Since the first description in 1989 of CD4‐Fc‐fusion antagonists that inhibit human immune deficiency virus entry into T cells, Fc‐fusion proteins have been intensely investigated for their effectiveness to curb a range of pathologies, with several notable recent successes coming to market. These promising outcomes have stimulated the development of novel approaches to improve their efficacy and safety, while also broadening their clinical remit to other uses such as vaccines and intravenous immunoglobulin therapy. This increased attention has also led to non‐clinical applications of Fc‐fusions, such as affinity reagents in microarray devices. Here we discuss recent results and more generally applicable strategies to improve Fc‐fusion proteins for each application, with particular attention to the newer, less charted areas.

The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (FcγRs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including twoN-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated thatO-glycans are located at Thr²²⁸, Ser²³⁰, and Ser²³², while O-glycan sites at Thr²²⁵ and Thr²³⁶ are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where <10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one FcN-glycosylation site affects the other. Neutrophil FcαR binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the Cα2 domain contribute to IgA1-neutrophil FcαR binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the FcγR. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S–S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα2N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG FcN-glycans, which are confined in the space between the two Cγ2 domains. This also suggests why in contrast to IgG Fc, the IgA1N-glycans are not undergalactosylated in rheumatoid arthritis.

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.

During experimental hut trials to assess the efficacy of insecticide-treated bednets against malaria mosquitoes, we observed that human subjects varied consistently in their attractiveness to mosquitoes. Attractiveness was assessed by estimating the numbers of wild Anopheles gambiae Giles mosquitoes entering a hut in which a man was sleeping, and the numbers of human-bloodfed An. gambiae sensu lato collected from each hut each morning. Five trials were carried out at Wali Kunda in rural Gambia during 2.5 yr. During each 6-wk trial a man slept under a bednet in each of the six huts. Morning collections of mosquitoes from the room, enclosed verandas, and window traps of each hut provided estimates of the number of mosquitoes that had entered during the night. Blood meals were analyzed using an ELISA technique to identify those mosquitoes feeding on humans. Specimens were collected by field workers, not the subjects; therefore, sampling was independent of the subjects' ability to catch mosquitoes. Moreover, the trials were designed to measure the relative attractiveness of individual sleepers to mosquitoes, allowing for other sources of variation (i.e., among huts, bednets, nights, and day of the week). Attractiveness of men to mosquitoes differed significantly among individuals as indicated by the consistent differences between the numbers of mosquitoes entering each man's hut and the numbers feeding on each man. However, the two measures of attractiveness were apparently independent of each other: subjects who attracted consistently high numbers of vectors into their hut did not necessarily have high numbers of mosquitoes feeding on them. These findings support the view that some individuals within a community are at greater risk from mosquito-borne pathogens than others.