Anticorpos IgG murinos de diferentes isotipos e região variável idêntica se ligam de forma diferente à cápsula de Cryptococcus neoformans e a receptores Fc-gama

Abstract

It is estimated there are 625,000 deaths worldwide per year caused by cryptococcal meningitis, the most affects people infected by HIV. Cryptococcus neoformans is responsible for the majority of these deaths. Since treatment of cryptococcosis using conventional antifungal has been less effective than ideal, monoclonal antibodies against fungus capsule were developed in the last three decades in order to create new therapeutic tools. However, treating mice with different isotypes of antibody were observed different protective responses. For example, treatment of C. neoformans infected mice with monoclonal antibody 3E5 IgG1 resulted in increased survival, whereas the disease was aggravated in animals treated with IgG3 antibody of identical variable region. Immunofluorescence assay showed that the binding patterns of these two 3E5 isotypes are diferentes to the capsule, which became questionable the classical concept of the antigen binding that defines it is exclusively determined by antibody variable region. In addition to these antigen binding differences, many studies published in 1980s suggest that the IgG3 isotype interacts with an Fc receptor on the surface of macrophages differently from other isotypes, which could also explain differences in protection provided by the 3E5 antibodies of different IgG isotypes. Considering the therapeutic importance of properly knowing the mechanisms by which the same IgG antibody may be protective or not, depending on their isotype, this study aimed to produce new recombinant monoclonal antibodies, assessing its binding standard antigen and its ability to mediate phagocytosis by Fc receptors. Two pairs of IgG1 and IgG3 antibodies were produced by heterologous expression in mammalian cells. The first pair was derived from monoclonal antibody 2H1, the antibody against C. neoformans capsule whose crystal structure has been resolved. The second pair was derived from the 4-4-20 antibody that recognizes the hapten FITC and was generated to evaluate whether receptor-mediated effector response is unique to antibodies against C. neoformans. The heavy and light chains variable domains of the antibodies were generated by chemical synthesis and cloned into expression vectors for IgG1 and IgG3 murine, then they were transfected into CHO and NSO cells. The transfection and purification conditions were optimized and led to the batch production of antibodies until it was sufficient for functional testing. The binding of the antibodies to C. neoformans cells was tested by indirect immunofluorescence and its ability to mediate phagocytosis by J774 macrophages and CHO-K1 cells. The 2H1 antibody showed binding pattern similar to that for 3E5, showing annular and punctate pattern for IgG1 and IgG3 respectively. Both 2H1 and 4-4-20 antibodies were also functional on phagocytosis assay by macrophages. The IgG1 isotype showed exclusive dependence on Fcγ receptor, whereas 2H1 IgG3 was able to mediate phagocytosis even when this receptor was blocked. Therefore, the produced recombinant antibodies were...