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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

• the invention relates to a suppressor of disease-associated autoreactive B lymphocytes in autoimmune disorders with three different ligands incorporated into one single molecule and manifesting affinity to one activating and to two inhibiting B cell receptors.
• autoimmune diseases develop in approximately two percent of the human population. In these pathological states, the patients' own tissues and organs are attacked by the immune system by autoantibodies or by autoreactive T lymphocytes. Serious damage or destruction of the targeted tissue results from the autoimmune attack. Depending on the number of organs targeted, autoimmune diseases are divided into two groups - systemic and organ-specific. The anti-inflammatory and immunosuppressive drugs used presently to treat them in most cases diminish or delay the injury to the targeted tissue, but do not affect the primary disease itself. Even more, such medicines have serious and even life-threatening side-effects.
• the synthetic DWEYSVWLSN peptide is shown to mimic antigenically native (double stranded) DNA and to be bound by disease-associated anti-DNA antibodies [Putterman, C. et al. (2000), J Immunol 164,2542].
• Patent application PCT/BG04/00010A1 describes an agent for selective suppression of disease-associated B-lymphocytes, the agent being a chimeric antibody molecule that cross-links the B cell immunoglobulin receptors with preselected anti-self specificity with the inhibitory Fc ⁇ RllB receptors.
• This agent is active only on IgG receptor-expressing B lymphocytes.
• WO03/030835A3 relates to a method for designing bispecific antibodies for regulating immune responses, wherein said bispecific antibodies bind to one activating and to one inhibiting receptor. As one of the possible inhibiting receptors Fc ⁇ RIIB is chosen. CD22 is not mentioned.
• the bispecific antibodies according to the above cited document, are only active if administered with an agent that stimulates the expression of an inhibiting receptor or one activating receptor or with additional therapeutics.
• WO9707218 describes a fusion protein that cross-links activating antigen- specific receptors with inhibiting receptors on targeted B cells.
• this fusion protein binds only to the human Fc ⁇ RIIB (CD32) and targets only B cells with IgG, but not these with immunoglobulin M antigen receptors [Wakabayashi, C. et al. (2002), Science 298,2392].
• the invention relates to a suppressor of disease-associated autoreactive B lymphocytes with affinity to inhibitory B-lymphocyte receptors possessing three different ligands, incorporated into one single molecule.
• the suppressor possesses affinity to one activating receptor and to two inhibiting B cell receptors and is build of an immunoglobulin G backbone and of two synthetic components.
• the first of the three ligands is a part of the immunoglobulin G backbone and the second and third ligands are the two synthetic components ( Figure 1).
• the first synthetic component coupled to the immunoglobulin G backbone, is a peptide epitope with affinity to the activating immunoglobulin receptors on targeted autoreactive B cells. It is a DNA-mimotope peptide made of ten aminoacids - DWEYSVWLSN (SEQ ID NO.1), where a hexamethylene linker is added to the C-end of the peptide.
• the second synthetic component coupled to the immunoglobulin G backbone is a synthetic peptide GGPGG (SEQ ID NO. 2) with a hexamethylene group at its C-end.
• a STN epitope with a free terminal 2,6 sialic acid is coupled to the N-end of the peptide ( Figure 2).
• an immunoglobulin G backbone a mouse monoclonal IgG antibody is used. It is obtained from IP 2-11-1 hybridoma cells grown in protein-free CHO medium. The immunoglobulin fraction of the synthetic medium is isolated after the end of the cultivation by ammonium sulfate precipitation and subsequent dialysis against phosphate-buffered saline pH 7.2.
• the monoclonal IgG antibody is coupled to the modified DWEYSVWLS N and to the STN-containing peptides using 1-ethyl- 3(3'-dimethylaminopropyl) carboimidine.HCI (EDC) [Bauminger, S. et al. (1980) Methods in Enzymology. 70,151].
• the components for the construction of the trispecific immunoglobulin molecule are prepared as follows. A 1.5 mg/ml solution of the monoclonal IgG in phosphate buffer pH 6.0, 0.3 mg/ml solution of EDC in PBS pH 6.0 and 0.2 mg/ml solutions of both peptides in dimethylphormamide/ phosphate (ratio of 1 :9) buffer are made. 7.5 ml of the mixed peptide solution is added to 3.75 ml EDC and to 10 ml of the IgG solution, the volume is brought to 150 ml with phosphate buffered saline pH 6.0. The mixture is incubated for 16 hours at +4 0 C with constant stirring.
• This preparation contains the trispecific immunoglobulin molecules, composed of the monoclonal IgG backbone, the modified CD22- binding STN epitope and the DNA-mimicking DWEYSVWLSN peptide.
• the protein content of chimera solution is determined spectrophotometrically at 280 nm.
• the purity of the constructed chimera is analyzed by SDS-PAGE under non-reducing conditions.
• the ability of the trispecific immunoglobulin molecule according to the invention to engage the CD22 and Fc ⁇ Rllb inhibitory receptors and to suppress selectively pathological DNA-specific B lymphocytes with IgM and with IgG antigen receptors is analyzed in vivo in animals that develop spontaneously an autoimmune disease, namely systemic lupus erythematosus.
• the study showed that the trispecific immunoglobulin molecule bound the inhibitory receptors on targeted disease-associated DNA-specific B cells and suppressed the activity of the latter.
• the healthy animals do not excrete proteins with the urine. Proteinuria levels correlate with the severity of renal involvement in lupus.
• the administration of the trispecific immunoglobulin molecule according to the invention resulted in the suppression of the IgM and IgG anti-DNA antibody levels, of proteinuria and in the increased survival time of the animals ( Figures 3-5).
• Figure 1 shows a schematic drawing of the trispecific immunoglobulin molecule, according to the invention.
• the DNA-mimicking and the STN epitopes are coupled to an IgG backbone.
• Figure 2 represents a scheme of the constructed modified STN epitope.
• Figure 3 shows the effect of the trispecific immunoglobulin molecule according to the invention on the levels of anti-DNA antibodies in treated lupus-prone MRL/lpr mice.
• Figure 4 shows the effect of trispecific immunoglobulin molecule, according to the invention on albuminuria levels in treated MRL/lpr mice (* p ⁇ 0.05; **p ⁇ 0.01 and ***p ⁇ 0.001 relative to controls, unpaired f-test).
• Figure 5 proves that the administration of the trispecific immunoglobulin molecule, according to the invention increases survival in female lupus- prone MLR/lpr mice (*p ⁇ 0.05 relative to controls, unpaired f-test).
• a decapeptide DWEYSVWLSN (SEQ ID NO. 1), that mimics antigenically DNA, was also coupled to the IgG backbone.
• a hexamethylene group was also added to the C-end of this peptide.
• Example 2 Construction of the chimeric immunoglobulin molecule.
• a 1.5 mg/ml solution of the monoclonal IgG in phosphate buffer pH 6.0, 0.3 mg/ml solution of EDC in PBS pH 6.0 and 0.2 mg/ml solutions of both peptides in dimethylphormamide/ phosphate (ratio of 1 :9) buffer are made.
• 7.5 ml of the mixed peptide solution is added to 3.75 ml EDC and to 10 ml of the IgG solution, the volume is brought to 150 ml with phosphate buffered saline pH 6.0. The mixture is incubated for 16 hours at +4 0 C with constant stirring.
• This preparation contains the trispecific immunoglobulin molecules, composed of the monoclonal IgG backbone, the modified CD22-binding STN epitope and the DNA-mimicking DWEYSVWLSN peptide. ( Figure 1).
• the protein content of the solution is determined spectrometrically at 280 nm.
• the purity of the constructed chimeric antibody is determined by SDS- PAGE under non-reducing conditions.
• Example 3 Determinantion of the effect of the administration of the chimeric antibody in autoimmune animals, in particular in lupus-prone mice.
• the constructed trispecific immunoglobulin molecule has been administered intravenously twice weekly (20 ug/dose) to groups of ten 7- and of 18-weeks old female MRL/lpr mice. Control group have been injected with the same amount of a similar modified antibody molecule that lacked the STN epitope and another control groups have been treated with PBS alone. It is known that female mice from this strain spontaneously develop at the age of 7-9 weeks a lupus-like disease accompanied by the appearance of high levels of disease-associated anti-double stranded DNA antibodies. These levels have been significantly reduced in the group treated with the trispecific immunoglobulin molecule, according to the invention ( Figure 3).
• the lupus glomerulonephritis results in kidney failure that is a major cause for the death of the animals.
• the quantity of proteins in the urine a measure of the severity of kidney disease, has been determined by using a standard dry strip test (from Bayer, UK).
• the administration of the trispecific immunoglobulin molecule according to the invention delays the increase of albuminuria that is seen in the control animals (Figure 4).
• the treated mice survive significantly longer than the control ones ( Figure 5).

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• 2005
  • 2005-01-05 BG BG109003A patent/BG65954B1/bg unknown
• 2006
  • 2006-01-04 EP EP06703172A patent/EP1844075A2/de not_active Withdrawn
  • 2006-01-04 WO PCT/BG2006/000001 patent/WO2006072152A2/en not_active Ceased

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