Generation of biologically active anti Cryptococcus neoformans isotype switch variant MAbs

Cryptococcus neoformans causes life threatening meningoencephalitis in approx. 10% of patients with AIDs. In the setting of severe immunosuppression, cryptococcal infections are usually incurable and often fatal. The difficulties encountered in treating these patients have renewed interest in passive antibody administration as an adjunct to antifungal therapy. Passive administration of MAbs to C. neoformans can alter the course of infection in mouse models. However, the effectiveness of these antibodies appears to depend on isotype and specificity. 3E5.y3 is an IgG3 secreting MAb to C. neoformans capsular glucuronoxylomannan. The antibodies do not prolong survival or reduce organ fungal burden. In contrast, the IgG1 switch variant of 3E5 significantly prolonged survival of infected animals. Comparison of isotype protection efficacy requires families of MAbs with identical fine specificity and different constant region. The generation of such families of hybridomas is not always possible either because of the immune response which allows the production of limited immunoglobulin classes or subclasses or because of the low frequency of isotype switching of such hybridomas in vitro. Using acridine orange and an ELISA spot assay we have been able to identify and isolate the entire set of isotype switch variants: y1, y2b;, y2a, e, a. All antibodies bind glucuronooxylomannan and share identical binding site specificity as determined by anti-idiotypic MAb. The functionality of these antibodies was demonstrated by their ability to enhance phagocytosis and anti-fungal efficacy of human THP-1 macrophage cells. The role of Ig subclass in conferring protection to C. neoformans will be discussed.