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  • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• compositions comprising a chimaeric TNF binding protein
• the present invention is directed to the use of a chimaeric TNF binding protein which comprises a soluble part of the human p55- or the p75-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human immunoglobulin or a pharmaceutically acceptable salt thereof for the method of treatment autoimmune diseases, which are frequently associated with inflammatory processes, e.g. rheumatoid arthritis, juvenile onset type I diabetes mellitus, systemic lupus erythematosus, thyroiditis, especially multiple sclerosis and for the preparation of a pharmaceutical composition for the treatment thereof.
• autoimmune diseases which are frequently associated with inflammatory processes, e.g. rheumatoid arthritis, juvenile onset type I diabetes mellitus, systemic lupus erythematosus, thyroiditis, especially multiple sclerosis and for the preparation of a pharmaceutical composition for the treatment thereof.
• MS Multiple Sclerosis
• CNS central nervous system
• MBP myelin basic protein
• MS is characterised by focal accumulations of inflammatory cells, edema and demyelination in CNS tissue. It is an old finding that at autopsy more focal lesions are found than would have been predicted from the clinical status.
• the lesions start as a perivenular infiltrate of mononuclear cells in white matter with ensuing edema.
• the infiltrating cells appear to mediate the selective destruction of myelin sheaths leaving the axons intact.
• a ⁇ trocyte proliferation leads to a gliotic scar, the so- called MS plaque, of 0.1 to several cm diameter.
• the clinical course of MS may be remitting/relapsing with a wide range in the duration of the disease free interval, or chronically progressive suggesting that the underlying pathophysiologic processes are under heterogeneous control.
• TNF producing cells in MS lesions.
• the majority of the TNF positive cells were morphologically classified as reactive fibrous astrocytes, and double imniuno ⁇ taining experiments confirmed that most reactive cells were astrocytes, the rest being monocyte / macrophages [Hofman et al., J. Exp. Med. 170. 607-612 (1989)].
• An independent study confirmed the positive immunoreactivity for TNF in astrocytes and monocytes in MS plaques, and also investigated lymphotoxin (LT) immunoreactivity.
• LT lymphotoxin
• TNF concentrations in serum and cerebrospinal fluid (CSF) of MS patients have been measured by a number of investigators. There is a consensus that a significant percentage number of MS patients has elevated TNF in CSF, and much less so in serum. The concentration of TNF does not directly relate to CSF cell content suggesting that CSF TNF content might reflect TNF production in and around MS lesions, especially in view of the frequent paraventricular location of lesions.
• EAE experimental encephalomyelitis
• the chimaeric TNF binding protein comprises the soluble part of the human p55-TNF-receptor is an object of the present invention.
• a furthermore preferred embodiment of the present invention is such a use wherein the chimaeric TNF binding protein comprises all domains except the first domain of the constant region of the heavy chain of human immunoglobulin which means that the soluble part of the human p55-TNF- receptor is fused at its C-terminal end to the hinge region of the immunoglobulin heavy chain.
• immunoglobulin can be either IgG, IgA, IgM or IgE, especially IgG, like IgGl or IgG3.
• a soluble part of the human p55- or the p75-TNF-receptor means in the context of the present invention either the complete extracellular domain of one of these receptors or a part thereof which still binds human TNF, e.g. in case of the p55-TNF-receptor the extracellular domain lacking the first 12 N-terminal amino acids.
• an object of the present invention to provide a method of treating an autoimmune disease in a mammal comprising administering to said mammal a therapeutically effective amount of a chimaeric TNF binding protein or a salt thereof as defined above which ameliorates the effects of such autoimmune disease, specifically such a method wherein the autoimmune disease is multiple sclerosis.
• TNF binding proteins or parts thereof as described in the following patent publications can be used for the preparation of such chimaeric TNF binding proteins : EP 308 378, EP 422339, GB 2 218 101, EP 393438, WO 90/13575, EP 398 327, EP 412486,
• chimaeric TNF binding protein as used throughout the specification comprises also such proteins in which any amino acid of the immunoglobulin part or the TNF binding part has been deleted or substituted by one or more amino acids or one or more amino adds have been added as long as the TNF binding part still binds TNF and the immunoglobulin parts shows one or more of its characteristic properties.
• the immunoglobulin part consists of all domains including the hinge region except the first domain of the constant region of the heavy chain such hinge region may lack the first five N-terminal amino acids.
• Such chimaeric TNF binding protein muteins can be prepared by methods known in the art and described e.g. by Sambrook et al.
• chimaeric TNF binding protein of the present invention can be also used in modified form, e.g., when coupled to chemical entities without altering its basic biological activity.
• a preferred and well known modification is the coupling to water soluble polymers, e.g., polyethylene glycols or polypropylene glycols, within a wide range of molecular weight of, e.g., from 500 to 20 * 000 daltons. This leads to protected proteins, which could be substantially non-immunogenic.
• chimaeric TNF binding proteins of the present invention can be in the form of a pharmaceutically acceptable salt.
• Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
• Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid and salts with organic acids such as, for example, acetic acid or oxalic acid.
• the chimaeric TNF binding protein to be used for the purpose of the present invention is preferably administered parenterally by injection, although other effective administration forms, such as intraarticular injection, or transdermal iontophoresis are also possible.
• One preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used.
• the primary solvent in such a carrier may be either aqueous or non-aqueous in nature.
• the carrier may contain other pharmacologically acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
• the carrier may contain still other pharmacologically acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the chimaeric TNF binding protein.
• excipients are those substances usually and customarily employed to formulate dosages for parental administration in either unit dose or multi-dose form.
• the therapeutic composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or in dehydrated form, e.g. as lyophilized powder.
• Such formulations may be stored either in a ready to use form or a form requiring reconstitution immediately prior to administration.
• the preferred storage of such formulations is at temperatures at least as low as 4°C and preferably at -70°C. It is also preferred that such formulations are stored and administered at or near physiological pH.
• a possible dosage range for the treatment of multiple sclerosis may be between about 0.001-5.0, preferably 0.1-5.0, more preferably 0.1-2.0 mg per kg of patient body weight per 1-4 weeks administered in a single dose and most preferably between about 0.1-2.0 mg per kg of patient body per 1-4 weeks and administered in a single dose of a chimaeric TNF binding protein which comprises a soluble part of the human p55-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human immunoglobulin or a pharmaceutically acceptable salt thereof, preferably wherein such chimaeric TNF binding protein comprises all domains except the first domain of the constant region of the heavy chain of human immunoglobulin such as IgG, IgA, IgM or IgE and most preferably wherein the human immunoglobulin is IgG, especially IgGl or IgG3.
• the frequency of dosing and the optimal dose will depend on pharmacokinetic parameters of the chimaeric TNF binding protein in
• the specific dose is calculated according to the approximate body weight of the patient. Further refinement of the calculations necessary to determine appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by the man skilled in the art.
• Groups of rats were dosed with a construct of the extracellular domain of the human p55-TNF-receptor (first 182 N- terminal amino acids of the receptor) fused to the hinge region of human IgG ⁇ l heavy chain and expressed in CHO-cells ("fusion construct") (i.p.) or with phosphate buffered saline (PBS) vehicle only.
• fusion construct i.p.
• PBS phosphate buffered saline
• Figure 1 shows the mean clinical score values of 10 animals in each group from day 10 to 21 and Figure 2 shows the corresponding mean body weight from day 8 to 20; p- values were determined by the Mann-Whitney-U-test (see statistical Methods in Biology, Bailey N.T.J., Hodder and Staughton, London)].
• mice Female mice were inoculated subcutaneously on the abdomen in two sites with 0.3 ml (total 0.6 ml) of an emulsion containing 6.6 mg/ml freeze- dried Biozzi spinal cord in phosphate-buffered saline (PBS) and 60 mg heat- killed mycobacteria [Mycobacterium tuberculosis H37Ra and M.
• PBS phosphate-buffered saline
• mice were dosed with the fusion construct (see Example I) (i.p.) or phosphate buffered saline (PBS) vehicle.
• fusion construct see Example I
• PBS phosphate buffered saline
• MBP myelin basic protein
• a monoclonal antibody against myelin oligodendrocyte glycoprotein [anti-MOG Mab 8-18C5, Schl ⁇ sener et al., J. Immunol. 139. 4016 (1987)] was produced from hybridoma supernatants and purified on protein A-sepharose beads (Sigma). Immunization
• Lewis rats were immunized into the hind footpads with either MBP (100 ⁇ g/rat), Cl (50 ⁇ g/rat), or SlOO ⁇ protein (50 ⁇ g/rat) emulsified in Freund's adjuvant (Gibco BRL AG, Basle, CH) completed with 4 mg/ml M. tuberculosis (H37Ra, Difco, Detroit, USA).
• T cell lines used in this study were specific for guinea pig MBP and/or Cl (F8, C1-C9, Cl, LMBP) or bovine SlOO ⁇ protein, a non-myelin, calcium binding protein (LSI). Two different protocols were used to establish antigen- specific T cell lines. Ten days after immunisation a single cell suspension was prepared from the draining popliteal, inguinal, and para-aortic lymph nodes.
• the primed cells were either cultured at a concentration of 10 7 cells ml (5 ml, petri dish) and antigen (10 ⁇ g/ml) to establish bulk T cell lines (LSI, Cl, LMBP) or alternatively serially diluted to provide a large number of pauciclonal T line cells (F8, C1-C9).
• lymph node cell suspensions were cultured in 96-well round-bottom microtiter plates at cell numbers ranging from 2 x 10 5 to 100 cells/well.
• the cells were supplemented with antigen and irradiated (4,000 rad) syngeneic thymus cells as source of antigen presenting cells (APC's) to give a final cell number of 2 x 10 5 /well in Eagle's medium (EA) enriched with L-asparagin (36 mg/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential aminoa ⁇ ds (1 % v/v), penicillin (100 U/ml), streptomycin (100 mg/ml; Gibco) and autologous rat serum (1 %). After 72 hrs the medium was removed and the remaining T cells expanded for further 7-10 days in media supplemented with IL-2.
• EA Eagle's medium
• L-asparagin 36 mg/ml
• sodium pyruvate (1 mM
• nonessential aminoa ⁇ ds (1 % v/v
• penicillin 100 U/
• the T cells were then restimulated with antigen in the presence of 2 x 10 5 irradiated APC's. After visual inspection of the cultures, those positive for growth were expanded in the presence of IL-2. Activated blastoid lymphocytes generated in bulk cultures were collected from the interface of Lymphoprep-gradients (density 1.077 g/ml, Nycomed Pharma AS, Oslo, Norway). The resting T cells were then restimulated in the presence of irradiated APC's (T cell to thymus cell ratio: 1:30). The cycle of propagation in the IL-2 containing medium and restimulation with antigen plus APC's was then repeated until stable T cell lines were established. Specificity assay
• T cell proliferation test was used. T cells (2 x 10 /ml) were restimulated in flat-bottom microtiterplates
• EAE Passive EAE was induced in syngeneic rats following intraperitoneally injection of freshly antigen-activated T cell blasts (5-7 x 10 6 /rat). If SlOO ⁇ specific T cells were used to induce EAE the rats received 10 7 cells followed by a single dose of an Mab 8-18C5 (3-5 mg/rat) at day five after the cell transfer. The weight and clinical score was monitored daily as described above. At appropriate time points animals were sacrificed and used for conventional central and peripheral nerve histology.
• Mouse anti-rat T cell-specific monoclonal antibodies pan-T (W 3.13), CD4 (W 3.25), CD8 (OX-8), a ⁇ -TcR [R73, Hunig et al., J. Exp. Med. 1£9_, 73 (1989)] were purified from hybridoma supernatant ⁇ on protein G-sepharose beads (Sigma, Taufkirchen, BRD) or purchased from Camon (Wiesbaden, BRD).
• TcR V ⁇ isotypespecific MAbs B73 (V 8.5), G101 (V ⁇ 10), and R78 (V ⁇ 8.2) see Torres-Nagel, Immunogenetics 31, 305 (1993).
• This model covers a relative broad spectrum of the pathogenesis including activation of autoaggressive T cells in vivo and various inflammatory stages of the disease within the central nervous system.
• the disease is normally limited only to perivascular infiltrations and not associated with primary large scale demyelination.
• T lymphocytes recovered from MBP or MBP- peptide immunized rats The cells are established and maintained in vitro as antigen specific lines. They are characterized as CD4 + , CD8-, TcR V ⁇ 8.2 + , MHC-class II restricted T cells which are able to predictively and reproducibly induce EAE upon transfer in vivo into normal naive recipients. Similar as in the active model tEAE is not characterized by demyelination but focuses on the recirculation and inflammatory effector phase of EAE including pathogenic interactions between the T cells and the blood brain barrier.

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• 1995
  • 1995-07-15 HU HU9700180A patent/HUT76666A/hu unknown
  • 1995-07-15 BR BR9508419A patent/BR9508419A/pt not_active IP Right Cessation
  • 1995-07-15 CZ CZ97193A patent/CZ283219B6/cs not_active IP Right Cessation
  • 1995-07-15 CA CA002195665A patent/CA2195665A1/en not_active Abandoned
  • 1995-07-15 WO PCT/EP1995/002788 patent/WO1996003141A1/en not_active Application Discontinuation
  • 1995-07-15 AU AU31119/95A patent/AU3111995A/en not_active Abandoned
  • 1995-07-15 EP EP95926902A patent/EP0772449A1/de not_active Withdrawn
  • 1995-07-15 PL PL95318501A patent/PL318501A1/xx unknown
  • 1995-07-15 JP JP8505421A patent/JPH09508140A/ja active Pending
• 1997
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