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

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.

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.

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria.

Human IgA is both a major serum immunoglobulin and the most abundant antibody class in seromucous secretions (1). The mucosal surfaces bathed by these secretions, such as those of the respiratory, gastrointestinal, and genitourinary tracts, are major potential sites of invasion due to their vast surface area. IgA therefore serves as a key first line of defense against many invading pathogens. Like all antibodies, IgA is capable of both recognizing the foreign invader and triggering its elimination. The latter process is frequently mediated by the interaction of the Fc region of IgA with Fc␣ receptors (Fc␣R) 1 present on the surface of neutrophils, macrophages, monocytes, and eosinophils (2, 3). The human myeloid Fc␣R, CD89, possesses two extracellular Ig-like domains and displays homology to the three classes of human IgG Fc receptors (Fc␥RI, Fc␥RII and Fc␥RIII) and the high affinity IgE receptor Fc⑀RI, albeit at a lower level than these receptors do to each other (4). Interaction of CD89 with IgA, aggregated either by binding to antigen or artificially, acts as a potent trigger for an array of myeloid cell functions including phagocytosis, antibody-dependent cellmediated cytotoxicity, superoxide generation, enzyme and inflammatory mediator release, and clearance of immune complexes (3). A detailed understanding of the molecular basis of the IgA-Fc␣R interaction is clearly important if the increasingly appreciated potential of recombinant IgA in numerous therapeutic applications (5-7) is to be fully realized.Although others have described expression of human IgA in insect (8) and plant cells (5), we have expressed hapten-specific recombinant human IgA of both subclasses, IgA1 and IgA2, in mammalian cell hosts (9 -11). Here, we have used an extensive panel of chimeric and site-directed mutant IgAs expressed in CHO K1 cells to identify residues critical for Fc␣R binding. We have constructed domain swap antibodies through exchange of the homologous C-terminal CH3 domains between human IgA1 and IgG1 in order to ascertain the contribution of each Fc domain to the interaction with Fc␣R. An IgA1 lacking the C-terminal 18 amino acid tailpiece has also been assayed for ability to bind the receptor. To allow more precise localization of the interaction site, a number of IgA1 mutants with single substitutions in loop regions lying at the interface of the CH2 and CH3 domains have been generated. The effects of such mutations on the ability to bind Fc␣R are consistent with the interdomain region of the Fc playing a critical role in binding to the receptor. Further support for this proposal is lent through correlation of the binding ability of IgAs derived from other species with sequence differences in these loops.To more readily assess receptor interaction, we have developed stable CHO cell transfectants expressing high levels of CD89, which have allowed, for the first time, comparison of the relative binding affinities of the different IgA molecules. As an additional, more physiologically relevant test for function, we hav...

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The binding of non-specific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Here we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped antibodies, IgM mutants and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM heavy chain and requires the IgM Cμ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cμ4 domain are equivalent to those regions of IgG and IgA recognised by Fc-binding proteins from bacteria, suggesting that this region of immunoglobulin molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and shows functional similarities with Fc-binding proteins from pathogenic bacteria.

The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to FcγRs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.