BG65715B1 - Device for selective suppression of pathologic dna-specific b cells - Google Patents

Abstract

The invention relates to a device for selective suppression of pathologic DNA-specific B cells, in particular in the system lupus erythematosus, which is a bi-specific chimerical monoclonal antibody.The invention also relates to a method for the preparation of the chimerical monoclonal antibody and to its administration for selective suppression of pathologic DNA-specific B cells.

Description

Technical field

The invention relates to a means of selectively suppressing pathological DNA-specific B cells, in particular in systemic lupus erythematosus.

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 these patients, the immune system begins to attack its own organs and tissues with specific autoantibodies or with autoreactive T lymphocytes. As a result of this autoimmune attack, an 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.

Systemic lupus erythematosus is a typical systemic autoimmune disease. In it, autoantibodies with specificity to double stranded (native) DNA, to chromatin, ribosomes and other nucleoprotein complexes (1,2) appear in the serum. As the disease progresses, the kidneys (glomerulonephritis), central nervous system (psychoses), joints (arthritis), skin and other organs and tissues are affected. Modern anti-inflammatory and immunosuppressive agents delay the development but do not affect the root cause of the disease. In addition, they have harmful side effects.

Pathologic DNA-specific B cells are a logical target for therapeutic intervention in patients with lupus erythematosus. The breakdown of DNA tolerance and the emergence of B cells with similar receptors is considered by most researchers to be a major cause of disease unlocking.

Because of the important role played by the cells secreting autoantibodies to native DNA in the induction of systemic lupus erythematosus, their selective removal or suppression is an important goal in disease control attempts.

Genetically predisposed to lupus erythematosus animals (US 6232522) are also known to monitor characteristic immune responses and critical human disease-like manifestations. It is also known that in mouse line (NZB x NZW) F1 that spontaneously develops a lupus-like disease, its associated autoantibodies are 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 (3). This approach is not effective in patients with lupus and MRL / lpr mice also developing spontaneously lupus-like disease because the anti-DNA specific B cells in them are of type B-2.

Studies by Coutts, S. M. et al., 1996 and Furie, R., et al., 2001 have shown that selective inactivation of pathologic B cells can produce one-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 results in suppression of renal impairment. LJP394 has already shown promising results in the second phase of clinical trials (4,5).

An approach is known in which administration of MRL / lpr mice with a lupus of phosphorylated peptide from the autoantigen ribonucleoprotein causes a decrease in the level of antibodies against IgG class DNA and protein content in urine (6). There is also an approach related to the administration of a monoclonal antibody agonist specifically to the CD 13 7 co-receptor suppresses lupus activity in the same mouse type (7).

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Publications of WO 9941285A1, WO 00011732 and EP 1210375A1, EP 1292621 A1 describe means and methods for the diagnosis and response of lupus by binding to the Fc receptor of immunoglobulin molecules, the receptors being selected from the group consisting of the Psaalpha receptor (Fcalpha receptor) FcraMaR).

WO 2001/009186 (PCT / US2000 / 020158) discloses a therapeutic agent comprising at least one portion that binds to the Fc receptor, e.g. The FcraMa receptor or the Psealpha receptor and at least one other moiety that binds to another target, such as an antigen or a tumor cell or pathogen. Therefore, these molecules can be used to induce or enhance the immune response, both in vivo and in vitro, against a normally non-immunogenic protein.

However, none of the cited patent documents mention FcraMaRI1B receptor binding.

An achievement in this respect is Haemerling U. (1975), which has the monoclonal antibody Lyl7.2 that binds specifically to the human FcramBPv receptor inhibitor but not the FcraMaRIIa receptor receptor (8).

It is known that when an immune complex composed of antigen and IgG binds simultaneously with the FcraMaR for the Fc fragment of IgG type Pb (FcraMaRIlB) and the membrane immunoglobulin receptor of the B cell, its activity is inhibited (9). This is the only receptor in the FcraMaRII group that B cells express on their surface (Fig. 1). It has also been found that FcraMaRIlB transfected into B lymphocytes, which do not express Fc receptors, effectively inhibits cell activity by the above mechanism (10). Mice defective in FcraMaRIlB respond with increased production of antibodies to T-dependent or T-independent antigens (11).

A peptide of the amino acid sequence DWEYSVWLSN is known, which resembles the native DNA in antigenic terms. It has been shown that it recognizes pathological murine IgG antibodies against native DNA; furthermore, immunization of mice with the peptide induces the synthesis of antibodies of the same specificity and lupus-specific renal lesions are observed (12).

It is also known that rat ATG hybrid registered in ATCC number HB-197 secretes a monoclonal antibody specific to both isoforms of the murine CD32 receptor (or FcraMaRII) -FcraMaRIIa and FcraMaRIlB.

SUMMARY OF THE INVENTION

In essence, the invention is a chimeric monoclonal antibody for the selective suppression of pathological DNA-specific B cells. As explained above, some of the disease autoimmune processes, including systemic lupus erythematosus, are characterized by the production of native antibodies specific to native DNA. For the selective suppression of pathological DNA-specific cells according to the invention, an agent is created that is a chimeric monoclonal antibody molecule that binds specifically to both the B-cell inhibitory receptor and the B-cell immunoglobulin receptors with anti-native DNA specificity . To prepare this agent according to the invention, a pre-modified synthetic peptide of 10 amino acids, in particular the peptide DWEYSVWLSN, which antigenically resembles native DNA and to which a hexamethylene group linker is added at the C-terminus (Figure 2).

At the same time, rat 2.4G2 hybrid (ATCC HB-197), a secreting monoclonal antibody against the murine CD32 receptor (FcraMaRII), was cultured in protein-free CHO medium (Gibco, Gaithersburg, MD) at 37 ° C in an atmosphere with a relative humidity of 90% and 5% carbon dioxide content. Upon completion of cultivation, the monoclonal antibody secreted in the medium was purified by precipitation with ammonium sulfate. The resulting monoclonal antibody contains no other specificity immunoglobulins and non-immunoglobulin impurities and has a purity of 90-95%.

After the purification step, the monoclonal antibody obtained in the manner described was coupled to the modified synthetic peptide DWEYSVWLSN by 1-ethyl-3 (3'-dimethylaminopropyl) carboimidine.

65715 Bl (EDC). To this end, a solution of 0.4 mg peptide in dimethylformamide / phosphate buffer in a ratio of 1: 9 and EDC in phosphate buffer pH 6.0-0.3 mg / ml are pre-prepared. A solution of the peptide and EDC was added to the antibody 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, on the one hand, of a monoclonal antibody against the murine CD32 receptor (FcraMaRIIa and PrgamaKPv isoforms) and, on the other hand, of the modified DWEYSVWLSN peptide, the chimeric molecule has the ability to bind specifically to the B-receptor. FcraMaRIIb cell receptor and B-cell immunoglobulin receptors with anti-native DNA specificity. This bispecificity of the chimera is manifested both when it includes the entire molecule of the monoclonal antibody and its F (ab ') ₂ or Fab fragments. It also manifests itself if a portion of the monoclonal antibody molecule of animal origin is replaced by sections of a human immunoglobulin molecule.

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.

FIG. 3 illustrates the binding of the DWEYSVWLSN peptide to the monoclonal antibody molecule as demonstrated by reverse phase HPLC.

Flow cytometry (Figure 4) demonstrates that there is no change in the ability to bind to the murine CD32 receptor after conjugation of the monoclonal antibody obtained from hybridoma 2.4O2 (ATCC HB-197) with the peptide.

The ability of the chimeric monoclonal antibody obtained according to the invention to bind specifically to the CD32 receptor (FcraMaRIIb) and to selectively inhibit pathologic DNA-specific B cells has been investigated, both in vitro and in vivo experiments.

To this end, the immunomodulatory effect of the engineered hybridoma is first determined in vitro under gastric cells derived from lupus mouse MRL / lpr mice, which synthesize and secrete DNA-specific IgG antibodies in the medium. Cultivation was carried out either in the presence of bacterial lipopolysaccharide or in the presence of the chimeric monoclonal antibody of the invention, as well as the same concentration of a control chimera composed of a 2.4G2 monoclonal antibody conjugated with the same amino acidsW, as well as N peptides but in sequence. The results show that the chimera according to the invention suppresses the secretion of IgG antibodies specific against native DNA (Figure 5).

The level of IgG class antibodies against double stranded DNA is an indicator of disease process activity.

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 secrete a protein in its urine and the presence of the latter testifies to the presence of a kidney disease process.

The in vivo studies are intended to demonstrate that the chimeric molecule of the invention specifically binds to the FcraMaRIIb receptor and deactivates pathologic DNA-specific B cells, thereby weakening the activity of the disease autoimmune process. The results show that administration of the chimera according to the invention to animals with systemic lupus leads to attenuation of the disease manifestations, inhibits the development of renal complication and prolongs their life.

Explanation of the annexed figures

Figure 1 shows the simultaneous engagement of the immunoglobulin antigen receptor and the inhibitory FcraMaRIIb on the Cell, leading to its deactivation.

Figure 2. Schematic view of the constructed chimeric molecule. The small circles represent the linker and the squares represent the decapeptide.

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Figure 3. Reverse-phase HPLC analysis of the unconjugated 2.4G2 antibody and of the chimeric molecule according to the invention.

Figure 4. Flow cytometry analysis of the ability of the chimeric molecule to bind to the FcraMaRIIb cell receptor:

A - negative control - cells only

B - commercially available 2.4G2 + anti-rat biotinylated antibody + streptavidinFITC

C - chimera according to the invention + anti-rat biotinylated antibody + streptavidin

D - commercial preparation of 2.4G2 antibody (0.5 microg / tube) + commercial preparation of 2.4G2-FITC (10 microg / tube)

E - chimera according to the invention (2.0 microg / tube) + commercial 2.4G2 antibodyFITC (10 microg / tube)

Figure 5 shows the in vitro inhibition of the production of anti-DNA antibodies:

Rhombuses indicate the level of antibodies in cell culture in the presence of 100 ng / ml chimeric molecule according to the invention.

Squared - in the presence of 100 ng / ml control chimeric molecule.

Triangles - in the presence of added buffered saline.

In circles - in the presence of 10 µg / ml bacterial lipopolysaccharide.

The standard deviations from the mean were below 5%.

Figure 6. Levels of anti-native DNA IgG antibodies in young 7-week-old MRL / lpr mice treated twice weekly for 6 weeks with 20 µg chimeric molecules:

Squares - injected with buffered saline.

Triangles - with control chimeric molecule.

Circles - with a chimeric molecule according to the invention.

Figure 7. Proteinuria level in young 7-week-old MRL / lpr mice treated twice weekly for 6 weeks with 20 µg chimeric molecules:

Squares - injected with buffered saline.

Triangles - with control chimeric molecule.

Circles - with the chimeric molecule of the invention.

Figure 8. Levels of anti-native DNA IgG antibodies in mature 17-week-old MRL / lpr mice treated twice weekly for 6 weeks with 20 μg per dose of chimeric molecules:

Squares - buffered saline.

Triangles - with control chimeric molecule.

Circles - with the chimeric molecule according to the invention.

Figure 9. Proteinuria level in mature 17-week-old MRL / lpr mice treated twice weekly for 6 weeks with 20 µg chimeric molecules:

Squares - injected with buffered saline.

Triangles - with control chimeric molecule.

Circles - with the chimeric molecule of the invention.

The invention is illustrated by the following exemplary embodiments.

Example 1. Preparation of a chimeric monoclonal antibody

10 ⁸ cells from rat hybridoma 2.4G2 (ATCC HB-197), a secreting monoclonal antibody against the murine CD32 receptor, in particular FcraMaRIIb, were cultured in protein-free CHO medium (Gibco, Gaithersburg, MD) and at 37 ° C, in an atmosphere with a relative humidity of 90% containing 5% carbon dioxide.

Upon completion of cultivation, the monoclonal antibody secreted in the medium was purified by precipitation with ammonium sulfate. For this purpose, equilibrate volumes and saturated ammonium sulfate solution are mixed and the mixture is left for 16 h at + 4 ° C with constant stirring. After centrifugation for 30 min at 5,000 x g, the supernatant was removed and the pellet resuspended 10 times less than the original volume of pH 7.0 phosphate-buffered saline and dialyzed twice against 500 volumes of the same buffer.

After the purification step, the binding of the monoclonal α5 obtained is accomplished

65715 B1 titanium with the synthetic peptide modified as described above using 1-ethyl3 (3'-dimethylaminopropyl) carboimidine.CH1 (EDC). The following solutions are pre-prepared: 1.5 mg / ml monoclonal antibody in phosphate buffer pH 6.0; 0.4 mg / ml peptide in dimethylformamide / phosphate buffer and 0.3 mg / ml EDC in phosphate buffer pH 6.0. To 10 ml of antibody solution was added 7.5 ml of peptide solution and 3.75 ml of EDC 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 solution of the resulting chimeric molecule was concentrated to a final volume of 20 ml using ultrafiltration through 5 kDa pore size filters (Amicon, Millipore Corp.).

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.

Figure 3 illustrates the fact of binding of the DWEYSVWLSN peptide to the monoclonal antibody molecule against the CD32 receptor as evidenced by the reverse phase HPLC method.

The result is a chimeric monoclonal antibody that has the ability to bind to its specific CD32 antigen receptor. The latter was demonstrated by flow cytometry (Figure 4), after which it was demonstrated that there was no change in the ability to bind to the murine CD32 receptor of either the parent monoclonal antibody or the chimeric obtained after chemical conjugation with the peptide.

Example 2. Determination of the immunomodulatory effect of engineered hybridoma under in vitro conditions

Glandular cells derived from lupus mouse MRL / lpr mice cultured in vitro synthesize and secrete DNA-specific IgG antibodies in the medium. The cultivation was carried out in the presence of 5 µg / ml bacterial lipopolysaccharide from E. coli, or in the presence of 100 ng / ml of the chimera according to the invention, or in the presence of the same concentration of the control chimera composed of the peptide conjugated 2.4G2 monoclonal antibody WSLDYWNESV, which contains the same amino acids as the DWEYSVWLSN peptide but is sequenced. The results show that the chimera according to the invention suppresses the secretion of IgG antibodies against native DNA (Figure 5).

Example 3. Methods for Measuring Levels of IgG Antibodies against Native DNA and Urinary Protein Concentration

The level of IgG class antibodies against native DNA was determined by immunoassay (ELISA) according to the method described in the literature (13). The appearance of these autoantibodies is associated with the onset of the disease and their levels correlate with the activity of the disease process.

Bovine thymus double-stranded DNA (Sigma) was treated with nuclease S1 to remove inevitably present regions of such strands from single-stranded DNA and biotinylated using a nucleic acid biotinylation kit (Vector Laboratories, UK). Dilutions of the test liquids (cell culture supernatants or sera) starting at 1:50 to 1: 2700 were mixed with a standard amount of biotinylated DNA solution and after incubation, the mixture was transferred to the wells of a plate loaded with avidin. After new incubation and washing, the presence of mouse IgG adsorbed on the plate was determined by adding an alkaline phosphatase anti-mouse IgG antibody conjugate.

The amount of urinary protein excreted as a measure of renal impairment in lupus was determined by a dry urine test (Bayer, UK).

Example 4. Investigation of the immunomodulatory activity of chimeric monoclonal in vivo in young animals

The ability of the chimeric monoclonal antibody obtained according to the invention to bind specifically to target receptors (FcraMaRIIb and immunoglobulin B-lymphocyte receptor with DNA specificity) was assayed in vivo.

For this purpose, a group of 15 females is formed

7-week-old MRL / lpr mice that are still healthy at this age, but the first signs of autoimmune lupus disease are

65715 Bl forthcoming. A group of five mice was treated twice weekly by intravenous route with 0.1 ml phosphate-buffered saline for 6 weeks. A second group of 5 animals was administered 20 [mu] g of the chimera according to the invention, and in another group of 5 animals 20 [mu] g of the control chimera according to Example 2. Blood was taken once every two weeks from the animals, the sera were frozen and stored at -20 ° C, after which the level of IgG class anti-DNA antibodies was determined.

The results show that intravenous administration according to the described scheme of administration of the chimera according to the invention leads to a delayed increase in the level of anti-DNA antibodies and the appearance of protein in urine (Figs. 6 and 7).

Example 5. Investigation of the immunomodulatory activity of chimeric monoclonal in vivo in adult animals with advanced lupus form

A group of 15 17-week-old MRL / lpr mice was divided into three subgroups of 5 animals each. At this age, they are already in an advanced stage of the disease. The first group of five mice was treated twice weekly by intravenous route as in Example 4 with 0.1 ml phosphate-buffered saline, 20 μg of the chimera according to the invention was administered to the second group of 5 animals, and to the third group of 5 animals, 20 [mu] g of the control chimera according to Example 2.

In animals treated with saline by intravenous route, kinetics at the level of anti-DNA antibodies showed an upward trend, maximum levels were reached by week 19, then a slight decrease followed, and only one animal in the group experienced the end of the experiment (23rd) week).

The intravenous administration of the chimera according to the invention results in the maintenance of a persistently low level of disease antiDNA antibodies in the first 4 weeks after initiation of infusions, whereas the control chimera shows intermediate results (Fig. 8).

The administration of the chimera according to the invention causes the proteinuria level to be kept at the same level until the end of the experiment, whereas in saline treated group it reaches very high values (15 g / Ι) before the death of animals from this group.

Claims (9)

Claims 1. A means of selectively suppressing pathologic DNA-specific B cells, in particular in systemic lupus erythematosus, characterized in that it is a chimeric monoclonal antibody that binds specifically to both the B cell receptor inhibitor and the membrane immunoglobulin autoreactive B cell receptor. 2. A pathological selective inhibitor of pathologic DNA-specific B cells according to claim 1, wherein the chimeric monoclonal antibody is composed of a native antibody and a pre-modified synthetic peptide mimicking antigenically double stranded DNA. 3. A method for producing a pathogen selective inhibitor of pathogenic DNA-specific B cells according to claims 1 and 2, characterized in that it comprises binding of a pre-modified synthetic peptide mimicking double stranded DNA with a monoclonal antibody against inhibitory B -cellular receptor in the presence of 1-ethyl-3 (3'-dimethylaminopropyl) carboimidine.CH1 and subsequent incubation, dialysis and concentration of the solution. A method for the preparation of an agent according to claims 1 to 3, characterized in that the pre-modified synthetic peptide mimicking antigenically double stranded DNA consists of 10 amino acids. A method for preparing an agent according to claims 1 to 4, characterized in that the pre-modified synthetic peptide has the amino acid sequence DWEYSVWLSN. Method for the preparation of an agent according to claims 1 to 5, characterized in that a hexamethylene group is added to its C-terminus for the preliminary modification of the synthetic peptide. A method for the preparation of an agent according to claims 1 to 3, characterized in that the monoclonal antibody against the B cell receptor inhibitor is derived from rat ATCC HB-197 hybridoma. 65715 Bl A method for the preparation of an agent according to claims 1 to 3 and 7, characterized in that the monoclonal antibody is in an intact form, fragmented to F (ab ') ₂ or Fab fragments, or a portion of its molecule is replaced by regions of human immunoglobulin molecule. Use of the chimeric monoclonal antibody according to claims 1 to 8 for the selective suppression of pathological DNA-specific B cells.