Linfomas são cânceres provenientes da transformação de um linfócito no sistema linfático, sendo que, o mais comum é o Linfoma Não-Hodgkin (LNH). Avanços na imunologia e na biologia molecular têm auxiliado na detecção desses tumores e aberto caminhos para novas estratégias de tratamento, como a Radioimunoterapia. O rituximab é um anticorpo monoclonal quimérico anti-CD20 já utilizado como imunoterápico no tratamento de LNH refratários ou recidivos. O objetivo deste trabalho foi estudar a conjugação deste anticorpo ao quelante bifuncional DOTA-NHS-éster e radiomarcar este imunoconjugado com o radioisótopo 177Lu, com o objetivo de desenvolver um radioimunoterápico para tratamento de LNH. Estudos de imunoconjugação com diferentes razões molares rituximab:DOTA foram estudadas (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 e 1:1000) afim de avaliar qual condição conferia maior pureza radioquímica. A estabilidade dos imuconjugados foi analisada por cromatografia de alta eficiência por até 240 dias em diferentes condições de armazenamento. A estabilidade do imuconjugado radiomarcado foi avaliada após incubação a 2-8 °C e em soro humano a 37 °C e a ligação às proteínas séricas foi determinada. Estudos de biodistribuição foram realizados em camundongos Swiss sadios a fim de caracterizar biologicamente o imunoconjugado radiomarcado. Com o objetivo de analisar se os processos de conjugação e de radiomarcação não danificaram a capacidade de reconhecimento do antígeno (imunorreatividade) deste anticorpo, realizou-se estudos preliminares de ligação às células de LNH (Raji). Os imuconjugados de razão molar baixa (1:5, 1:10 e 1:20) mostraram-se estáveis quando armazenados por até 240 dias em diferentes condições. A análise em cromatografia em camada delgada e CLAE, revelou que o Acm conjugado na razão molar 1:50 foi radiomarcado com alta pureza radioquímica (superior a 95%) quando purificado em coluna PD-10. Este mesmo radioimunoconjugado apresentou razoável estabilidade a 2-8° C. A análise da estabilidade em soro humano não indicou grande metabolismo pelas enzimas do soro. O radioimuconjugado apresentou alta ligação às proteínas séricas indicando clareamento sanguíneo lento, o qual foi confirmado pelos estudos in vivo. O radioimunoconjugado apresentou alta captação no fígado o que é característico de anticorpos monoclonais. Os estudos preliminares de competição indicaram que o processo de obtenção do radioimunoconjugado não prejudicou sua ligação às células Raji sendo esta ligação específica. / Lymphomas are tumors origened from the transformation of a lymphocyte in the lymphatic system. The most common lymphoma is the Non-Hodgkin Lymphoma (NHL). Advances in immunology and molecular biology have been improving NHLs detection and treatment strategies development, such as Radioimmunotherapy (RIT). Rituximab is an anti-CD20 monoclonal antibody used as immunotherapeutic to treat refractory or relapsed NHL. The goal of the present work was to conjugate this antibody to DOTA-NHS-ester bifunctional chelator and to radiolabel it with 177Lu radioisotope in order to develop a radioimmunotherapeutic agent for NHLs treatment. Different rituximab to DOTA molar ratios (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 and 1:1000) were evaluated in order to determine the best condition for obtaining the highest radiochemical purity of radioimmunotherapeutic. The stability of the unlabeled immunoconjugated was evaluated by high performance liquid chromatography (HPLC) for up to 240 days in different storage conditions. The stability of the labeled preparations was evaluated either after storing at 2-8 °C or incubation in human serum at 37 °C. The binding to serum proteins was also determined. In vivo studies were performed in healthy Swiss mice, in order to characterize the biological properties of labeled conjugate. Finally, preliminary studies of radioimmunoconjugated competitive binding to CD20 positive Raji cells were carried out in order to analyze if the process of conjugation and radiolabeling compromises the immunoreactivity of the antibody. The conjugation applying lower antibody to chelator molar ratios (1:5, 1:10 and 1:20) showed high stability when stored for up to 240 days in different conditions. The HPLC analysis showed that the monoclonal antibody conjugated in molar ratio 1:50 was labeled with higher radiochemical purity (> 95%) when purified in PD-10 column. This conjugate showed reasonable stability at 2-8 ° C. The analysis of the stability in human serum did not suggest high metabolic degradation by serum enzymes. The labeled conjugate showed high serum protein binding, suggesting slow blood clearance, which was confirmed by in vivo studies. The labeled conjugate presented high uptake in the liver, in accordance to biodistribution pattern of monoclonal antibodies. The preliminary competitive binding studies indicated a specific binding and suggest that the synthesis of 177Lu-DOTA-rituximab did not compromise its binding to CD20 positive tumor cells.

Linfomas são cânceres provenientes da transformação de um linfócito no sistema linfático, sendo que, o mais comum é o Linfoma Não-Hodgkin (LNH). Avanços na imunologia e na biologia molecular têm auxiliado na detecção desses tumores e aberto caminhos para novas estratégias de tratamento, como a Radioimunoterapia. O rituximab é um anticorpo monoclonal quimérico anti-CD20 já utilizado como imunoterápico no tratamento de LNH refratários ou recidivos. O objetivo deste trabalho foi estudar a conjugação deste anticorpo ao quelante bifuncional DOTA-NHS-éster e radiomarcar este imunoconjugado com o radioisótopo 177Lu, com o objetivo de desenvolver um radioimunoterápico para tratamento de LNH. Estudos de imunoconjugação com diferentes razões molares rituximab:DOTA foram estudadas (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 e 1:1000) afim de avaliar qual condição conferia maior pureza radioquímica. A estabilidade dos imuconjugados foi analisada por cromatografia de alta eficiência por até 240 dias em diferentes condições de armazenamento. A estabilidade do imuconjugado radiomarcado foi avaliada após incubação a 2-8 °C e em soro humano a 37 °C e a ligação às proteínas séricas foi determinada. Estudos de biodistribuição foram realizados em camundongos Swiss sadios a fim de caracterizar biologicamente o imunoconjugado radiomarcado. Com o objetivo de analisar se os processos de conjugação e de radiomarcação não danificaram a capacidade de reconhecimento do antígeno (imunorreatividade) deste anticorpo, realizou-se estudos preliminares de ligação às células de LNH (Raji). Os imuconjugados de razão molar baixa (1:5, 1:10 e 1:20) mostraram-se estáveis quando armazenados por até 240 dias em diferentes condições. A análise em cromatografia em camada delgada e CLAE, revelou que o Acm conjugado na razão molar 1:50 foi radiomarcado com alta pureza radioquímica (superior a 95%) quando purificado em coluna PD-10. Este mesmo radioimunoconjugado apresentou razoável estabilidade a 2-8° C. A análise da estabilidade em soro humano não indicou grande metabolismo pelas enzimas do soro. O radioimuconjugado apresentou alta ligação às proteínas séricas indicando clareamento sanguíneo lento, o qual foi confirmado pelos estudos in vivo. O radioimunoconjugado apresentou alta captação no fígado o que é característico de anticorpos monoclonais. Os estudos preliminares de competição indicaram que o processo de obtenção do radioimunoconjugado não prejudicou sua ligação às células Raji sendo esta ligação específica. / Lymphomas are tumors origened from the transformation of a lymphocyte in the lymphatic system. The most common lymphoma is the Non-Hodgkin Lymphoma (NHL). Advances in immunology and molecular biology have been improving NHLs detection and treatment strategies development, such as Radioimmunotherapy (RIT). Rituximab is an anti-CD20 monoclonal antibody used as immunotherapeutic to treat refractory or relapsed NHL. The goal of the present work was to conjugate this antibody to DOTA-NHS-ester bifunctional chelator and to radiolabel it with 177Lu radioisotope in order to develop a radioimmunotherapeutic agent for NHLs treatment. Different rituximab to DOTA molar ratios (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 and 1:1000) were evaluated in order to determine the best condition for obtaining the highest radiochemical purity of radioimmunotherapeutic. The stability of the unlabeled immunoconjugated was evaluated by high performance liquid chromatography (HPLC) for up to 240 days in different storage conditions. The stability of the labeled preparations was evaluated either after storing at 2-8 °C or incubation in human serum at 37 °C. The binding to serum proteins was also determined. In vivo studies were performed in healthy Swiss mice, in order to characterize the biological properties of labeled conjugate. Finally, preliminary studies of radioimmunoconjugated competitive binding to CD20 positive Raji cells were carried out in order to analyze if the process of conjugation and radiolabeling compromises the immunoreactivity of the antibody. The conjugation applying lower antibody to chelator molar ratios (1:5, 1:10 and 1:20) showed high stability when stored for up to 240 days in different conditions. The HPLC analysis showed that the monoclonal antibody conjugated in molar ratio 1:50 was labeled with higher radiochemical purity (> 95%) when purified in PD-10 column. This conjugate showed reasonable stability at 2-8 ° C. The analysis of the stability in human serum did not suggest high metabolic degradation by serum enzymes. The labeled conjugate showed high serum protein binding, suggesting slow blood clearance, which was confirmed by in vivo studies. The labeled conjugate presented high uptake in the liver, in accordance to biodistribution pattern of monoclonal antibodies. The preliminary competitive binding studies indicated a specific binding and suggest that the synthesis of 177Lu-DOTA-rituximab did not compromise its binding to CD20 positive tumor cells.

A radioimunoterapia tem se mostrado uma modalidade terapêutica promissora, especialmente para terapia de tumores hematológicos o que tem impulsionado o desenvolvimento deste tipo de radiofármaco. Existe hoje apenas um radioimunoconjugado aprovado pelo Food and Drug Administration (FDA), ibritumomabe-tiuxetan-90Y (Zevalin®), e ele apresenta maior taxa de resposta global e de remissão completa quando comparados aos tratamentos convencionais. Entretanto, nenhum deles é comercialmente disponível no Brasil. Neste contexto, o objetivo deste trabalho foi estudar as etapas envolvidas no processo de conjugação e radiomarcação com Lu-177 do anticorpo monoclonal anti-CD20, de forma que fosse possível consolidar nacionalmente a metodologia para desenvolvimento de outros radioimunoconjugados. Nos estudos realizados para determinar a melhor razão molar anticorpo:quelante (DOTA), a razão molar 1:50, apresentou pureza radioquímica elevada (superior a 95%, após a purificação) e imunorreatividade superior a muitos estudos publicados. Além disto, o imunoconjugado apresentou estabilidade de, no mínimo 3 meses, sob refrigeração quando conjugado por dois métodos diferentes. O estudo dos parâmetros de radiomarcação permitiu a obtenção de um radioimunoconjugado com atividade específica de 740 MBq/mg, com estabilidade suficientemente longa que permitirá seu transporte às clínicas médicas. Os perfis de biodistribuição e farmacocinético foram compatíveis com outros radioimunoconjugados encontrados na literatura. O radioimunoconjugado apresentou captação tumoral e estabilidade in vivo apreciáveis, esta última evidenciada pela baixa captação óssea. Realizaram-se estudos de liofilização da formulação aperfeiçoada do imunoconjugado que promoveram a liofilização sem dano estrutural evidenciado por eletroforese em gel de poliacrilamida com manutenção da imunorreatividade. A pureza radioquímica foi acima de 95% (após purificação) quando radiomarcado com atividade específica de 740 MBq/mg, com estabilidade relevante quando armazenado à -20 °C por até 48 horas. Foi possível não somente padronizar as metodologias de conjugação e radiomarcação de anticorpos monoclonais, mas também aprimorá-las de forma que o radioimunoconjugado produzido foi superior em muitos aspectos quando comparado com a literatura publicada. Conclui-se, portanto, que o radioimunoconjugado anti-CD20-DOTA-177Lu é uma ferramenta promissora para o tratamento de tumores linfáticos que expressam o receptor CD20. / The radioimmunotherapy (RIT) has proven to be a promising therapeutic modality, especially for therapy of hematological malignancies, which has stimulated the development of this type of radiopharmaceutical. Currently, there is one radioimmunoconjugated approved by Food and Drug Administration (FDA) for refractory or relapsed non-Hodgkin lymphoma (NHL) therapy, 90Y-ibritumomab tiuxetan (Zevalin®), and it has higher overall response and complete remission rates compared to conventional treatments. However, Zevalin® is not commercially available in Brazil. In this context, the goal of this work was to study the steps involved in the process of conjugation and radiolabeling with Lu-177 of anti-CD20 monoclonal antibody, in order to consolidate the in house methodology for development of this radioimmunoconjugate, contributing for the treatment of patients with NHL and also contributing for the future development of other radioimmunoconjugates. In the studies performed to determine the best antibody: chelator (DOTA) molar ratio, the molar ratio 1:50, showed high radiochemical purity (greater than 95% after purification) and the immunoreactivity was higher than many published studies. Additionally, the immunoconjugate was stable for, at least, 3 months under refrigeration when conjugated by two different methods. The study of radiolabeling parameters, produced a radioimmunoconjugate with specific activity of 740 MBq/mg, with adequate stability that allowed the transportation of the radiopharmaceutical to nuclear medicine centers. The biodistribution and pharmacokinetic profiles were consistent with other radioimmunoconjugates in the literature. The radioimmunoconjugate showed tumor uptake and in vivo stability appreciable, the latter evidenced by low bone uptake. The lyophilization studies were performed for the optimized formulation of immunoconjugate that allowed the lyophilization without structural damage, evidenced by polyacrylamide gel electrophoresis, and with maintenance of immunoreactivity. The radiochemical purity was above 95% (after purification) when radiolabeled with a specific activity of 740 MBq/mg, with significant stability when stored at -20 ° C for 48 hours. It was possible, not only to standardize the conjugation of the monoclonal antibody and radiolabelling methodologies, but also improve them so that the radioimmunoconjugate produced was superior in many aspects when compared with the published literature. In conclusion, the radioimmunoconjugate 177Lu-DOTA-anti-CD20 is a promising tool for the therapy of tumors that express CD20.

<p>Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. </p><p>In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate.</p><p>In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36.</p><p>In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice.</p><p>In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.</p>

Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

Radioimmunotherapy is becoming important as a new therapeutic strategy for treatment of tumour diseases. Lately monoclonal antibodies tagged with radionuclides have demonstrated encouraging results in treatment of hematological malignancies. The progress in treatment of solid tumours using radioimmunotherapy, however, has been slow. New strategies to improve the treatment response need to be evaluated. Such new strategies include the combination of radioimmunotherapy with other treatment modalities but also elucidation and exploration of the death effector mechanisms involved in tumour eradication. As the combination of radioimmunotherapy and radiotherapy provides several potential synergistic effects, we started out by optimising a treatment schedule to detect benefits combining these treatment modalities. An anti-cytokeratin antibody labelled with 125I administered before, after, or simultaneously with radiotherapy, indicated that the highest dose to the tumour was delivered when radiotherapy was given prior to the antibody administration. The optimised treatment schedule was then applied therapeutically in an experimental study on HeLa Hep2 tumour bearing nude mice given radiotherapy prior to administration of 131I-labelled monoclonal antibodies. Combining these treatment regimes enhanced the effect of either of the treatment modalities given alone, and a significant reduction in tumour volumes could be demonstrated. This treatment caused a dramatic change in tumour morphology, with increased amounts of connective tissue, giant cells and cysts. Furthermore cellular alterations like heterogeneity of nuclear and cytoplasmic size and shape were observed, and at least a fraction of the tumour cells presented some characteristics of apoptosis. The induced sequential events in Hela Hep2 cells exposed to 2.5-10 Gy of ionizing radiation were studied further, with special emphasis on cell cycle arrest, mitotic aberrations and finally cell death. Following radiation HeLa Hep2 cells initiated a transient G2/M arrest trying to repair cellular damage. This arrest was followed by a sequence of disturbed mitoses with anaphase bridges, lagging chromosomal material, hyperamplification of centrosomes and multipolar mitotic spindles. These mitotic disturbances produced multinuclear polyploid cells and cells with multiple micronuclei, cells that were destined to die via mitotic catastrophes and delayed apoptosis. Induction of apoptosis in HeLa Hep2 cells following radiation doses and dose-rates equivalent to those delivered at radioimmunotherapy was concurrently studied in vitro. Significant induction of apoptosis was obtained and found to be induced relatively slowly, peaking 72-168 hours post irradiation. Caspases from the intrinsic pathway as well as the extrinsic pathway were found to be activated in response to ionizing radiation. Furthermore caspase-2, which has recently been acknowledged for its role as an initiator caspase was found to be activated following radiation and seems to play an important role in this delayed apoptosis.

Objective: CSL360 is a chimeric IgG1 mAb recognizing CD123+/CD131- LSCs responsible for acute myeloid leukemia (AML). The in vitro targeting properties of 111In-labeled CSL360 modified with nuclear localization sequence (NLS) were evaluated in AML cells. Methods: 111In-NLS-CSL360 was constructed and its binding affinity, cellular uptake and nuclear importation were analyzed on CD123+ cells. Cytotoxicity was evaluated by clonogenic assays on AML cells (CD123+/CD131-). Results: 111In-NLS-CSL360 exhibited preserved binding to CD123. High cellular and nuclear uptake was observed at 266 nM after 24 hour of incubation. Nuclear uptake of 111In-NLS-CSL360 (266 nM) was 2.0-fold higher than 111In-CSL360 (266 nM) after 24 hour of incubation. Clonogenic survival (CS) of AML cells was reduced to 27.5 ± 4.1%. The nuclear uptake and cytotoxicity were reduced when pre-exposed to unlabeled CSL360, indicating 111In-NLS-CSL360 was CD123-specific. Conclusion: 111In-NLS-CSL360 could be a promising radioimmunotherapeutic agent specific for LSCs.

Objective: CSL360 is a chimeric IgG1 mAb recognizing CD123+/CD131- LSCs responsible for acute myeloid leukemia (AML). The in vitro targeting properties of 111In-labeled CSL360 modified with nuclear localization sequence (NLS) were evaluated in AML cells. Methods: 111In-NLS-CSL360 was constructed and its binding affinity, cellular uptake and nuclear importation were analyzed on CD123+ cells. Cytotoxicity was evaluated by clonogenic assays on AML cells (CD123+/CD131-). Results: 111In-NLS-CSL360 exhibited preserved binding to CD123. High cellular and nuclear uptake was observed at 266 nM after 24 hour of incubation. Nuclear uptake of 111In-NLS-CSL360 (266 nM) was 2.0-fold higher than 111In-CSL360 (266 nM) after 24 hour of incubation. Clonogenic survival (CS) of AML cells was reduced to 27.5 ± 4.1%. The nuclear uptake and cytotoxicity were reduced when pre-exposed to unlabeled CSL360, indicating 111In-NLS-CSL360 was CD123-specific. Conclusion: 111In-NLS-CSL360 could be a promising radioimmunotherapeutic agent specific for LSCs.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.