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Orientador: Elenice Deffune / Banca: Rosana Rossi Ferreira / Banca: Ana Paula Costa Nunes da Cunha Cozac / Resumo: A tecnologia de produção de anticorpos monoclonais revolucionou a investigação diagnóstica, especialmente a análise dos grupos sanguíneos em imuno-hematologia. O polimorfismo das hemácias humanas torna a produção de novos insumos extremamente necessária. Dentre os sistemas de grupos sanguíneos descritos, o sistema Rh tem se mostrado um dos mais complexos pela vasta composição de antígenos. Analisando aspectos estruturais do antígeno D associado às técnicas de biologia molecular, identificam-se numerosas variantes deste antígeno por trocas de DNA entre genes similares levando a alteração de epítopos e consequentemente de padrão de expressão fenotípica na superfície das hemácias. Desvendar e identificar estas variantes com insumos biotecnológicos desenvolvidos no país foi o desafio desta pesquisa, onde anticorpos monoclonais contra antígenos eritrocitários foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados com hemácias fenótipo D categoria IVa. Nove fusões foram realizadas e os sobrenadantes de cultura foram triados por teste de hemaglutinação em microplacas. Foram congelados e mantidos em nitrogêncio líquido 41 híbridos. Estes híbridos foram clonados e mantidos em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 12 clones IgG1, 5 IgG2a e 10 IgM. Os clones foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. Os anticorpos monoclonais produzidos, inicialmente não demonstraram a especificidade foco do presente trabalho que foi a produção de anticorpos anti-D, no entanto, os hibridomas podem ter secretado diferentes especificidades de anticorpos monoclonais, de acordo com os antígenos presentes na membrana das hemácias utilizadas para a imunização dos camundongos. Uma segunda fase da pesquisa está sendo ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The blood groups analyses in immunohematology have been highly benefited based on the advance of the monoclonal antibody technology improvement on diagnostics investigation. The human red blood cell polymorphism increases the need for new alternatives. The Rh system blood group is the most complex of all, due to the vast antigenic composition. When analyzing the structural aspects of the D antigen in combination with the molecular techniques, based upon DNA exchange between similar genes it is possible to identify a huge number of partial D variants, this situation leads to a change of epitopes and hence the red blood cell phenotypic expression pattern. Unveil and identify these variables through the use of local biotechnology was the challenge of this research. Monoclonal antibodies against human red blood cell were produced by cell fusion, using spleen cells from immunized BALB/c mice and DIVa red blood cells. Nine fusions had been made and the culture supernatants were selected to the hemaglutination test using microplates. Forty-one hybrids had been frozen and kept in liquid nitrogen. These hybrids had been cloned and kept in the culture to collect monoclonal antibodies, resulting in 12 IgG1, 5 IgG2a e 10 IgM. The clones had been selected for ascitic fluid production, by injection in BALB/c mice. The monoclonal antibodies produced initially had not produced the anti-D as expected by the research. However, the hibrydomas could have created different specificities of monoclonal antibodies, according to the antigen located in the donor red blood cells membrane used in the mice's immunization. The second phase of the research is being done to confirm the monoclonal antibodies specificities produced through the deployment of new tests to immunoglobulin identification / Mestre

Cancer immunotherapy has revolutionized the field of oncology by giving the possibility to ligands (e.g., antibodies) to selectively target tumor antigens and accumulate at the site of the disease while sparing normal tissues. During the past years, the number of patient eligible for immune-based cancer treatments has seen an exponential increase as these therapies are becoming first line treatment for many cancer indications. A promising anticancer strategy consists in the targeted delivery of bioactive compounds (e.g., cytokines) to the tumor microenvironment with high-affinity ligands specific for tumor-associated antigens. This approach improves the efficacy of the drug by reducing related side effects and increasing the therapeutic index of the payload. Currently, antibodies represent one of the most successful class of ligands used for this purpose as they can be generated against virtually any antigen. Many methodologies have been described for the generation and isolation of antibodies with high antigen-binding specificity. Among these, phage display technology has emerged as a powerful and versatile tool for the in vitro discovery of antibodies and peptides. Since it was invented in mid 1980s, phage-display has paved the way to the generation of more than 70 phage–derived monoclonal antibodies (mAbs) that entered clinical studies, and 14 of which have been approved in the market. Cytokines are proteins capable of modulating the activity of the immune system and some cytokine-products have gained marketing authorization for the treatment of cancer. In order to increase the therapeutic index of cytokine payloads, the generation of fusion proteins with tumor-homing antibodies has been proposed. These so-called “immunocytokine” products constitute a class of “armed” antibody products, in which a tumor-targeting immunoglobulin is fused with a cytokine. In this thesis, we present the generation and characterization of antibodies specific for two tumor microenvironment-associated antigens: Tenascin C and Fibroblast Activation Protein. Both antigens are undetectable in healthy tissues but abundantly expressed in the tumor stroma. In the first part of the thesis, we have isolated antibodies specific for the spliced domain D of Tenascin C from the synthetic phage library “ETH2Gold”. Antibodies were affinity matured randomizing key residues of CDR1 of heavy and light chains. The highest affinity clone, R6N, was characterized in vitro and in vivo showing selective accumulation at the tumor site in mouse models of cancer. An immunocytokine featuring IL12 as payload has been generated and its therapeutic activity evaluated in tumor bearing mice. R6N-IL12 exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. In the second part of this thesis, a monoclonal antibody has been isolated against Fibroblast Activation Protein. After affinity maturation of the CDR2 of heavy and light chains of the parental antibody C5, the selected 7NP2 antibody showed improved affinity and excellent tumor targeting properties in SKRC52-hFAP tumor bearing mice. When fused to IL12, 7NP2 was able to induce tumor growth retardation and tumor remission in mouse models of cancer. Collectively, in this thesis we have isolated and validated two monoclonal antibodies selective for tumor microenvironment-associated antigens. Both antibodies when fused to IL12 induced tumor growth retardation and remission in immunodeficient and immunocompetent mouse models providing a rationale for possible future applications of R6N and 7NP2 antibodies for the treatment of cancer patients.

The causative agent of the most deadly form of malaria, P. falciparum, was identified over 130 years ago, yet this disease still causes 430,000 deaths each year. Although naturally-acquired immunity exists, it requires a heavy and sustained exposure to the parasite, with most succumbing as young children, before this immunity has fully developed. Effective treatments exist but with small-molecule drug resistance on the rise and little in the way of affordable alternatives, the need for an efficacious malaria vaccine is as great as ever. A successful malaria vaccine is likely to necessitate targeting each stage of the parasite's lifecycle. Immunity directed to the blood-stage, the stage which causes all the symptoms of malaria, is unique in that it would allow for a concomitant development of naturally-acquired immunity along with a reduction in morbidity and mortality. To date, antibody-mediated immunity to the blood stage requires intractably high levels of antibody and this problem is compounded by a paucity of viable candidates with which to effectively target different strains. Other fields of vaccinology, over the past decade, have been employing various structure-based strategies to increase the specific activity of the immune response thus lowering the antibody levels required for protection. However, very few detailed investigations of this kind have been conducted on a P. falciparum vaccine candidate, and certainly none as promising as PfRH5. In a world's first, fully-human antibodies raised in response to PfRH5 vaccination were isolated and extensively characterised, both functionally and structurally with the intention of elucidating the important features necessary to inform the design of an improved PfRH5-based vaccine. Synergistic and antagonistic effects of antibody combinations were noted and highlight new complexities of the immune response to PfRH5, opening the door to unanticipated potential for rational vaccine design.

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T20:50:03Z : No. of bitstreams: 1 cardoso_ra_me_botfm.pdf: 896730 bytes, checksum: 97ac1a0ea8e45d9861c31c7feaac2ecb (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / A tecnologia de produção de anticorpos monoclonais revolucionou a investigação diagnóstica, especialmente a análise dos grupos sanguíneos em imuno-hematologia. O polimorfismo das hemácias humanas torna a produção de novos insumos extremamente necessária. Dentre os sistemas de grupos sanguíneos descritos, o sistema Rh tem se mostrado um dos mais complexos pela vasta composição de antígenos. Analisando aspectos estruturais do antígeno D associado às técnicas de biologia molecular, identificam-se numerosas variantes deste antígeno por trocas de DNA entre genes similares levando a alteração de epítopos e consequentemente de padrão de expressão fenotípica na superfície das hemácias. Desvendar e identificar estas variantes com insumos biotecnológicos desenvolvidos no país foi o desafio desta pesquisa, onde anticorpos monoclonais contra antígenos eritrocitários foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados com hemácias fenótipo D categoria IVa. Nove fusões foram realizadas e os sobrenadantes de cultura foram triados por teste de hemaglutinação em microplacas. Foram congelados e mantidos em nitrogêncio líquido 41 híbridos. Estes híbridos foram clonados e mantidos em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 12 clones IgG1, 5 IgG2a e 10 IgM. Os clones foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. Os anticorpos monoclonais produzidos, inicialmente não demonstraram a especificidade foco do presente trabalho que foi a produção de anticorpos anti-D, no entanto, os hibridomas podem ter secretado diferentes especificidades de anticorpos monoclonais, de acordo com os antígenos presentes na membrana das hemácias utilizadas para a imunização dos camundongos. Uma segunda fase da pesquisa está sendo... / The blood groups analyses in immunohematology have been highly benefited based on the advance of the monoclonal antibody technology improvement on diagnostics investigation. The human red blood cell polymorphism increases the need for new alternatives. The Rh system blood group is the most complex of all, due to the vast antigenic composition. When analyzing the structural aspects of the D antigen in combination with the molecular techniques, based upon DNA exchange between similar genes it is possible to identify a huge number of partial D variants, this situation leads to a change of epitopes and hence the red blood cell phenotypic expression pattern. Unveil and identify these variables through the use of local biotechnology was the challenge of this research. Monoclonal antibodies against human red blood cell were produced by cell fusion, using spleen cells from immunized BALB/c mice and DIVa red blood cells. Nine fusions had been made and the culture supernatants were selected to the hemaglutination test using microplates. Forty-one hybrids had been frozen and kept in liquid nitrogen. These hybrids had been cloned and kept in the culture to collect monoclonal antibodies, resulting in 12 IgG1, 5 IgG2a e 10 IgM. The clones had been selected for ascitic fluid production, by injection in BALB/c mice. The monoclonal antibodies produced initially had not produced the anti-D as expected by the research. However, the hibrydomas could have created different specificities of monoclonal antibodies, according to the antigen located in the donor red blood cells membrane used in the mice’s immunization. The second phase of the research is being done to confirm the monoclonal antibodies specificities produced through the deployment of new tests to immunoglobulin identification

No description available.

The need for an unlimited source of islets and a safer method of immunosuppression has limited the widespread application of islet transplantation. To remedy the shortage of donor tissue, xenotransplantation of neonatal porcine islets (NPI) has been proposed. In this study we sought to determine if combining co-transplantation of NPI with Sertoli cells (SC) with a short-term monoclonal antibody (mAb) therapy would prevent NPI xenograft rejection. We hypothesize that this combination of treatments will lead to long-term NPI xenograft survival. Our result show a significant increase in the proportion of mice achieving long-term graft survival compared to untreated mice transplanted with NPI alone, as 7/7 mice treated with anti-LFA-1 mAb (p=0.001), 7/8 mice treated with anti-CD154 mAb (p=0.003), and 4/9 mice treated with anti-CD45RB mAb (p=0.020) achieved and maintained normoglycemia long-term. Therefore, we conclude that the combination of mAb therapy with SC is highly efficacious in preventing NPI xenograft rejection. / Experimental Surgery

The ability to formulate monoclonal antibodies at high concentration in a low-viscosity form is of broad interest in drug delivery, as monoclonal antibody-based drugs are now prescribed for cancer, autoimmune disorders, and many other diseases. Herein, we create highly concentrated antibody dispersions (up to 260 mg/mL) via three different methods, utilizing proline as an interacting depletant or trehalose as a non-interacting depletant. These dispersions are able to achieve viscosities an order of magnitude lower than similarly concentrated antibody solutions over a range of formulation pHs. When diluted, these antibody dispersions return to monomer. The proline acts to minimize protein zeta potential, thus reducing the electrostatic repulsion on the protein, even when formulated 3 pH units away from the antibody pI. In addition, it acts as a depletant, forcing the monomers into cluster via osmotic effects / text