CZ19397A3 - Use of chimeric protein binding tnf in the preparation of a pharmaceutical preparation for treating auto-immune diseases - Google Patents

Abstract

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 of a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the treatment of autoimmune diseases.

Description

(57)

The use of a chimeric TNF binding protein comprising a soluble fraction of human p55TNF receptors or p75-TNF receptors and all or part of the human or immunoglobulin heavy or light chain constant domains or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical composition for treating autoimmune diseases, particularly multiple sclerosis.

CZ 193-97 A3

01-2786-96-Ho i

Use of a chimeric TNF binding protein for the manufacture of a pharmaceutical composition for the treatment of autoimmune diseases

Technical field

The invention relates to the use of a chimeric TNF binding protein comprising a soluble portion of the human p55- or p75-TNF receptor and all or part of the human or immunoglobulin heavy chain or light chain constant domains or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical agent for the treatment of autoimmune diseases. associated with inflammatory processes such as rheumatoid arthritis, juvenile onset diabetes mellitus type I, systemic lupus erythematosus, thyroiditis, particularly multiple sclerosis.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and appears to originate from autoimmune disorders with myelin components as a shield. The suggestion of autoimmune soil MS is supported by the fact that myelin basic protein (MBP) reactive T lymphocytes are found in humans and which are phenotypically similar to MBP specific encephalitogenic T cells in experimental allergic encephalomyelitis (Sun, Acta. Neurol. Scand. Suppl. 142, 1993, Voskuhl et al., Autoimmunity 15, 137-143, 1993) and it is clear that the frequency of MBP reactive T cell clones in MS patients is increased (Allegretta et al., Science 247, p. 718-721 (1990). In addition, various experimental models of autoimmune encephalomyelitis animals have been developed that mimic the pathophysiological aspects of human MS (as discussed below). However, there are environmental factors controlling MS occurrence as evidence of the existence of geographically well-defined endemic areas.

MS is characterized by focal accumulation of inflammatory cells, edema and demyelination in CNS tissue. It is an ancient finding that autopsy reveals more focal lesions than would be predicted from the clinical condition.

Typically, the lesions start as a perivullary mononuclear cell infiltrate with the following edema. The infiltrated cells mediate the selective destruction of myelin shells while leaving asons intact. In a further procedure, astrocyte proliferation leads to a glyotic scar, a so-called MS plate with a diameter of 0.1 to several cm. The clinical course of MS may have intervals with the disease with varying duration, or the disease may progress chronically, indicating that pathophysiological processes are subject to heterogeneous control.

Clinical pathology studies demonstrate the presence of TNF producing cells in MS lesions. Most TNF-positive cells are morphologically classified as reactive filamentous astrocytes, and dual immunostaining experiments confirmed that most reactive cells are astrocytes, with the remainder being monocytes / macrophages (Hofman et al., J. Exp. Med. 170, pp. 607-612). 1989). An independent study confirmed positive immunoreactivity for TNF in atrocytes and monocytes in MS plates and also examined lymphotoxin (LT) immunoreactivity. Surprisingly, in addition to the expected lymphocytes, microglial cells were found to be positive for LT (Selmaj et al., J. Clin. Invest., 87: 949-95 (1991A)). In addition, investigations of cytokine mRNA expression in MS lesions by in situ hybridization show that high levels of TNF, IL-6, and INF-garaa (and other cytokine) mRNA are present in all inflammatory perivascular lesions. TNF, IL-L and IFN-gamma mRNA levels are particularly high in lesions that have sustained demyelination (Woodroofe and Cuzner, Cytokine 5, 583-588, 1993).

The direct cytotoxic effect of TNF, but not IFN-gamma, IL-2, T-cell supernatants, or antigalactocerebroside antisera, on igodendrocytes in myelinated cultures of mouse spinal tissue has been described (Selmaj and Raine, Ann. Neurol. 23, 339-346). 1988). Treatment of TNF of these cultures causes 1 igodendrocyte necrosis; some nerve fibers show progressive demyelination.

The concentration of TNF in serum and CSF of MS patients has been measured by numerous researchers. It is agreed that a significant percentage of MS patients show an increased TNF content in CSF and much less in serum. TNF concentrations are not directly related to CSF cell content, suggesting that CSFTNF content may reflect TNF production in and around MS lesions, particularly with regard to frequent paraventricular placement of lesions.

Animal studies in experimental encephalomyelitis (EAE) significantly elucidated the view of autoimmune origin and the role of cytokines in human MS. EAE is induced either by active sensitization with myelin antigens or by adoptive transfer of specific T cell clones to a syngeneic susceptible host animal. The finding that experimentally inducing an immune response to myelin antigens leads to a disease similar to that of human MS is important evidence for the autoimmune origin of MS. The role of TNF in generating the evolutionary mechanisms responsible for the characteristic pathology of the disease is exemplified by the protective action of anti-TFN monoclonal antibody therapy (Ruddle et al., J. Exp. Med., 172, 1193-100, 1990, Selmaj et al. Ann. Neurol., 30, 694-700 (1991b). Elimination of TNF-producing cells (and other cytokines) similarly suppresses EAE (Huitinga et al., J. Exp. Med., 172, 1025-1033 (1990)). Further support for the important role of TNF in EAE is the finding that encepalitogenics in adoptive transfer strongly depend on TNF and LT production ability of different T cell clones, all of which are observed on the same MBPM peptide (Powell et al., Intern. Immunol., 2 1990, 539-544).

SUMMARY OF THE INVENTION

The present invention provides the use of a chimeric TNF binding protein comprising a soluble fraction of the human p55-TNF receptor or the p75-TNF receptor and all or part of the human immunoglobulin heavy or light chain constant domains or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical composition for treating autoimmune diseases.

The use of soluble proportions of the human p55-TNF receptor and the p7,5 TNF receptor for the treatment of experimental encephalomyelitis (EAE) rats is already described in European Patent Specification No. 512528. However, there are no data on the protective action of TNF-binding chimeric proteins and their pharmaceutically acceptable salts. Therefore, the use of a chimeric TNF binding protein comprising a soluble fraction of the human p55-TNF receptor and the p75-TNF receptor and all or part of the human or immunoglobulin heavy or light chain constant domains or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for treating autoimmune diseases, for example, multiple sclerosis, particularly where the chimeric TNF binding protein contains soluble portions of the human p55-TNF receptor is therefore the subject of the present invention. A preferred embodiment of the invention is that a chimeric TNF binding protein comprising all domains except the first domain of the human immunoglobulin heavy chain constant region is used, meaning that the soluble portion of the human p55-TNF receptor is fused to the C terminus to the immunoglobulin heavy chain hinge region. . Such mimunoglobulin may be IgG, IgA, IgM or IgE, particularly IgG similar to IgG1 or IgG3. The term soluble portion of the human p55-TNF receptor and the p75-TNF receptor refers to either the complete extracellular domain of one of these receptors, or a portion thereof that still binds the TNF, for example in the case of the p55-TNF-receptor extracellular domain 12 N-terminal amino acids.

defined, thereby illness, in particular

The invention also relates to a method of treating auto-immune diseases in a mammal comprising administering to the mammal a pharmaceutically effective amount of a TNF-binding chimeric protein or a salt thereof, from the above alleviating the action of such autoimmune diseases when the autoimmune disease is multiple sclerosis.

The process for preparing TNF-binding chimeric proteins used in accordance with the invention is described in detail in European Published Application EP 417563 and also described by Loetscher et al. (J. Biol. Chem. 266: 18324-18329, 1991). In addition, TNF binding proteins or portions thereof according to the following patent literature can be used to prepare such TNF binding proteins: EP 308378, EP 422339, GB 2

No. 393438, WO 90/13575, EP 398327, EP 412486, WO91 / O3553, EP 418014, JP 127,800 / 1991, EP 433900, U.S. Pat. No. 5,160,221, GB 2 246569, EP 464533, WO 92/01002, WO 92/13095, WO 92/16221, EP 512528, EP 526905, WO 93/07863, EP 568928, WO 93/21946, WO 93/19777, EP 417563 and WO 94/06476.

chimeric 218101, EP

As used herein, the chimeric TNF binding protein also includes proteins in which any amino acid of the immunoglobulin moiety or TNF binding moiety can be deleted or replaced by one or more amino acids, or one or more amino acids are added if the TNF binding moiety still binds TNF and immunoglobulin moieties show one or more. several of its characteristics. Where the immunoglobulin moiety comprises all domains including a hinge region except the first heavy chain constant region domain, such a hinge region need not have the first five N-terminal amino acids. Such TNF-binding chimeric protein muteins can be prepared by methods known in the art, such as described, for example, by Sambrook et al. (Molecular Cloning, Second Edition, 1989, Cold Spring Harbor Laboratory Press, New York) or as described in at least one of the aforementioned patents.

In addition, the chimeric TNF-binding protein of the invention can also be used in its modified form, for example, coupled to a chemical unit without altering its basic biological activity. A preferred and well known modification is the coupling to water-soluble polymers such as polyethylene glycols or polypropylene glycols with a wide range of molecular weights, for example 5000 to 20,000 daltons. This provides protected proteins which may be substantially non-immunogenic. Some models of coupling polymers to proteins through various linkers are known in the art and are described in the literature (for example, Perspectives in Bioconjugate Chemistry, C. F. Meares, American Chemical Society, Washington 1993, and in particular U.S. Patent 4,179,337).

In addition, the TNF-binding chimeric proteins of the invention may be in the form of pharmaceutically acceptable salts. Salts of the carboxyl group can be formed in a manner known per se, and inorganic salts such as sodium, calcium, ammonium, iron or zinc, but also salts with organic bases such as amines such as triethanolamine, arginine or lysine, piperidine and procaine. Acid addition salts include, for example, salts with mineral acids such as hydrochloric or sulfuric acid and salts with organic acids such as acetic or oxalic acid.

The TNF-binding chimeric protein for the purposes of the invention is preferably administered parenterally by injection, however other effective forms of administration, for example intra-articular injection or transdermal iontophoresis, are also possible. A preferred carrier is a physiologically saline solution, but other pharmaceutically acceptable carriers may also be used. The primary solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmaceutically acceptable excipients to modify or maintain the pH, osmolarity, viscosity, clarity, color, sterility, stability, degree of dissolution or odor of the formulation. Similarly, the carrier may contain other pharmaceutically acceptable excipients to modify or maintain the stability, degree of dissolution, release, or absorption of the TNF-binding chimeric protein. Such excipients are those commonly used for formulating doses for parenteral administration in man in unit or multiple unit form. The finished therapeutic composition may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid or in a dehydrated form, for example, as a lyophilized powder. Such compositions may be stored in a form ready for use or in a form requiring reconstitution immediately prior to administration. The pharmaceutical compositions are stored at a low temperature of at least 4 ° C, preferably at -70 ° C. It is also preferred to maintain such pharmaceutical compositions and administer them at a pH close to the physiological pH.

The possible dosage in the treatment of multiple sclerosis may be about 0.001 to 5.0, preferably 0.1 to 5.0 and even more preferably 0.1 to 2.0 mg per kg body weight of the patient for 1 to 4 weeks administered in a single dose and more preferably at a dose of about 0.1 to 2.0 mg / kg of patient body weight for 1 to 4 weeks and administering in a single dose a chimeric TNF-binding protein comprising a soluble fraction of the human p55-TNF receptor and all or a human immunoglobulin light chain or a pharmaceutically acceptable salt thereof, preferably such a chimeric TNF binding protein comprises all domains except the first domain of a human immunoglobulin heavy chain constant region such as IgG, IgA, IgM or IgE, most preferably the human immunoglobulin is IgG, especially IgG1 or IgG3. Dosage frequency and optimal dose depend on the pharmacological parameters of the chimeric TNF binding protein in the pharmaceutical composition used.

Regardless of the route of administration, the specific dose is calculated according to the approximate weight of the patient. Calculation accuracy to be determined to determine the appropriate dosage for treatment with each of the above pharmaceutical compositions is conventional to those of skill in the art.

The following examples and attached figures illustrate the invention.

Picture list

Fig. 1 shows the mean clinical sign for 10 animals in each group from day 10 to day 21.

The x-axis shows the days, the y-axis shows the clinical scale. Individual lines have the following meaning:

and— controls

- f uzní construct 10 mg / kg IP -AND-  fuzn í construct 5 mg / kg if f u η í construct 1 mg / kg IP (*) P <. 05 / (**}? <. 01

Figure 2 shows the corresponding mean body weight from day 8 to day 21.

The x-axis shows days, the y-axis shows body weight. Individual lines have the following meaning:

—A— controls

f uzní construct 10 mg / kg IP f acknowledges construct 5 mg / kg if fuzní construct 1 mg / kg IP

Fig. 3 shows a clinical evaluation mark of 10 animals from day 10 to day 20 with p-values determined by the Mann9 U-test

Whitney.

The x-axis shows the days, the y-axis shows the clinical scale.

Individual lines have the following meaning:

—3— controls ® fusion construct * P <.05

Figure 4 shows the corresponding mean body weight with p-values determined by an unpaired T-test.

The x-axis shows days, the y-axis shows body weight.

Individual lines have the following meaning:

gg control

-9 * P <fusion construct. 05 *** P <.001

In Fig. 5, drawing 5a relates to a control experiment with pure brine only (carrier) and drawing 5b of the fusion construct situation, where the bars show clinical scale values and the curve is an indicator for body weight change (in g]).

The x-axis shows the days.

Student's t-test: * p <0.05, Taank sum = p <0.1

Figure 6 shows the effect of the fusion construct on active experimental autoimmune encephalomyelitis (EAE), the development of rat weight.

Immunization: 50 µg Cl s.c. (# 1-6)

Fusion construct: 10 mg / kg day 2, 7, 8, 9, 10 i.p. (# 4-6)

Figure 7 shows the effect of the fusion construct on T cell-mediated EAE (tEAE).

cell transfer: 6 x 10 ⁶ ip, rat 16

Fusion construct: 10 mg / kg ip, day 0, 2, 3, rat # 4-6 Figure 8 shows the effect of the fusion construct on the transmission of experimental autoimmune panencephalomye itis (tEAP) rats passively treated with S100J3 protein specific T cells (Figure 8). # 1-3), rats after administration of anti-MOG monoclonal antibody (Fig. 8 # 4-6), rats that were fused with a fusion construct in addition to Mab-8-18C5 (Figs. 8, # 7-9).

Cell transfer: 1x10 ⁷ LSI (Sβ-specific), day 0, rat # 1-9

Rat # 1-3: LSI only

Rat # 4-6: α-MOG-Ab, Day 5 (3 mg / rat)

Rat # 7-9: α-MOG-Ab, day 5, fusion construct 10 mg / kg, day 1, 3, 5

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Acute EAE model (AHH / R rats)

Female AHH / R rats (Bloxham et al., J. Pharmacol. Exp. Ter. 252: 1331-1340 (1990)) were inoculated with an emulsion (0.05 ml) containing guinea pig spinal cord juvant and Freund's complete adjuvant (1: 1). ) containing Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI, USA) (10 mg / ml) into the planter surface of both hind paws. Animals are weighed and scored daily for neurological signs according to a four-point scale (clinical scale): 0 = normal, 1 = flaccid tail, 2 = partial hind paw paralysis, 3 = full hind paw paralysis, 4 = tetraplegia. Groups of rats are dosed with the human β-55-TNF receptor extracellular domain construct (the first 182 N-terminal receptor amino acids) fused to the human IgG1 gamma 1 heavy re11 hinge region heavy and expressed into CHO cells (fusion construct) (ip) or phosphate only. buffered brine carrier (PBS). Administration of the encephalitogenic suspension in rats leads to the onset of neurological symptoms (day 10) which then culminates on day 14. Neurological symptoms are accompanied by a significant weight loss. Treatment fusion constructs (1, 5 and 10 mg / kg, ip, administered daily, post-induction of EAE, days 8-14) significantly reduce EAE symptoms and changes in body weight in a dose-dependent manner (Fig. 1 shows mean clinical sign at 10 animals in each group from day 10 to day 21 and Figure 2 shows the corresponding mean body weight from day 8 to day 21. p-values are determined by Mann-Whitney U-test [Statistical Methods in Biology, Bailey NTJ, Hodder and Staughton, London ]). In the second series of tests, a fusion construct of 10 mg / kg, ip) administered after induction during the disease window (day 8 to 20) again significantly reduces the symptoms of EAE and changes in body weight up to day 14 (Fig. 3 shows a clinical evaluation score of 10 animals). from day 10 to day 20 with p-values determined by MannWhitney U-test [Statistical Methods in Biology, Bailey NTJ, Hodder and Staughton, London] and Fig. 4 shows the corresponding mean body weight with p-values determined by unpaired T-test [Statistical Methods in Biology, Bailey, NTJ, Hodder and Staughton, London].

Example 2

Chronic EAE model (Biozzi mice)

Inbred Biozzi selection I AB / H mice (Baker et al., J. Neuroimmunol., 28, 261-270, 1990) are maintained on a standard pelleted diet and water ad libitum. Male mice are inoculated subcutaneously on the abdomen at two sites with 0.3 ml (total 0.6 ml) emulsion containing 6.6 mg / ral freeze-dried Biozzi spinal cord in phosphate buffered saline (PBS) and 60 mg heat-killed mycobacterium (Mycobacterium tuberculosis H) 37 Ra and M. butyricum [8: 1], Difco

Laboratories, Detroit, MI st. a.) in 0.15 ml Freund's incomplete adjuvant (Baker et al., J. Neuroimmunol., 28, 261-270, 1990). The injection is repeated after 7 days. Animals are weighed and scored daily for neurological signs according to a five-point scale (clinical scale): 0 = normal, 1 = drooping tail, 2 = impaired righting reflex (IRR), 3 = partial hind paw paralysis, 4 = complete paralysis hind paws, 5 = death. Groups of mice are dosed with a fusion construct (according to Example 1) (i.p.) or a phosphate buffered saline carrier. In the chronic EAE model, Biozzi mice show an acute disease incidence in the vehicle-treated group in 90% with neurological symptoms manifesting on day 14 after EAE induction. Pretreatment with a fusion construct (7 mg / kg, ip dosed every 3 days during the experimental protocol) reduces the number of animals showing paralysis (number 1 to 4 in the 5-point evaluation grid) during both the acute phase (3/9) and disease recurrence (1 / 7) compared to control (9/10 and 6/8) (Table I and Fig. 5, where drawing 5a relates to a control experiment with pure brine only (carrier) and drawing 5b of the fusion construct situation, the columns showing clinical scale and curve are indicators of weight change (in g]) In a subsequent dose-response study, the fusion construct (1 to 14 mg / kg, ip) is administered after disease induction (day 10 to 30), also observed the beneficial effect and the minimum effective dose is 7 mg / kg.

Example 3

General ways

Animal, reagents and cell lines

Lewis inbred rats weighing 120-150 g are obtained from Charles River Wiga (Sulzfeld, Germany). Guinea pig myelin basic protein (MBP) is isolated from whole brains (Eylar et al., J. Methods Enzymol. 32B, p. 323, 1979). C1, the encephalitogenic peptide MBP (amino acids 68-88) is a commercial product of Peptide Products (Porton Down, United Kingdom). The bovine S100ft protein is obtained from Sigma Chemical Co. (St. Louis, MO, USA). The protein pure M. tuberculosis derivative (PPD) is obtained from the Statens Serum Institute (Kopenhagen, Denmark). The monoclonal antibody against myelin of 1 igodentrocyte glycoprotein (anti-MOG Mab 8-18C5, Schlusener et al., J. Immunol. 139, p. 4016, 1987) is produced from hydromide supernatants and purified on A-sepharose bead protein ( Sigma).

Immunization

Lewis rats are immunized in the hind legs of either MBP (100 Hg / rat), Cl (50 µg / rat), or S100 (3 (50 pg / rat), protein emulsified in Freund's adjuvant (Gibco BRL AG, Basel, Switzerland) assembled 4 mg / ml M. tuberculosis (H37 Ra, Difco, Detroit, Sp. St. A.).

Anrigen specific T cells

The T cell lines used in this study are specific for guinea pig MBP and / or Cl (F8, C1-C9, Cl, LMBP) or bovine SlOOP protein, nemyelin, calcium binding protein (LSI). Two different protocols are used to determine antigen-specific T cell lines. Ten days after immunization, a single cell suspension is prepared from depleted knees, inguinal and para-aortic lymph nodes. Primary immunized cells are either cultured at 10 ⁷ cells / ml (5 ml, petri dishes) and antigen (10 µg / ml) to determine T cell line mass (LSI, Cl, LMBP) or serially diluted to produce a large number of pauciclonal T cells. cell lines (F8, C1-C9). In this case, the lymph node cell suspensions are cultured in 96-well round bottom microtiter plates at a cell number of 2 x 10 ⁵ to 100 cells / length. Cells are supplemented with antigen and irradiated (4000 rad) syngeneic thymic cells as a source of antigen presenting cells (APC) to achieve a final cell count of 2 x 10 ⁵ / well in Eagle (EA) enriched with L-asparagine (36 mg / ml) L-glutamine (2 mM), sodium pyruvate (1 mM), non-essential amino acids (1% v / v), penicillin (100 J / ml), treptomycin (100 mg / ml, Gibco) and autologous rat dusk (1%) . After 72 hours, the medium is removed and the remaining T cells are expanded for an additional 7-10 days in an IL-2 supplemented medium. T cells are then restimulated with antigen in the presence of 2 x 10 5 irradiated APCs. After visual assessment of the cultures, the growth positive cultures expand in the presence of IL-2. Activated blastoid lymphocytes generated in cultures are separated from the interface of Lmphoprep gradients (density 1.077 g / ml, Nycomed Pharma AS, Oslo, Norway). The remaining T cells are then restimulated in the presence of irradiated APCs (T cells relative to thymus cells 1:30). The cycle of propagation in IL-2 containing media and restimulation with antigen plus APC is then repeated until a stable T cell line is formed.

Specificity test

Standard T cell proliferation assays are used to assess specificity of T cells. T cells (2 x 10 ⁴ / ml) are restimulated in flat bottom microtiter plates (200 µg / well) in the presence of APC (6 x 10 ⁵ / ml) and relevant MBP, Cl or S100 antigens (3 (20 µg / ml) ) and irrelevant PPG antigen (10 µg / ml) and ConA (2.5 µg / ml) After 72 hours of culture, cells are harvested, followed by 16 hours of pulse with 1 µCi / well tritiated thymidine ( ³ H-dT,

Ci / mmol, Amesham-Buchler, Braunschweig, Germany). Radiolabeled cells are collected on glass fiber filters and radioactivity on washed and dried filters is quantitated with a matrix 96 counter (Canberra-Packard, Frankfurt, Germany).

Active EAE

Active EAE is induced in rats after immunization with MBP or Cl. Weight and clinical trials are monitored on a daily basis by clinical inspection. Clinical disease is scored as follows: 0 = normal, 1 = tail flaccid, 2 = non-motorized paraparesis, 3 = hind limb paralysis, 4 = hind limb paralysis, 5 = death. The study is performed in a blank test. This means the observer does not know the protocol.

Passive EAE

Passive EAE is induced in syngeneic rats following intraperitoneal injection of fresh antigen-activated T cell blasts (5-7 x 10 ⁶ / rat). When S10OP-specific T cells are used to induce EAE, 10 ⁷ cells are administered to rats followed by a single dose of Mab-8-18C5 (3-5 mg / rat) on the fifth day after cell transfer. Weight and clinical signs are monitored daily as described above. At the appropriate time, the rats are sacrificed and used for the normal histology of central and peripheral nerves.

EAE treatment

Active or passively induced EAE is treated by repeated or single intraperitoneal injection of 10 mg / kg fusion construct (according to Example 1).

FACS analysis

Mouse anti-rat T cell specific monoclonal antibodies (MAb) pan-T (W 3.13), CD4 (W 3.25), CD8 (OX-8), and (3-TcR (R73, Hunig et al., J Exp. Med., 169, 73 (1989)) are separated from hybridoma supernatants on G-sepharose protein beads (Sigma, Taufkirchen, Germany) or Camon (Wiesbaden, Germany) for TcR V (3 isotype specific MAb B73 ( In 8.5, G101 (νβΙΟ) and R78 (Vβ 8.2), see Torres-Nagel, Immunogenetics 37, 305 (1993), and specific MAb labeling was determined using DTAF labeled F (ab ') 2 fragments of goat anti-mouse IgG (H +). Immunofluorescence of living cells with the exception of propidium iodate (10 µg / ml, Sigma) was measured by FACScan (Becton Dickinson, Heidelberg, Germany).

Example 3.1

Effect of fusion construct on active experimental autoimmune encephalomyelitis (EAE)

This model covers a relatively broad spectrum of pathogenesis, including in vivo activation of autoaggressive T cells and various inflammatory conditions of the central nervous system. However, the disease is normally restricted to per ivascular infiltration only and is not related to large-scale primary demyelination.

Groups of Lewis rats (n = 3) are immunized with a Cl peptide having a span of the encephalitogenic domain of guinea pig MBP for Lewis rats from amino acid 68-88. All rats increased in weight for the next 8 days (Fig. 6), followed by a straightening of the weight curve from day 9. At the beginning of the ninth day, the control group, untreated by the fusion construct, showed a rapid weight loss with a slack tail, followed by a marked weakening of the hind paws and a roughening of the fur. Partial loss of righting reflex and incontinence is also observed. These clinical signs of the disease are present until the sixteenth day when rats begin to recover and begin to recover weight. Rats receiving intraperitoneal injections of the fusion construct show a weighting curve from day 9, but treatment results in a dramatic reduction in the severity of clinical symptoms. In total, they behave normally clinically. Only a slight partial loss of tail tonicity, which occurs on the thirteenth day and lasts 2 to 3 days, is observed without showing any further increase.

Example 3.2

Effect of fusion construct on T cell mediated EAE (tEAE)

The basis of this model is T lymphocytes derived from MBP or MBP-peptide immunized rats. Cells are established and maintained in vitro as antigen specific lines. They are characterized as CD4 ⁺ , CD8 -, TcR V | 38.2 ⁺ , MHC class II restricted T cells, which are capable of predictably and reproducibly inducing EAE upon transfer in vivo to normal primitive recipients. As in the active tEAE model, it is not characterized by demyelination but focuses on recirculation and inflammatory effector phases of EAE, including pathogenic interactions between T cells and the blood brain barrier.

A group of nine Lewis rats (n = 3) are passively treated with Cl-specific T cells (C1 to C9) introperitoneally. The control group, which is not treated with the fusion construct (Fig. 7), develops typical severe tEAE traits accompanied by severe weight loss starting on the second day after cell transfer. The onset of clinical symptoms begins markedly on the second day and is similar to that of active EAE. Rats recover approximately six days. Rats treated with the fusion construct show only slight weight loss from day 3 at three different time points. However, clinical signs are greatly reduced to only a partial loss of tail tonicity on the third to sixth day.

Example 3.3

Influence of fusion construct on transmission of experimental autoimmune panencephalomye1 itis (tEAP)

In this model, S1ΟΟβ-specific T cells, derived from immunized rats, mediate an inflammatory lesion throughout the central nervous system, spinal cord, optic nerve, retina, and midbrain. In addition, similarly in human multiple sclerosis (MS), the eyes are usually attacked and show retinal periphlebitis. Cell infiltrates are observed per ivascularly but also in the surrounding parenchyma, generally fewer ED1 ⁺ macrophages are present compared to MBP induced by EAE. Only mild clinical signs such as weight loss are observed. A clinically more severe condition appears after injection of Mab 8-18C5 (Linington et al., J. Immunol. 23, 1364, 1993). Histologically, large foci of demyelination arranged around the infiltrate bridging this model to certain cases of human MS are observed.

Groups of Lewis rats (n = 3) are passively treated with S10OP protein specific T cells (LSI). These cells induce a strong inflammatory response in the nervous system, but only minimal neurological dysfunction in the primitive syngenic recipient (Fig. 8 # 1-3). However, clinical signs occur when rats are administered (Figure 8 # 4-6) anti-MOG monoclonal antibodies on the fifth day after cell transfer. All rats show a total loss of tail tonicity lasting 48 hours. In contrast, rats that were fused with a fusion construct in addition to the Mab-8-18C5 fusion show no clinical symptoms and behave quite normally (Figs. 8, # 7 to 9).

Industrial applicability

The use of a chimeric TNF-binding protein comprising a soluble fraction of human p55-TNF receptor or p75-TNF receptor and all or part of the human or immunoglobulin heavy chain or light chain constant domains or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical composition for treating autoimmune diseases particularly multiple sclerosis.

Claims (5)

PATENT CLAIMS Use of a chimeric TNF binding protein comprising a soluble fraction of human p55-TNF receptors or p75-TNF receptors and all or part of the human immunoglobulin heavy or light chain constant domains or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical composition for the treatment of auto-immune diseases. The use of claim 1, wherein the autoimmune disease is multiple sclerosis. Use according to claims 1 and 2, wherein the chimeric TNF binding protein is a soluble fraction of human p55-TNF receptors. Use according to claims 1 to 3, wherein the chimeric TNF binding protein comprises all domains except the first domain of the human immunoglobulin heavy chain constant region, such as IgG, IgA, IgM or IgE. Use according to claim 4, wherein the human immunoglobulin is IgG, in particular IgG1 or IgG3. o bro ím bro bro bro bro bro bro bro bro bro á á á á á á Ίρ 'n'j, mares 5 ^c' i> set ί ζiá, -savcům serves therapists icky- efficacious / amount of a chimaeric TNF binding kproťéinu ^. or is! ./ i— · i. '——', -. A salt of a suitable salt according to claims 1 to 3 to 5, which ameliorates the incidence of tertiary autoimmune diseases. L · 7. , ti-m rq-si s, The method according to Crk-tt-6, characterized by the treatment of putoimmune disease is multiple scle-