BG65954B1 - Means for selective suppression of the activity of pathologic autoreactive b-cells - Google Patents

Abstract

The invention relates to a means for selective suppression of the activity of pathologic autoreactive B-cells in cases of autoreactive diseases. By their nature it is in the form of a chimerical molecule, comprising immunoglobulin G carrier and two synthetic components, the first one of which is specifically bound to the inhibition surfactant receptor CD22, and the second one -ûto the immunoglobulin receptors of the B-cells, identifying a selected autoantigene.

Description

Technical field

The invention relates to a means of selectively inhibiting the activity of pathologic autoreactive B cells in autoimmune diseases.

BACKGROUND OF THE INVENTION

It is known that due to a combination of poorly understood genetic factors and environmental influences, about 2% of people develop various autoimmune diseases. In them, the immune system begins to attack its own organs and tissues with specific autoantibodies or autoreactive 'Tymphocytes'. As a result of this autoimmune attack, the inflammatory process is unlocked, which in most cases leads to destruction or serious damage to the target organ or tissue. Depending on the number of organs attacked, autoimmune diseases are divided into systemic and organ-specific. Modern anti-inflammatory and immunosuppressive agents only delay the development of these diseases but do not affect their root cause. In addition, these medications have harmful side effects.

Pathologic autoreactive B cells are a logical target for therapeutic intervention in autoimmune diseases. In mouse (NZB x NZW) F1 mice that spontaneously develop lupus-like disease, autoantibodies associated with it are known to be secreted by B-1 cells. Prolonged and selective removal of these cells from an early age prevents the development of glomerulonephritis and significantly increases the life span of these animals (1). This approach is not effective in patients with lupus and MRL / Ipr mice also developing spontaneously lupus-like disease, since the anti-DNA specific B cells in them are of type B-2.

Studies by Courts, S. M. et al., 1996 and Furie, R., et al., 2001 show that selective inactivation of pathologic B cells can induce single-signal energy in an antigen-specific manner. For this purpose, the B cell surface immunoglobulin receptor engages a structure that contains oligovalent B cell epitopes immobilized on a non-immunogenic carrier. Such a structure should not contain T cell epitopes. An example of such a B cell tolerogen is LJP394, which in vivo inactivates B cells specific for native DNA in BXSB mice developing spontaneous, lupus-like disease. Administration of this immunomodulator prolongs their survival and suppresses renal impairment. LJP394 has already shown promising results in the second phase of clinical trials / 2,3 /.

BG 108155 describes an agent for the selective inhibition of pathologic autoreactive B cells, which is a chimeric antibody molecule that binds both to B-lymphocyte immunoglobulin receptors with specificity for DNA and to FcraMaRIIB-type inhibitors. The action of this agent is limited to B cells expressing immunoglobulin receptors of the IgG isotype. However, none of the documents cited refers to the CD22 receptor used as a target molecule for immunotherapy.

On the other hand, the suppressive effect of the CD22 surface receptor on B-lymphocyte function is well known (4-6). It is not yet clear what its physiological ligand is, but the receptor has been found to bind to polysaccharides with the final 2,6 sialic acid. When the B lymphocyte is in the inactive state, CD22 is in the inactive (masked) form (7). DNA-specific B cells are undoubtedly activated in autoimmune disease (lupus) and their surface receptor CD22 is able to interact with their natural or purposefully synthesized ligands. The receptor is composed of seven immunoglobulin-type domains expressed on the cell surface, and in the cytoplasmic tail contains signaling motifs responsible for the transmission of an inhibitory intracellular signal. It is known that when acting simultaneously

65954 Bl but on CD22 by its ligand or monoclonal antibody and membrane immunoglobulin receptor of the IgM isotype of the same B cell, its activity is inhibited (8, 9).

The decapeptide of the amino acid sequence DWEYSVWLSN is known to resemble native DNA in antigenic terms. It has been shown that it recognizes pathological mouse IgG antibodies against native DNA, and upon immunization of mice with the peptide induces the synthesis of antibodies of the same specificity and lupus-specific renal lesions are observed (10).

SUMMARY OF THE INVENTION

In essence, the invention is a chimeric molecule that selectively inhibits the activity of pathologic autoreactive cells by acting on their CD22 receptor inhibitory activity.

For the selective suppression of pathologic DNA-specific cells according to the invention, an agent is created that is a chimeric mouse immunoglobulin G molecule molecule consisting of two separate synthetic components, one of which binds specifically to the CD22 surface receptor inhibitor and the other to immunoglobulin receptors of B cells with anti-DNA specificity (Fig. 1).

The first of these two synthetic components is a pre-modified synthetic peptide of 10 amino acids, in particular DWEYSVWLSN (SEQ IDNO. 1) / 10 /, which antigenically resembles native DNA and to which a linker representing the hexamethylene group is added to the C-terminus.

The second synthetic component involved in the chimeric molecule according to the invention is also a pre-modified synthetic peptide, in particular the pentapeptide GGPGG (SEQ ID NO. 2) with an oligosaccharide SIN epitope (11) added to its N-terminus containing a free terminal sial residue. To the end of this synthetic peptide was added a linker representing a hexamethylene group. For its part, the pentapeptide GGPGG is covalently linked via a hexamethylene diamine linker to a solid-phase synthesis resin. For the pentapepide, the epitope SIN (commercial product of Calbiochem, Darmstadt, DE) is bound via the serine residue of the latter, leaving the sial residue free. The newly synthesized synthetic peptide has the following schematic view (Figure 2).

As carrier molecules, a mouse monoclonal IgG2a antibody derived from hybridoma IP 2-11-1 grown on synthetic serum-free culture medium CHO or a pool of mouse immunoglobulins G, obtained from a mixture of sera from healthy mice (obtained by Prof.

G. Lazslo, Otvos University, Budapest).

The immunoglobulin fraction of the synthetic culture medium was purified by precipitation with ammonium sulfate and that of the mixed serum by precipitation with ammonium sulfate and subsequent fractionation on an immunoaffinity column containing immobilized protein G (commercially available from Pharmacia).

The binding of monoclonal or polyclonal mouse immunoglobulins G to the modified synthetic peptides DWEYSVWLSN and the STN-bearing peptide is accomplished by known methods using the binding reagent 1-ethyl-3 (3'-dimethylaminopropyl) carboimidine.CH1 (EDC) /.

To this end, prepare a solution of each peptide in dimethylformamide / phosphate buffer and EDC in phosphate buffer pH 6.0. A solution of peptides and EDC was added to the immunoglobulin solution, the reaction mixture was incubated with constant stirring and dialyzed against phosphate-buffered saline at pH 7.0. The resulting solution was concentrated using ultrafiltration. It contains a chimeric antibody molecule composed of the murine immunoglobulin carrier, the STN epitope binding to CD22, and the modified peptide DWEYSVWLSN.

The protein content of the solution was determined spectrophotometrically at 280 nm. The purity of the resulting chimera was analyzed using SDS-PAGE under non-reducing conditions.

The ability of the resulting chimeric monoclonal antibody of the invention to bind specifically to the CD22 receptor and to selectively inhibit pathologic DNA-specific B cells (Fig. 3) was tested in in vivo ex.

65954 Bl periments on animals developing spontaneous autoimmune disease, in particular systemic lupus. The amount of urine excreted protein (measure of renal impairment in lupus) was determined by standard dry tests (Bayer, UK). A healthy animal does not excrete a protein in its urine and its presence indicates the presence of a kidney disease process. In vivo studies have shown that the chimeric molecule of the invention specifically binds to the CD22 receptor and inhibits pathologic DNA-specific B cells, thereby weakening the activity of the disease autoimmune process. Administration of the chimera according to the invention to animals with systemic lupus leads to a later onset of the disease, suppression of the development of renal complication and prolongation of their life (Figs. 4 and 5).

Explanation of the annexed figures

Figure 1 is a schematic view of the engineered chimeric molecule.

Figure 2 presents a schematic of the modified STN epitope.

Figure 3 illustrates the cellular activity inhibiting intracellular signals after simultaneous binding of the immunoglobulin to the CD22 receptor at the surface of target B lymphocytes.

Figure 4 shows proteinuria levels in young MRL / Ipr mice treated with chimeric molecules according to the invention. The white bars indicate the mean levels and standard deviation of the chimera-treated mice and the black ones of the controls (** p <0.01; ♦ p <0.05, non-digit t-test).

Figure 5 shows that administration of the chimeric molecule leads to a significant prolongation of the survival time in MRL / Ipr mice with autoimmune disease (spontaneous lupus). The squares denote the survival of the animals in the experimental group and the triangles indicate the survival of the controls (* p <0.05, non-digit t-test).

Examples of carrying out the invention

The invention is characterized by the following non-limiting embodiments.

Example 1. Preparation of modified synthetic peptides

For the preparation of the selective inhibitor of the activity of pathologic autoreactive B cells according to the invention, a pre-modified synthetic peptide GGPGG (SEQ ID NO. 2) with an oligosaccharide STN epitope attached to its N-terminus (purchased from Calbiochem, Darmstadt, DE) with free terminal sial residue. A linker representing the hexamethylene group is attached to the C-terminus of the synthetic peptide. The synthesis was performed using the Fmoc method (13).

The synthetic peptide of 10 amino acids DWEYSVWLSN (SEQ ID NO. 1) (10), which is antigenically similar to double stranded DNA, is used. A linker representing the hexamethylene group is also added to its C-terminus.

Example 2. Construction of a chimeric immunoglobulin molecule

The two modified peptides were purified by HPLC (14) and then coupled to the monoclonal or polyclonal mouse immunoglobulins obtained by the described methods. A solution of 0.4 mg of each peptide in dimethylformamide / phosphate buffer was prepared in advance. at a ratio of 1: 9 and EDC in phosphate buffer pH 6.0-0.3 mg / ml. A solution of peptides and EDC was added to the immunoglobulin solution and the volume was adjusted to 150 ml with phosphate buffer pH 6.0. The reaction mixture was incubated for 16 h at 4 ° C with constant stirring. It was then dialyzed at 4 ° C for 16 h against 200 volumes of pH 7.0 phosphate-buffered saline. The resulting solution was concentrated to a final volume of 20 ml using ultrafiltration through 5 kDa pore size filters (from Amicon, Millipore Corp.). It contains a chimeric antibody molecule composed of the murine immunoglobulin carrier, the STN epitope binding to CD22, and the modified peptide DWEYSVWLSN (Figure 1).

The protein content of the solution was determined spectrophotometrically at 280 nm. The purity of the resulting chimera was analyzed using SDS-PAGE under non-reducing conditions.

Example 3. Determining the effect of at

65954 The administration of the chimeric molecule on mice with advanced autoimmune disease, in particular with spontaneous lupus.

The resulting chimeric molecule was treated with a group of female MRL / Ipr mice at 6 weeks of age. Each mouse received 20 [mu] g of the chimera twice weekly by intravenous route. The control group received intravenous injections with phosphate-buffered saline. Mice in this inbred line develop spontaneously at about 7-9 weeks of age a disease very close to systemic autoimmune lupus erythematosus in humans. As a rule, animal disease ends with irreversible kidney damage that causes them to die.

The amount of urine excreted protein (measure of renal impairment in lupus) was determined by standard dry tests (Bayer, UK). A healthy animal does not excrete a protein in its urine and its presence indicates the presence of a kidney disease process. The administration of the chimeric molecule according to the invention slows the onset and weakens the level of albuminuria (Fig. 4). Administration of the same agent also showed a significant lengthening of the lupus mice (Fig. 5).

Claims (5)

Claims 1. A selective inhibitor of the activity of pathologic autoreactive B cells, characterized in that it is a chimeric molecule consisting of carrier immunoglobulin G and two synthetic components, the first of which binds specifically to the CD22 surface receptor inhibitor, and the second to immunoglobulin receptors of B cells recognizing a selected autoantigen. An agent according to claim 1, characterized in that the first of the two synthetic components is composed of the peptide DWEYSVWLSN (SEQ ID NO. 1) and the hexamethylene group at the C-terminus. The agent according to claim 1, characterized in that the second synthetic component is composed of the peptide GGPGG (SEQ ID NO. 2) with the STN epitope at the N-terminus and hexamethylene group at the C-terminus. The agent according to claim 3, characterized in that the STN epitope has a free terminal sial residue. Use of an agent according to claims 1 to 4 for the manufacture of a medicament for selectively suppressing the activity of pathologic autoreactive B cells.