage of caspase-8, -9 and -3.By transfecting CLL cells with anti-Bcl-xL siRNA using a sonoporation method, we found that reduction of BclxLcontent was associated with increased sensitivity to these antibodies. Our results suggest that apoptoticsignalization pathways differ between rituximab and GA101 with a greater involvement of the mitochondrialpathway for GA101. Inhibition of Bcl-xL could constitute an approach to sensitize CLL cells to the apoptoticeffects of anti-CD20 antibodies

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Linfomas são cânceres que se iniciam a partir da transformação maligna de um linfócito no sistema linfático. Os linfomas são divididos em duas categorias principais: os linfomas de Hodgkin e todos os outros linfomas, denominados linfomas não-Hodgkin (LNH). Os pacientes com LNH são comumente tratados com radioterapia apenas ou combinada com quimioterapia utilizando-se de anticorpo monoclonal anti-CD20, principalmente o rituximab (MabThera®). O uso de anticorpos monoclonais (Acm) conjugados à quelantes bifuncionais radiomarcados com radionuclídeos metálicos ou lantanídeos é uma realidade de tratamento para portadores de LNH pelo princípio de radioimunoterapia (RIT). Este estudo concentrou-se nas condições de conjugação do anticorpo monoclonal rituximab (MabThera®) com grupamentos quelantes bifuncionais DOTA e DTPA. Na marcação dos Acm conjugados com lutécio-177, foram estudadas as condições de pré-purificação do Acm, condições de conjugação, determinação de número de quelantes acoplados à molécula do anticorpo, purificação do anticorpo conjugado, radiomarcação do anticorpo conjugado, com lutécio-177, purificação do anticorpo marcado, a ligação específica in vitro dos compostos marcados às células Raji, e distribuição biológica em camundongos BALB/c sadios. As três metodologias empregadas na pré-purificação do anticorpo (diálise, cromatografia de exclusão molecular com coluna Sephadex G-50 e ultrafiltração) demonstram-se eficientes e proporcionaram recuperação da amostra superior a 90%. A metodologia de ultrafiltração foi considerada a mais simples e prática, podendo ser aplicada a procedimentos rotineiros de produção de radiofármacos. Além disso, proporcionou a recuperação final de amostra de 97% em microlitros. Nas conjugações do anticorpo com os quelantes DOTA e DTPA em razões molares diferentes do Acm:quelante, observou-se número de grupamentos quelantes acoplados à molécula do Acm proporcional à razão molar estudada. Quando foi avaliada a influência de condições diferentes de conjugação no número de quelantes acoplados à molécula do Acm, não foram observadas diferenças significativas, com resultados de pureza radioquímica (PR) inferior a 80% em todas as condições estudadas. Na comparação de métodos de purificação do Acm conjugado, a abordagem inédita apresentada neste estudo, na qual a cromatografia de exclusão molecular foi combinada com a ultrafiltração resultou em maior eficiência na purificação e preservação da estrutura do anticorpo. Nos estudos de radiomarcação do anticorpo conjugado com DOTA e DTPA, os imunoconjugados de DTPA apresentaram, de forma geral, maior eficiência de marcação com resultados reprodutíveis quando comparados com os imunoconjugados de DOTA, considerando-se as diferentes razões molares utilizadas. As metodologias cromatográficas empregadas no controle de pureza radioquímica do composto radiomarcado proporcionaram a discriminação das diferentes espécies radioquímicas no meio de marcação. A metodologia de purificação do composto conjugado e radiomarcado utilizada proporcionou a obtenção de compostos com alta pureza radioquímica, 97,4±1,3% (DOTA 1:50) e 98,7±0,2% (DTPA 1:50). Nos estudos de ligação específica às células tumorais Raji, o anticorpo conjugado com quelante DTPA nas razões molares de 1:50 e 1:20 apresentaram perfil semelhante de ligação, com aumento da porcentagem de ligação específica proporcional à concentração celular, enquanto que o imunoconjugado na razão molar de 1:10 apresentou alta porcentagem de ligação não específica. Os resultados obtidos nos estudos de biodistribuição in vivo do anticorpo conjugado e radiomarcado nem sempre se mostraram compatíveis com a biodistribuição de anticorpos radiomarcados íntegros. No caso do quelante DOTA, o imunoconjugado obtido a partir da razão molar 1:20, apresentou melhores características de biodistribuição. No caso do quelante DTPA, a razão molar utilizada pareceu refletir diretamente no clareamento sanguíneo do anticorpo e todas as razões molares utilizadas apresentaram instabilidade in vivo.
Lymphomas are malignancies or cancers that start from the malign transformation of a lymphocyte in the lymphatic system. Lymphomas are divided in two major categories: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Patient with NHL are generally treated with radiotherapy alone or combined with immunotherapy using monoclonal antibody rituximab (MabThera®). Currently, monoclonal antibodies (Mab) conjugated with bifunctional chelate agents and radiolabeled with metallic or lanthanides radionuclides are a treatment reality for patients with NHL by the principle of radioimmunotherapy (RIT). This study focused on the conditions of conjugation of Acm rituximab (MabThera®) with bifunctional chelating agents DOTA and DTPA and labeling with 177-luthetium. Various parameters were studied: method of Acm purification, conditions of Acm conjugation and the determination of the number of chelate coupled to the Acm, the purification of the conjugated Mab, labeling conditions with lutetium-177, purification of the radiolabeled immunoconjugate, radiochemical purity (RP), in vitro specific binding determination to Raji cells (Human Burkitt) and biological distribution performed in normal BALB/c mouse. The three methodologies employed in pre-purification of Acm (dialysis, size exclusion chromatograph and ultrafiltration) demonstrated to be efficient; they provided sample recovery exceeding 90%. However, the methodology of ultrafiltration resulted in greater sample recovery and in microliters. The number of chelate attached to the Mab molecule was proportional to the molar ratio studied. When the influence of different conditions of conjugation in the number of chelate bounded to the Mab was studied, no notable differences were observed. The RP < 80% was observed in all the methods applied. Purification of the conjugated antibody by different methods showed that the innovative combination of Sephadex and ultrafiltration methods resulted in higher efficiency of purification. The optimized conditions for purification of the conjugated antibody preserved the protein integrity. Radiolabelling studies of DOTA and DTPA immunoconjugated showed that DTPA derivatives presented, in general, radiochemical yield superior than DOTA conjugated Mab, considering the different molar ratios studied. The chromatographic methods employed in the RP determination were efficient to separate the different radiochemical species presented in the reaction medium. The methodology used in the purification of the labeled Mab resulted in labeled compounds with high radiochemical purity, 97.4±1.3% (DOTA 1:50) and 98.7±0.2% (DTPA 1:50). Considering specific cell binding assays (Raji cells), the Mab conjugated to DTPA at 1:50 and 1:20 molar ratios presented similar results, and the percent of cell binding were proportional to the cell concentration, whereas the cell binding for 1:10 molar ratio showed high percent of nonspecific cell binding. The results of in vivo biodistribution studies of labeled Mab not always were compatible with the biodistribution of intact radiolabelled antibody. The DOTA immunoconjugated produced at 1:20 molar ratio, showed better performance in biodistribution studies. In the case of DTPA immunoconjugated, the blood clearance seems to be influenced by the molar ratio applied and the immunoconjugated produced with DTPA chelate at different molar ratio resulted in high in vivo instability compounds.

Background: Epstein-Barr virus(EBV)-associated posttransplant lymphoproliferative disease (PTLD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). Especially in cases with involvement of the central nervous system (CNS) treatment is difficult because the efficacy of most chemotherapeutic agents as well as EBV-specific cytotoxic donor T cells in liquor is uncertain. In the last years the anti-CD20 monoclonal antibody Rituximab was intensively investigated in the treatment of EBV-PTLD. However, only 8 patients with B-cell lymphoma and CNS involvement treated with Rituximab were reported. Case Report: A 24-year-old female patient with acute T-lymphoblastic leukemia in second complete remission had received allogeneic, unrelated, T-cell depleted HSCT. 10 months later an EBV-associated PTLD was diagnosed. Beside peripheral lymphomas and B symptoms the patient showed neurological symptoms. Examination of the cerebrospinal fluid (CSF) revealed a meningeosis lymphoblastica caused by the EBV lymphoma. Treatment with Rituximab and the antiviral drug Cidofovir led to complete remission with regression of the peripheral lymphomas and disappearance of the neurological symptoms. In addition, the PCR control on EBV DNA became negative in the plasma as well as in CSF. Conclusion: The combination of Rituximab and Cidofovir appears as an interesting alternative treatment in patients with EBV-associated PTLD and CNS involvement
Hintergrund: Die Epstein-Barr-Virus(EBV)-assoziierte Posttransplantations-lymphoproliferative Disease (PTLD) ist eine gefürchtete Komplikation nach allogener hämatopoetischer Stammzelltransplantation (HSCT). Insbesondere bei Befall des zentralen Nervensystems (ZNS) ist die Behandlung auf Grund der unsicheren Liquorwirksamkeit der meisten Chemotherapeutika als auch von EBV-spezifischen zytotoxischen T-Spenderzellen schwierig. Der monoklonale Anti-CD20-Antikörper Rituximab wurde in den letzten Jahren bei Patienten mit EBV-PTLD intensiv untersucht. Allerdings wurde bislang lediglich von 8 Patienten mit ZNS-Befall eines B-Zell-Lymphoms berichtet, bei denen eine Therapie mit Rituximab erfolgte. Kasuistik: Eine 24-jährige Patientin hatte wegen einer akuten T-lymphoblastischen Leukämie in zweiter kompletter Remission eine allogen-unverwandte, T-Zelldepletierte HSCT erhalten. 10 Monate später wurde eine EBV-assoziierte PTLD diagnostiziert. Neben peripheren Lymphomen und B-Symptomen zeigte die Patientin neurologische Symptome. Die Liquoruntersuchung erbrachte den Befund einer Meningeosis lymphoblastica im Rahmen des EBV-Lymphoms. Die Behandlung mit Rituximab und dem Virustatikum Cidofovir führte zu einer kompletten Remission mit Rückbildung der peripheren Lymphome und Verschwinden der neurologischen Symptomatik. Außerdem wurde die PCR-Kontrolle auf EBV-DNA sowohl im Plasma als auch im Liquor negativ. Schlussfolgerung: Die Kombination von Rituximab und Cidofovir erscheint als eine interessante Therapiealternative für Patienten mit EBV-assoziierter PTLD und ZNS-Befall
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Background: Epstein-Barr virus(EBV)-associated posttransplant lymphoproliferative disease (PTLD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). Especially in cases with involvement of the central nervous system (CNS) treatment is difficult because the efficacy of most chemotherapeutic agents as well as EBV-specific cytotoxic donor T cells in liquor is uncertain. In the last years the anti-CD20 monoclonal antibody Rituximab was intensively investigated in the treatment of EBV-PTLD. However, only 8 patients with B-cell lymphoma and CNS involvement treated with Rituximab were reported. Case Report: A 24-year-old female patient with acute T-lymphoblastic leukemia in second complete remission had received allogeneic, unrelated, T-cell depleted HSCT. 10 months later an EBV-associated PTLD was diagnosed. Beside peripheral lymphomas and B symptoms the patient showed neurological symptoms. Examination of the cerebrospinal fluid (CSF) revealed a meningeosis lymphoblastica caused by the EBV lymphoma. Treatment with Rituximab and the antiviral drug Cidofovir led to complete remission with regression of the peripheral lymphomas and disappearance of the neurological symptoms. In addition, the PCR control on EBV DNA became negative in the plasma as well as in CSF. Conclusion: The combination of Rituximab and Cidofovir appears as an interesting alternative treatment in patients with EBV-associated PTLD and CNS involvement.
Hintergrund: Die Epstein-Barr-Virus(EBV)-assoziierte Posttransplantations-lymphoproliferative Disease (PTLD) ist eine gefürchtete Komplikation nach allogener hämatopoetischer Stammzelltransplantation (HSCT). Insbesondere bei Befall des zentralen Nervensystems (ZNS) ist die Behandlung auf Grund der unsicheren Liquorwirksamkeit der meisten Chemotherapeutika als auch von EBV-spezifischen zytotoxischen T-Spenderzellen schwierig. Der monoklonale Anti-CD20-Antikörper Rituximab wurde in den letzten Jahren bei Patienten mit EBV-PTLD intensiv untersucht. Allerdings wurde bislang lediglich von 8 Patienten mit ZNS-Befall eines B-Zell-Lymphoms berichtet, bei denen eine Therapie mit Rituximab erfolgte. Kasuistik: Eine 24-jährige Patientin hatte wegen einer akuten T-lymphoblastischen Leukämie in zweiter kompletter Remission eine allogen-unverwandte, T-Zelldepletierte HSCT erhalten. 10 Monate später wurde eine EBV-assoziierte PTLD diagnostiziert. Neben peripheren Lymphomen und B-Symptomen zeigte die Patientin neurologische Symptome. Die Liquoruntersuchung erbrachte den Befund einer Meningeosis lymphoblastica im Rahmen des EBV-Lymphoms. Die Behandlung mit Rituximab und dem Virustatikum Cidofovir führte zu einer kompletten Remission mit Rückbildung der peripheren Lymphome und Verschwinden der neurologischen Symptomatik. Außerdem wurde die PCR-Kontrolle auf EBV-DNA sowohl im Plasma als auch im Liquor negativ. Schlussfolgerung: Die Kombination von Rituximab und Cidofovir erscheint als eine interessante Therapiealternative für Patienten mit EBV-assoziierter PTLD und ZNS-Befall.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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Linfomas são cânceres que se iniciam a partir da transformação maligna de um linfócito no sistema linfático. Os linfomas são divididos em duas categorias principais: os linfomas de Hodgkin e todos os outros linfomas, denominados linfomas não-Hodgkin (LNH). Os pacientes com LNH são comumente tratados com radioterapia apenas ou combinada com quimioterapia utilizando-se de anticorpo monoclonal anti-CD20, principalmente o rituximab (MabThera®). O uso de anticorpos monoclonais (Acm) conjugados à quelantes bifuncionais radiomarcados com radionuclídeos metálicos ou lantanídeos é uma realidade de tratamento para portadores de LNH pelo princípio de radioimunoterapia (RIT). Este estudo concentrou-se nas condições de conjugação do anticorpo monoclonal rituximab (MabThera®) com grupamentos quelantes bifuncionais DOTA e DTPA. Na marcação dos Acm conjugados com lutécio-177, foram estudadas as condições de pré-purificação do Acm, condições de conjugação, determinação de número de quelantes acoplados à molécula do anticorpo, purificação do anticorpo conjugado, radiomarcação do anticorpo conjugado, com lutécio-177, purificação do anticorpo marcado, a ligação específica in vitro dos compostos marcados às células Raji, e distribuição biológica em camundongos BALB/c sadios. As três metodologias empregadas na pré-purificação do anticorpo (diálise, cromatografia de exclusão molecular com coluna Sephadex G-50 e ultrafiltração) demonstram-se eficientes e proporcionaram recuperação da amostra superior a 90%. A metodologia de ultrafiltração foi considerada a mais simples e prática, podendo ser aplicada a procedimentos rotineiros de produção de radiofármacos. Além disso, proporcionou a recuperação final de amostra de 97% em microlitros. Nas conjugações do anticorpo com os quelantes DOTA e DTPA em razões molares diferentes do Acm:quelante, observou-se número de grupamentos quelantes acoplados à molécula do Acm proporcional à razão molar estudada. Quando foi avaliada a influência de condições diferentes de conjugação no número de quelantes acoplados à molécula do Acm, não foram observadas diferenças significativas, com resultados de pureza radioquímica (PR) inferior a 80% em todas as condições estudadas. Na comparação de métodos de purificação do Acm conjugado, a abordagem inédita apresentada neste estudo, na qual a cromatografia de exclusão molecular foi combinada com a ultrafiltração resultou em maior eficiência na purificação e preservação da estrutura do anticorpo. Nos estudos de radiomarcação do anticorpo conjugado com DOTA e DTPA, os imunoconjugados de DTPA apresentaram, de forma geral, maior eficiência de marcação com resultados reprodutíveis quando comparados com os imunoconjugados de DOTA, considerando-se as diferentes razões molares utilizadas. As metodologias cromatográficas empregadas no controle de pureza radioquímica do composto radiomarcado proporcionaram a discriminação das diferentes espécies radioquímicas no meio de marcação. A metodologia de purificação do composto conjugado e radiomarcado utilizada proporcionou a obtenção de compostos com alta pureza radioquímica, 97,4±1,3% (DOTA 1:50) e 98,7±0,2% (DTPA 1:50). Nos estudos de ligação específica às células tumorais Raji, o anticorpo conjugado com quelante DTPA nas razões molares de 1:50 e 1:20 apresentaram perfil semelhante de ligação, com aumento da porcentagem de ligação específica proporcional à concentração celular, enquanto que o imunoconjugado na razão molar de 1:10 apresentou alta porcentagem de ligação não específica. Os resultados obtidos nos estudos de biodistribuição in vivo do anticorpo conjugado e radiomarcado nem sempre se mostraram compatíveis com a biodistribuição de anticorpos radiomarcados íntegros. No caso do quelante DOTA, o imunoconjugado obtido a partir da razão molar 1:20, apresentou melhores características de biodistribuição. No caso do quelante DTPA, a razão molar utilizada pareceu refletir diretamente no clareamento sanguíneo do anticorpo e todas as razões molares utilizadas apresentaram instabilidade in vivo.
Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

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.