Tchavdar L. Vassilev

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.

Summary: Targeting autoreactive B lymphocytes at any stage of their differentiation could yield viable therapeutic strategies for treating autoimmunity. All currently used drugs, including the most recently introduced biological agents, lack target specificity. Selective silencing of double-stranded DNA-specific B cells in animals with spontaneous lupus has been achieved previously by the administration of a chimeric antibody molecule that cross-links their DNA-reactive B cell immunoglobulin receptors with inhibitory FcγIIb (CD32) receptors. However, long-lived plasmacytes are resistant to this chimeric antibody as well as to all conventional treatments. Bortezomib (a proteasome inhibitor) depletes most plasma cells and has been shown recently to suppress disease activity in lupus mice. We hypothesized that the co-administration of non-toxic doses of bortezomib, that partially purge long-lived plasma cells, together with an agent that selectively silences DNA-specific B cells, should have additive effects in an autoantibody-mediated disease. Indeed, our data show that the simultaneous treatment of lupus-prone MRL/lpr mice with suboptimal doses of bortezomib plus the chimeric antibody resulted in the prevention or the delayed appearance of the disease manifestations as well as in a prolonged survival. The effect of the combination therapy was significantly stronger than that of the respective monotherapies and was comparable to that observed after cyclophosphamide administration.

T. Vassilev1, I. Djoumerska-Alexieva1, J. Dimitrov2, L. Roumenina2, E. Voynova1, I. Nina Ivanovska1, K. Srini2; 1Stefan Angelov Institute of Microbiology, Sofia, Bulgaria, 2INSERM Unit 872, Paris, France. The exposure of some IgG antibodies to ferrous ions is known to enhance their polyspecificity that includes the ability to bind at least one pro-inflammatory cytokine (J.Biol.Chem.2006:281,439). Sepsis is a medical disaster that responds poorly to available drugs including intravenous immunoglobulin (IVIg) preparations. IVIg, pre-exposed in vitro to Fe(II) ions was used for passive immunotherapy of mice with sepsis induced by the injection of LPS, of live E.coli, of zymosan or by the CLP technique. A single dose of 50 to 250 mg/kg of the modified immunoglobulin preparation, but not of the native, commercially available IVIg significantly increased survival in all sepsis models. The mechanisms of the protective activity of modified IVIg were studied in LPS sepsis. Its therapeutic effect was not due to a more efficient LPS neutralization and was still present when administered 6 hours after LPS. In the treated animals the serum levels of pro-inflammatory cytokines were decreased, IL10 levels were increased, the coagulation abnormality was overcome and the complement exhaustion was prevented. The exposure to Fe(II) ions induced structural changes in the IgG molecules as demonstrated by fluorescent spectroscopy, kinetic and thermodynamic analyses. These changes did not result in their denaturation as the modified preparations still met the strict Pharmacopoeia requirements for IVIg. We suggest that modified IVIg with additionally enhanced polyspecificity, induced by a brief exposure to pro-oxidative ferrous ions, has a clinical potential in sepsis and other variants of the SIRS syndrome (post-traumatic, in avian flu, etc.).

Polyspecificity (polyreactivity) is currently considered an intrinsic property of a subset of antibodies, primarily of naturally occurring autoantibodies. Polyspecificity is no longer viewed as a biologically irrelevant stickiness. Furthermore, the capacity to bind defined sets of unrelated antigens finds its structural explanation. What is most intriguing, the elucidation of the role of polyspecificity may promote a better understanding of specific recognition as a function of the entire immune system. The early events of immune recognition depend on polyspecific binding. Thus, the completeness of the naïve repertoires of antigen receptors is ensured. The process of immunologically-relevant antigen recognition that is initiated goes beyond simple molecular interaction with the antigenic determinants. It involves cellular cooperation and culminates in antibody response maturation. Recent findings also pave the way for the clinical application of posttranslationally induced polyspecificity.

IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen‐binding polyreactivity. The molecular mechanisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self‐antigens, enhanced binding to non‐self‐antigens (bacterial), an increased ability to engage in F(ab′)₂/F(ab′)₂(idiotype/anti‐idiotype) interactions and an increased functional antigen‐binding affinity are reported here. The newly acquired ‘induced polyreactivity’ of low‐pH buffer‐exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low‐pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen‐binding behavior and biological activity. Low‐pH buffer‐treated pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer‐exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide‐induced septic shock.

Intravenous immunoglobulin (IVIg) preparations are known to modulate autoimmune/inflammatory diseases through several F(ab')₂- and Fc-dependent mechanisms. In this study, we show that the in vitro and the in vivo exposure of B lymphocytes from lupus-prone and from healthy mice to IVIg results in an increased expression of their surface inhibitory FcγIIB receptors. Further, this exposure enhanced the ability of a chimeric antibody, cross-linking FcγRIIB and immunoglobulin receptors on DNA-specific B lymphocytes, to suppress IgG anti-DNA antibody production. F(ab')₂ fragments of IVIg had a similar activity as the intact preparation, whereas Fc fragments had no effect. This study describes a novel approach with clinical relevance for modulating B lymphocyte activity.

Natural polyreactive IgG antibodies are found in the sera of all healthy individuals. The in vitro exposure of pooled human IgG to protein-destabilizing chemical or physical factors has been previously shown to result in the exposure of their “hidden” polyspecificity. We hypothesize that such an enhancement of their pre-existing immunoreactivity may occur in vivo in the aggressive microenvironment of inflammation sites. An increase in the antigen binding intensity as well as of the number of recognized antigens was observed in the sera of IgG-infused immunodeficient SCID mice with induced local inflammation. The expansion of the IgG pathogen-binding repertoire may have important biological consequences.

The pathological DNA‐specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross‐links their surface immunoglobulins with the inhibitory FcγIIb surface receptors. A hybrid molecule was constructed by coupling the DNA‐mimicking DWEYSVWLSN peptide to a monoclonal anti‐mouse FcγRIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/lpr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti‐DNA but not of anti‐diphtheria IgG antibody‐producing cells. Intravenous infusions with the DNA‐peptide antibody chimera to 7‐wk‐old animals prevented the appearance of IgG anti‐DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA‐peptide chimeric antibody to 18 wk‐old mice with full‐blown disease resulted in the maintenance of a flat level of IgG anti‐DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis. The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease‐associated B cells in autoimmune diseases.