Abs exert several of their effector functions by binding to cell surface receptors. For murine IgG3 (mIgG3), the identity of its receptors (and the very existence of a receptor) is still under debate, as not all mIgG3 functions can be explained by interaction with FcgRI. This implies the existence of an alternate receptor, whose identity we sought to pinpoint. We found that blockage of integrin b1 selectively hampered binding of mIgG3 to macrophages and mIgG3-mediated phagocytosis. Manganese, an integrin activator, increased mIgG3 binding to macrophages. Blockage of FcgRI or Itgb1 inhibited binding of different mIgG3 Abs to variable extents. Our results are consistent with the notion that Itgb1 functions as part of an IgG receptor complex. Given the more ancient origin of integrins in comparison with FcgR, this observation could have far-ranging implications for our understanding of the evolution of Ab-mediated immunity as well as in immunity to microorganisms, pathogenesis of autoimmune diseases, and Ab engineering.

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host’s antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern.The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

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...

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host’s antifungal immunity. In this work we show the generation of and promising results with a mAb against HSP90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM.Author summaryParacoccidioidomycosis (PCM) is a severe disease caused by fungi, common in Latin America. It is treatable, but some of the drugs that are available are very toxic or not very effective, and the treatment can take as long as two years to clear the infection. To address the need for improved therapeutic alternatives, we have been developing drug candidates based on antibody technologies against Paracoccidioides brasiliensis and P. lutzii, which cause PCM. In this work, we produced monoclonal antibodies (mAbs) that bind to the fungal protein HSP90, which is essential for fungal cells to survive. One mAb, 4D11, recognized the HSP90 target on the surface of fungal cells. These antibody-covered cells were ingested more efficiently by immune cells called macrophages, suggesting they could improve the host resistance to infection by Paracoccidioides. Future improvements on these antibodies could thus lead to more effective and safer PCM treatments.