Elisaveta Voynova

Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice

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.

IgM‐enriched human intravenous immunoglobulin suppresses T lymphocyte functions in vitro and delays the activation of T lymphocytes in hu‐SCID mice

Previous studies of an experimental human immunoglobulin preparation for intravenous use, containing normal pooled IgM (IVIgM), have shown its beneficial therapeutic effect in experimental autoimmune diseases. The mechanisms of its immunomodulatory activity remain however, poorly understood. In the experiments reported here, IVIgM inhibited the proliferation of various autonomously growing human lymphoid cell lines in vitro, as well as of MLR‐ and of PHA‐stimulated human T‐lymphocytes. These effects of IVIgM were observed at non‐apoptotic concentrations and were stronger on a molar basis than those of normal pooled IgG for intravenous use (IVIg). Both preparations, when administered to SCID mice, repopulated with human peripheral blood mononuclear cells, delayed the expression of the early activation marker CD69 on both human CD4+ and CD8+ T‐lymphocytes, activated by the mouse antigenic environment. The data obtained show that normal pooled human IgM exerts a powerful antiproliferative effect on T‐cells that is qualitatively similar but quantitatively superior to that of therapeutic IVIg. Our results suggest that infusions with IVIgM might have a significant beneficial immunomodulating activity in patients with selected autoimmune diseases.

Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis

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.

An antibody-based construct carrying DNA-mimotope and targeting CR1(CD35) selectively suppresses human autoreactive B-lymphocytes.

There is an urgent and unmet need for therapeutic agents targeting selectively disease-associated B-lymphocytes in autoantibody-mediated diseases. We have constructed a chimeric molecule able to cross-link cell surface immunoglobulin with the inhibitory complement receptor type 1 (CD35) on DNA-specific B cells from SLE (systemic lupus erythematosus) patients with the aim of selectively silencing them. This engineered molecule is made of copies of the DNA-mimotope peptide DWEYSVWLSN coupled to a monoclonal anti-CD35 antibody. We found that the DNA-like peptide chimera induced a dose-dependent decrease in the number of IgG anti-dsDNA antibody producing cells when PBMCs of lupus patients were cultured in its presence. Our data present evidence that clustering BCR and the inhibitory CR1 on disease-associated autoreactive B-lymphocytes selectively suppresses autoantibody production.

Simultaneous engagement of FcγIIb and CD22 inhibitory receptors silences targeted B cells and suppresses autoimmune disease activity

All B cell targeting therapeutic approaches used at present are unspecific and there is an urgent need for agents that silence selectively pathological autoreactive B lymphocytes only. We hypothesized that this aim could be achieved by chimeric antibodies that cross-link B cell immunoglobulin receptors with inhibitory receptors on the surface of the same targeted disease-associated cell. A hybrid molecule was constructed by coupling copies of the DNA-mimicking DWEYSVWLSN peptide and of the CD22-binding STN epitope with a free terminal sialic acid to a mouse monoclonal IgG antibody backbone. The DNA mimotope peptide binds to the immunoglobulin B cell receptor of pathological DNA-specific B cells of lupus mice, the STN epitope - to CD22 and the IgG by its Fc fragment - to FcgammaIIb on the surface of the same cell. Mass-spectra analysis showed that 4 STN epitopes plus 5 DNA mimotope peptides were coupled to a single light immunoglobulin chain and 4 STN - and 2 DNA mimotopes - to a heavy chain. Both FcgammaIIb and CD22 receptors on spleen cells from lupus MRL/lpr mice were phosphorylated after exposure to the chimeric antibody, indicating the involvement of both inhibitory pathways. The constructed chimera suppressed specifically in vitro as well as in vivo anti-DNA IgM and IgG antibody production and delayed the development of glomerulonephritis in the lupus-prone animals. The use of chimeric antibodies targeting two independent inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of pathological autoreactive B cells in autoimmune diseases.

Selective silencing of autoreactive B lymphocytes—Following the Nature's way

A novel approach for the selective silencing of targeted autoreactive B lymphocytes is reviewed that mimics the physiological mechanisms for suppressing B cell activity. It is based on the use of bi- or tri-specific chimeric antibodies that cross-link BCRs with a pre-selected antigen-binding specificity with one or more inhibitory types of receptors on the surface of the same disease-associated B lymphocyte. The effect of these engineered antibodies was proved to be specific as they only suppressed the production of the targeted pathological antibodies while sparing those with other specificities. The administration of the chimeric molecules to lupus-prone MRL/lpr mice resulted in decreased levels of disease-associated IgG autoantibodies and of proteinuria, in the prevention of cutaneous lesions, in decreased sizes of the lymphoid organs and in prolonged survival. These results prove that it is indeed possible to selectively silence unwanted B lymphocytes as well as to significantly delay the natural course of a spontaneous antibody-mediated autoimmune disease.

Re-establishing tolerance to DNA in humanized and murine models of SLE

DNA-specific B cells in SLE represent a logical target for therapeutic intervention. We hypothesize that it is possible to re-establish tolerance to native DNA in SCID mice with cells transferred from SLE patients or from lupus-prone MRL/lpr mice by administering chimeric molecules, containing a monoclonal antibody against inhibitory B cell receptors coupled to a peptide that antigenically mimics DNA. These protein-engineered molecules are able to co-crosslink selectively the antigen receptors of B cells possessing anti-native DNA specificity with the inhibitory surface receptors, thus delivering a strong suppressive signal.