Richard S. McIntosh

The Importance of Human FcγRI in Mediating Protection to Malaria

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.

Treatment of Erdheim-Chester disease with cladribine: a rational approach

Erdheim-Chester disease is a rare, life threatening lipoid granulomatosis1 with fewer than 100 cases described in the world literature. The disease typically affects the long bones and symmetrical sclerosis of the diaphyseal and metaphyseal regions is pathognomonic. Extraskeletal manifestations may affect the lungs, pericardium, aorta, retroperitoneum, skin, and orbits and diabetes insipidus occurs in approximately 30% of cases. Erdheim-Chester disease is characterised microscopically by an infiltrate of lipid laden foamy macrophages (histiocytes), scattered Touton giant cells, chronic inflammatory cells, and fibrosis. The foamy macrophages can be distinguished from Langerhans cells on the basis of negative results on staining for S-100 protein and CD1a. Treatment of the disease has been on an ad hoc basis and no treatment regimen has been shown to be clearly superior. This study documents the clinical findings in a patient with Erdheim–Chester disease, investigates the pathogenesis, and provides a rational basis for effective treatment. This white man, aged 45, developed aching in his legs, night sweats, lethargy, and impotence in October 1988, for which no cause was found. His night sweats resolved by July 1989 and he was discharged. He presented in November 1990 with reduced vision (6/9) in the left eye, bilateral proptosis of 12 months’ duration, chemosis, ophthalmoplegia, and optic disc oedema. He still had sexual dysfunction and lethargy and now also had leg oedema and thrombocythaemia. At that time his thyroid function was normal, but erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) were moderately elevated. A computed tomography (CT) scan of the orbits showed bilaterally enhancing masses lying predominantly within the muscle cone and encasing both optic nerves. An orbital biopsy in November 1990 showed an inflammatory picture. There was no evidence of vasculitis on muscle biopsy and a clinical diagnosis of orbital pseudotumour was made. He was initially treated …

A Novel Human IgA Monoclonal Antibody Protects against Tuberculosis

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.

Mutant forms of tumour necrosis factor receptor I that occur in TNF‐receptor‐associated periodic syndrome retain signalling functions but show abnormal behaviour

Tumour necrosis factor (TNF)‐receptor‐associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal‐dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS‐related mutations, we transfected HEK‐293 cells to produce lines stably expressing high levels of either wild‐type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine‐rich domain (CRD1) were produced both as full‐length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (Δsig). High‐level expression of either WT or mutant full‐length TNFRSF1A spontaneously induced apoptosis and interleukin‐8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full‐length TNFRSF1A formed cytoplasmic aggregates that co‐localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant Δsig forms of TNFRSF1A did not induce apoptosis or interleukin‐8 production. However, whereas the WT full‐length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell‐surface expression. The WT and mutant Δsig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co‐localized with BiP. Furthermore, the mutant forms of surface‐expressed Δsig TNFRSF1A were defective in binding TNF‐α. We conclude that TRAPS‐related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

The binding of nonspecific 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. Herein 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 Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H 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 recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig 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 that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.

Polymeric human Fc-fusion proteins with modified effector functions

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.

Structural requirements for the interaction of human IgM and IgA with the human Fcα/μ receptor

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcα/μ receptor (hFcα/μR). Ligand polymerization status was crucial for the interaction, because hFcα/μR binding did not occur with monomeric Ab of either class. hFcα/μR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcα/μR binding. IgM binding required contributions from both Cμ3 and Cμ4 Fc domains, whereas for dIgA, an exposed loop in the Cα3 domain was crucial. This loop, comprising residues Pro440–Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcαRI and polymeric Ig receptor (pIgR), as well as IgA‐binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440–Phe443 loop resulted in loss of hFcα/μR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA‐binding proteins were shown to inhibit the dIgA–hFcα/μR interaction. Therefore, we have identified a motif in the IgA–Fc inter‐domain region critical for hFcα/μR interaction, and highlighted the multi‐functional nature of a key site for protein–protein interaction at the IgA Fc domain interface.

Inhibition of Erythrocyte Invasion and Plasmodium falciparum Merozoite Surface Protein 1 Processing by Human Immunoglobulin G1 (IgG1) and IgG3 Antibodies

ABSTRACT Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP119) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP119 can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab′)2 fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human γ1 and γ3 constant regions, retain the ability to bind to both parasites and recombinant MSP119, and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcγ receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.

Glycans as targets for therapeutic antitumor antibodies.

Glycans represent a vast class of molecules that modify either proteins or lipids. They exert and regulate important and complex functions in both normal and cancer cell metabolism. As such, the most immunogenic glycans have been targeted in passive and active immunotherapy in human cancer for the past 25 years but it is only recently that techniques have become available to uncover novel glycan targets. The main focus of this review article is to highlight why and how monoclonal antibodies (mAbs) recognizing glycans, and in particular the glycans expressed on glycolipids, are being used in various strategies to target and kill cancer cells. The article reports on the historical use of mAbs and on very recent progress made in antitumor therapy using the anti-GD2 mAb and the antiganglioside mAbs, anti-N-glycolylneuraminic acid mAb and anti-Lewis mAb. Anti-GD2 is showing great promise in Phase III clinical trials in adjuvant treatment of neuroblastoma. Racotumomab, an anti-idiotypic mAb mimicking N-glycolylneuraminic acid-containing gangliosides, is currently being tested in a randomized, controlled Phase II/III clinical trial. This article also presents various strategies used by different groups to develop mAbs against these naturally poorly immunogenic glycans.

Antibody- and Fc-receptor-based therapeutics for malaria.

Abs (antibodies) are complex glycoproteins that play a crucial role in protective immunity to malaria, but their effectiveness in mediating resistance can be enhanced by genetically engineered modifications that improve on nature. These Abs also aid investigation of immune mechanisms operating to control the disease and are valuable tools in developing neutralization assays for vaccine design. This review explores how this might be achieved.