NO328228B1 - Use of a mixture of polypeptides for the preparation of a pharmaceutical composition for the treatment of an autoimmune disease other than MS. - Google Patents

Description

INTRODUCTION

The present invention relates to the use of a mixture of polypeptides which have an average molecular weight of 2-40 kDa, where each polypeptide consists of glutamic acid, tyrosine, alanine and lysine. Said mixture of polypeptides has been shown to be effective in the treatment of a variety of autoimmune diseases and binds to class II tissue type complex (MHC) molecules as well as to antigen presenting cells. Such peptides are referred to below as "copeptides" to emphasize their relationship to copolymer 1.

Copolymer 1 is a heterogeneous mixture of synthetic, random, linear copolymers of tyrosine, alanine, glutamic acid and lysine. If such mixtures of synthetic, random, linear copolymers mainly consist of three of the four amino acids found in copolymer 1, they are termed terpolymers.

BACKGROUND OF THE INVENTION

Autoimmune diseases occur when an organism's immune system fails to recognize some of the organism's own tissues as "own" and attacks them as "foreign". Normally, self-tolerance develops early during the development of the immune system, which prevents the organism's own T cells and B cells from reacting with the organism's own tissues. MHC cell surface proteins help regulate these early immune responses by binding to and presenting processed peptides to T cells.

This self-tolerance process breaks down in the development of autoimmune diseases. Now the organism's tissues and proteins are recognized as "autoantigens" and attacked by the organism's immune system. For example, multiple sclerosis is believed to be an autoimmune disease that occurs when the immune system attacks the myelin layer, whose function is to insulate and protect nerves. Multiple sclerosis is a progressive disease characterized by demyelination, followed by loss of neuron and muscle function. Rheumatoid arthritis ("RA") is also believed to be an autoimmune disease involving chronic inflammation of the synovial joints and infiltration of activated T cells, macrophages and plasma cells, leading to progressive destruction of articular cartilage. Rheumatoid arthritis is the most serious form of joint disease. Which autoantigen(s) are attacked in rheumatoid arthritis is poorly understood, although collagen type II is a candidate.

A tendency to develop multiple sclerosis and rheumatoid arthritis is hereditary - these diseases occur more frequently in individuals who carry one or more characteristic MHC class II alleles. For example, inherited susceptibility to rheumatoid arthritis is strongly associated with the MHC class II alleles DRB1 <*>0401, DRB1<*>0404, DRB<*>405 or DRB*0101 alleles. The histocompatibility locus antigens (HLA) are found on cell surfaces and help to give tissues from different people their distinctive character. Genes for the histocompatibility locus antigens are located in the same region of chromosome 6 as the tissue type complex (MHC) genes. The MHC region expresses a number of distinct molecular classes in different cells of the body, where these genes are, in the order they appear along the chromosome, the class I, II and IH MHC genes. The class I genes include HLA genes, which are further divided into A, B and C subregions. The class II genes are further divided into the subregions DR, CQ and DP. The MHC-DR molecules are the best known, these appear on the surface of antigen-presenting cells such as macrophages, dendritic cells from lymphatic tissue and epidermal cells. The class III MHC products are expressed in various components of the complement system as well as in some non-immune-related cells.

A number of therapeutic agents have been developed for the treatment of autoimmune diseases, including steroidal and non-steroidal anti-inflammatory drugs, for example, methotrexate, various interferons and certain inhibitors of prostaglandin synthesis. However, these agents can be toxic if they are used over more than a short period of time, or they can produce unwanted side effects. Other therapeutic agents bind to and/or inhibit the inflammatory activity of tumor necrosis factor (TNF), for example anti-TNF-specific antibodies or antibody fragments, or a soluble form of the TNF receptor. These agents target a protein on the surface of a T cell and generally prevent its interaction with an antigen presenting cell (APC). However, therapeutic preparations containing naturally folded proteins are often difficult to manufacture, design, store and administer. Furthermore, the inherent heterogeneity of the immune system can limit the effectiveness of the drugs and complicate the long-term treatment of autoimmune diseases.

MS is an autoimmune disease where the myelin layer that surrounds the nerve cells is attacked by the body's own immune system. A number of therapeutic solutions have been proposed in the art, of which the use of copolymer 1, a heterogeneous mixture of synthetic random linear copolymers of tyrosine, alanine, glutamic acid and lysine, is one of these (Israel Journal of Medical Sciences, vol. 33,1997, p 280-284; Proe. Nati. Acad. Sei. USA, vol. 91, 1994, pages 4872-4876; European Journal of Pharmacology, vol. 342, 1998, pages 303-310).

In order to effectively treat autoimmune diseases and other immune conditions, there is thus a need for new drugs that do not have the side effects of current therapeutic agents and that satisfactorily deal with the inherent heterogeneity of the immune system.

REFERENCES

Aharoni, et al., 58 Immunology Letters 79 (1997).

Allison in IMMUNOSUPRESSION AND ANTI-INFLAMMATORY DRUGS,

ANNALS OF THE NEW YORK ACADEMY OF SCIENCE 696:XI (1993). Ben-Nun A et al., 243 J NEUROL (SUPPL 1) S14-S22 (1996).

Dorlig et al., 6 CUR OPINIONS IMMUNOL. 765 (1994).

Ferrara et al., 324 NEW ENGLAND J. OF MEDICINE 667 (1991).

Fridkis-Hareli, et al., 63 J. NEUROCHEM. 63 (SUPPL. 1) (1994).

Fridkis-Hareli et al., 163 CELL. IMMUNOL. 229 (1995).

Fridkis-Hareli et al., 160 J. IMMUNOL. 4386 (1998).

Johnson, 1 NEUROLOGY 65-70 (1995).

Kay et al., 22 TRANSPLANTATION PROCEEDINGS 96 (1990).

Kelemen, et al., 102 INT ARCH ALLERGY IMMUNOL. 309 (193).

Mengle-Gaw, The Major Histocompatibility Complex (MHC), in ENCYCLOPEDIA OF MOLECULAR BIOLOGY 602-06 (Oxford: Blackwell Science Ltd., 1994). Rothbard, J.B. et al., 9 ANNU. FOX. IMMUNOL. 527 (1991).

Schlegel, et al., 84 BLOOD 2802 (1994).

Sela M et al., 88 BULL INST PASTEUR 303-14 (1990).

Stazl, 22 TRANSPLANTATION PROCEEDINGS 5 (1990).

Sykes, 10 THE FASEB JOURNAL 721 (1996).

Teitelbaum et al., 1 EUR. J. IMMUNOL. 242-48 (1971).

Teitelbaum et al., 3 EUR. J. IMMUNOL. 273-79 (1973).

Teitelbaum et al., 64 J. NEUROIMMUNOL. 209-17 (1996).

Thomson, 10 IMMUNOLOGY TODAY 6 (1988).

Van den Bogaerde, et al., 52 TRANSPLANTATION 15 (1991).

Webb et al. 13 IMMUNOCHEM. 33 (1976).

SUMMARY OF THE INVENTION

Surprisingly, the copeptides can be used for the preparation of a pharmaceutical composition for the treatment of a number of autoimmune diseases in a heterogeneous patient population. These copeptides can inhibit some of the physiological responses of T cells that attack self-antigens as these diseases develop. Furthermore, the copeptides used in the present invention bind with high affinity to antigen-presenting cells with different genetic backgrounds and to several class II MHC molecules so that the immune cell's recognition and attack is blocked. In addition, the copeptides can stimulate the growth and function of copolymer 1-specific T cells for further treatment and prevention of various autoimmune diseases.

Accordingly, the present invention relates to the use of a mixture of polypeptides having an average molecular weight of 2-40 kDa, where each polypeptide consists of glutamic acid, tyrosine, alanine and lysine, for the preparation of a pharmaceutical composition for the treatment of an autoimmune disease, except for multiple sclerosis, wherein said mixture of polypeptides is in an amount effective to treat a human suffering from an autoimmune disease, and wherein said pharmaceutical composition is suitable for administration to an individual suffering from an autoimmune disease.

BRIEF DESCRIPTION OF THE FIGURES

As applied below is

"GAL" a random terpolymer of glutamic acid, alanine and lysine,

"TGA" or "YEA" a random terpolymer of tyrosine, glutamic acid and alanine,

"TAL" or "YAK" a random terpolymer of tyrosine, alanine and lysine,

"GTL" or "YEK" a random terpolymer of glutamic acid, tyrosine and lysine, and "YEAK" copolymer 1.

Figure 1 shows the effect of copolymer 1 and terpolymers on the proliferation of T cells that were selective for certain peptides from myelin basic protein (MBP). Several of the terpolymers inhibited proliferation of T cells specific for antigen from myelin basic protein. MBP 84-102 (SEQ. ID. NO. 1). The following symbols were used: 1 (•); CRAZY (□); TGA (A); NUMBER (O); GTL (X). Figure 1A shows inhibition of the proliferation of the mouse T-cell clone MBP-sp-1, which is specific for MBP 84-102 peptide (0.5 g/well). As a control, the proliferation of the mouse T-cell clone MBP-sp-1 stimulated by MBP 84-102 (SEQ ID NO: 1) without inhibitor was 21,145 cpm. Figure 1B shows inhibition of the response of the human T cell clone GP-25 to MBP 84-102 peptide (0.125/well). Proliferation of the human T-cell clone GP-25 stimulated by MBP 84-102 (SEQ ID NO: 1) peptide without inhibitor was 11,442 cpm.

Figure 2A compares binding of certain terpolymers with binding of copolymer 1 to class II tissue type complex molecules of type DR1 (top), DR2 (middle), and DR4 (bottom). Binding of copolymer 1 (■) was compared to binding of the following biotinylated terpolymers: GAL (□); TGA ( Å) ; GTL (A); and NUMBER (♦). Amount of class II tissue type complex molecule was kept constant at 0.5 µg/sample and the concentration of polypeptide was varied between 0-8 µM as indicated on the y-axis. The binding took place at pH 5.0 for 40 hours at 37 °C. The binding was detected by capturing the polypeptide class II complexes with an LB33.1 antibody and determining the amount of biotinylated polypeptide bound by measuring the absorbance at 410 nm after reaction with streptavidin-conjugated alkaline phosphatase. Figure 2B shows Lineweaver-Bruke plots of the binding results provided in Figure 2A. Figure 3 shows competitive inhibition of binding of copolymer 1 to class II tissue type complex molecules. Purified HLA-DR1 molecules (top), HLA-DR2 molecules (middle) and HLA-DR4 molecules (bottom) were incubated with a constant amount (1.5 µM) of biotinylated copolymer 1 either alone or in the presence of one of the following unlabeled polypeptides: copolymer 1 (0); CRAZY (□); TGA'(^; GTL (A); and TAL (♦). The inhibition by these polypeptides was compared with the inhibition of the myelin basic protein antigen HA 306-318 (O) (SEQ: ID NO. 2). The binding occurred at pH 5.0 for 40 hours at 37°C. The amount of unlabeled polypeptide varied between 0.1-1000, as indicated on the y-axis. Specific binding is expressed as percent inhibition using Equation 1:

signal with competitor - background

percent inhibition = 100 - x 100 1

signal without competitor - background

Figure 4A shows competitive inhibition of TAL binding to class II tissue type complex myolecules by the bacterial superantigens SEA, SEB and TSST-1. Purifying HLA-DR1 molecules (top) and HLA-DR2 molecules (middle) and HLA-DR4 molecules (bottom) were incubated with a constant amount of biotinylated TAL, in the presence of increasing amounts of an unlabeled bacterial superantigen SEA ( ); SEB (■); or TSST-1 (#. Binding occurred at pH5.0 for 40 hours at 47 °C. The amount of superantigen was varied between 0.1-1000 µM, as indicated on the y-axis. Specific binding is expressed as percent inhibition using Eq. 1. Figure 4B shows competitive inhibition of TGA binding to class II tissue-type complex molecules by the bacterial superantigens SEA, SEB and TSST-1 Purified HLA-DR1 molecules (top), HLA-DR2 molecules (middle) and HLA-DR4 molecules (bottom) were incubated with a constant amount of biotinylated polypeptide TGA in the presence of increasing amounts of unlabeled bacterial superantigen SEA ( ; SEB (ji); or TSST-1 (•). Binding occurred at pH 5.0 for 40 h at 37 ° C. The amount of superantigen was varied between 0.1-1000 µM, as indicated on the y-axis. Specific binding is expressed as percent inhibition, calculated according to equation 1 above. Figure 4C shows the competitive inhibition of GAL binding to class II tissue type complex molecules by the bacterial superantigens SEA, SEB and TSST-1. Purified H LA-DR1 molecules (top) and HLA-DR2 molecules (bottom) were incubated with a constant amount of biotinylated GAL in the presence of increasing amounts of unlabeled bacterial superantigen SEA ( ; SEB (■); or TSST-1 (•). The binding took place at pH 5.0 for 40 hours at 37 °C. The amount of superantigen was varied between 0.1-1000 µM as indicated on the y-axis. Specific binding is expressed as percent inhibition using equation 1 above. Figure 5 shows inhibition of binding of labeled molecules to purified MHC class II molecules by terpolymers. Fig. 5 A shows inhibition of binding to MHC HLA-DR1 protein. Fig. 5B shows inhibition of binding to MHC HLA-DR4 protein. Unlabeled competitors include terpolymers, influenza virus hemagglutinin (HA) peptide 306-318 (SEQ ID NO: 2) and type II collagen (CII) peptide 261-273 (SEQ ID NO: 3). The concentration of unlabeled competitor is indicated on the abscissa. In each field, inhibition at CII 261-273 (SEQ ID NO: 3) is shown as open circles (O), inhibition at HA 306-318 (SEQ ID NO: 2) is shown as filled circles (•), inhibition at one of the terpolymers is indicated by open or filled triangles or squares. The extent of inhibition by GTL is shown by open triangles (A), and by TAL as filled triangles (▲), by TGA as open squares (□) and by copolymer 1 as filled squares P). The observed binding shown on the ordinate was calculated as percent inhibition using equation 1 above. Figure 6 shows inhibition of IL-2 production by DR1-restricted-CII-specific T-cell hybridomas in the presence of different polypeptides. Irradiated L57.23 cells (fibroblasts transfected with a gene encoding HLA-DR1) were incubated in duplicate with collagen peptide CII 261-273 (40 µg/ml) and varying concentrations, shown on the abscissa, of one of the respective polypeptides in 2 hours at 37 °C. Then T cells (clone 3.19 or 19.3, as indicated) were added and the mixtures were further incubated for 24 hours at 37°C. The supernatants (30 µl) were then removed and assayed for activity in IL-2-induced proliferation of IL-2-dependent cytotoxic T lymphocytes (CTL-L). The extent of inhibition by TAL is shown as filled circles (•), by TGA as filled triangles '(£), by GTL as open triangles (A) and by polymer 1 as filled squares <0). Percent inhibition of CTL-L proliferation shown on the ordinate was calculated according to equation 1. Figure 7 shows inhibition of IL-2 production by DR4-restricted Cll-specific T cell hybridomas (3838 and D3) in the presence of different polypeptides. Fig. 7A shows the effects of incubating irradiated 3838 or D3-Priess cells with collagen peptide CII 261-273 (SEQ ID NO:3) at a fixed concentration of 40 µg/ml, and with varying concentrations of each of the respective polypeptides for 2 hours at 37 °C. Fig. 7B

shows the effect of incubating L cells transfected with a gene encoding HLA-DR4 with collagen peptide CII 261-373 (SEQ ID NO:3) at a fixed concentration of 40 µg/ml, and with varying concentrations of each of the respective polypeptides for 2 hours at 37 °C. T cells were then added (clone 3838 or D3 as indicated), the samples were further incubated for 24 hours at 37°C, and the supernatants were analyzed as described in Figure 6. Each polypeptide was analyzed in duplicate. The concentration of the respective polypeptides is indicated on the abscissa. The same symbols that were used in figure 6 are used in this figure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a mixture of polypeptides which have an average molecular weight of 2-40 kDa, where each polypeptide consists of glutamic acid, tyrosine, alanine and lysine, for the production of a pharmaceutical composition for the treatment of an autoimmune disease, excluding multiple sclerosis. Autoimmune diseases occur when the immune system attacks certain tissues or cells in an unwanted way. The application according to the present invention can prevent the immune system from attacking, for example by suppressing the proliferation or function of T or B cells responsible for the attack, or by protecting the tissue from attack by binding to an MHC protein on the surface of the cells that make up the tissue.

Amino acids according to the present invention include, but are not limited to, the 20 commonly occurring amino acids. Furthermore, naturally occurring and synthetic derivatives, for example selenocysteine, are included. Amino acids also include amino acid analogues. An amino acid "analogue" is a chemically related compound of the amino acid with a different configuration, such as an isomer or a D configuration rather than an L configuration, or an organic molecule of approximately the same size and shape as the amino acid, or an amino acid in which the atoms that form part of the peptide bond are modified so that the peptide bond is protease resistant when the analogue forms part of a polypeptide.

The terms "amino acid" and "amino acid sequence" as defined herein may include one or more constituents that are amino acid derivatives and/or amino acid analogs that make up part or all of the amino acid residues for one or more of the 20 naturally occurring amino acids indicated in the sequence. In an amino acid sequence with one or more tyrosine residues, for example, a proportion of one or more of these residues can be substituted with homotyrosine. Furthermore, an amino acid sequence with one or more non-peptide bonds or peptide-like bonds between two neighboring residues is covered by this definition.

The one-letter code and the three-letter code for the amino acids (and the amino acid each represents) are as follows: A stands for ala (alanine), C stands for cys (cysteine), D stands for asp (aspartic acid), E stands for glu (glutamic acid), F stands for phe (phenylalanine), G stands for gly (glycine), H stands for his (histidine), I stands for ile (isoleucine), K stands for lys (lysine), L stands for leu (leucine), M stands for for met (methionine), N stands for asn (asparagine), P stands for pro (proline), Q stands for gin (glutamine), R stands for arg (arginine), S stands for ser (serine), T stands for thr (threonine), V stands for val (valine), W stands for trp (tryptophan) and Y stands for tyr (tyrosine).

The term "hydrophobic" amino acid is defined herein to include the aliphatic amino acids alanine (a or ala), glycine (G or gly), isoleucine (I or ile), leucine (L or leu), proline (P or pro) and valine (V or val), the terms in parentheses are the standard one-letter code and three-letter code for each amino acid, and the aromatic amino acids tryptophan, (W or trp), phenylalanine (F or phe), and tyrosine (Y or tyr). The amino acids provide hydrophobicity as a function of the length of aliphatic and the size of aromatic side chains when they are present as amino acid residues in a protein.

The term "charged" amino acid is defined herein as including the amino acids aspartic acid (D or asp), glutamic acid (E or glu), histidine (H or his) arginine (R or arg) and lysine (K or lys) which give a positive (his, lys and arg) or negative (asp and gly) charge at physiological pH values in aqueous solutions of proteins containing these amino acid residues.

Applications covered by the invention

The present invention relates to the use of a mixture of polypeptides that have an average molecular weight of 2-40 kDa, where each polypeptide consists of glutamic acid, tyrosine, alanine and lysine, for the production of a pharmaceutical composition for the treatment of an autoimmune disease, excluding multiple sclerosis, wherein said mixture of polypeptides is in an amount effective to treat a human suffering from an autoimmune disease, and wherein said pharmaceutical composition is suitable for administration to an individual suffering from an autoimmune disease.

Said polypeptides may consist of 1- or d-amino acids. As known to those skilled in the art, 1-amino acids occur in most natural proteins. However, D-amino acids are commercially available and can replace one or all of the amino acids. Said polypeptides can consequently be formed from mixtures of d- and 1-amino acids as well as essentially consist of either 1- or d-amino acids.

In a first embodiment according to the first aspect of the invention, said autoimmune disease is autoimmune hemolytic anemia, autoimmune oophoritis, autoimmune thyroiditis, autoimmune uveoretinitis, Crohn's disease, chronic immune thrombocytopenic purpura, colitis, contact sensitivity disease, diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis, or systemic lupus erythematosus.

In another embodiment, the autoimmune disease is rheumatoid arthritis. In a third embodiment, the autoimmune disease is myasthenia gravis. In a fourth embodiment, the autoimmune disease is Crohn's disease. In a fifth embodiment, the autoimmune disease is colitis. In a sixth embodiment, the autoimmune disease is autoimmune uveoretinitis.

According to a seventh embodiment according to the invention, glutamic acid is present in the mixture of polypeptides in a mole fraction of approx. 0.14; tyrosine is present in the mixture of polypeptides in a mole fraction of approx. 0.10; alanine is present in the mixture of polypeptides in a mole fraction of approx. 0.43; and lysine is present in the mixture of polypeptides in a mole fraction of approx. 0.34.

According to the eighth embodiment according to the invention, the mixture of polypeptides has an average molecular weight of approx. 4-12 kDa, such as an average molecular weight of approx. 4-9 kDa.

In a further embodiment of the present invention, the amount of the mixture of polypeptides is at least 10 mg, for example at least 15 mg or at least 20 mg.

In a further embodiment according to the present invention, the amount of the mixture of polypeptides is 25-400 µg per kg of body weight per day.

The above pharmaceutical composition is preferably administered orally, topically, by inhalation or by injection. More preferably, it is administered orally. In one embodiment, said pharmaceutical composition is lyophilized.

In a further embodiment according to the present invention, said autoimmune disease is a B-cell mediated autoimmune disease, a T-cell mediated autoimmune disease, an arthritic condition, a demyelination disease, or an inflammatory disease.

The term "arthritic condition" as used herein is a condition in which at least one symptom of rheumatoid arthritis is observed in at least one joint in a mammal, for example in the shoulder, knee, hip, back or a finger joint in the mammal. Examples of arthritic conditions include "polyarthritis", which is an arthritic condition that affects more than one joint, "juvenile arthritis" an arthritic condition in people under the age of 21, and Felty's syndrome, which can include the symptoms of neutropenia, splenomegaly, weight loss, anemia, lymphadenopathy and pigment spots in the skin.

RA is a T-cell mediated autoimmune disease that can be treated according to the present invention. The pathological process in autoimmune diseases and immune rejection is mediated by T cells. After binding to and recognizing an antigen, T cells will proliferate, secrete cytokines and recruit additional inflammatory and cytotoxic cells to the site. The polypeptides used according to the present invention prevent T-cell proliferation and T-cell functions, for example cytokine secretion and recruitment of inflammatory and cytotoxic cells to the site. If the autoimmune disease is an arthritic condition, the autoantigens can be collagen, and the polypeptides used according to the present invention can inhibit the proliferation and function of collagen-responsive T cells.

The application according to the present invention may involve binding the polypeptides to an antigen-presenting cell, for example a macrophage, a dendritic cell from the lymphatic tissue or an epidermal cell. A T cell's proliferation and functions are activated when an appropriate antigen is presented to the cell. By binding to antigen-presenting cells, the polypeptides used according to the present invention can block or otherwise interfere with T-cell activation.

The use of the present invention may involve binding the polypeptides to a class II tissue type complex protein associated with an autoimmune disease. The class II MHC proteins are mainly expressed on the surface of B-lymphocytes and antigen-presenting cells, such as macrophages. These class II MHC proteins have a peptide-binding cleft that is the site of presentation of antigenic peptides to T cells. By binding the aforementioned polypeptides to a tissue type complex protein of class II, the polypeptides can block or otherwise interfere with antigen presentation and/or T-cell activation.

The application according to the present invention may involve binding of the polypeptides to copolymer 1-reactive B cell antibodies and/or copolymer 1-reactive T cells. Copolymer 1-reactive Th2/3 T cells promote the therapeutic effects of copolymer 1. Upon binding to copolymer 1-reactive T cells, said polypeptides stimulate these T cells to proliferate, secrete anti-inflammatory cytokines and promote the therapeutic benefits of treatment according to the present applications. Said polypeptides can also bind to autoantigen-reactive antibodies, which can prevent the antibody from attacking the target tissue and thereby help prevent the development of the autoimmune disease. If the said polypeptides are, for example, bound to MBP-specific antibodies, the antibodies cannot bind to MBP and thereby lead to the destruction of MBP in the myelin layer.

The polypeptides used according to the present invention can be supplied in any conventional manner. In one embodiment, the present polypeptides can be administered by injection to promote delivery to tissues affected by the autoimmune disease. Thus, for example, the said polypeptides can be injected, ingested, inhaled or applied topically. The polypeptides can be applied in a cream, solution or suspension for topical application. The polypeptides can further be administered preferably orally, topically or by injection without the addition of an adjuvant.

Synthesis of terpolymers

The terpolymers can be prepared by any method available to a person skilled in the art. The terpolymers can, for example, be prepared under condensation conditions by using the desired mole fraction of amino acids in solution, or by methods that include syntheses in the solid phase. Condensation conditions include appropriate temperature, pH and solvent conditions for condensation of the carboxyl group of one amino acid to the amino group of another amino acid to form a peptide bond. Condensing agents, for example dicyclohexylcarbodiimide, can be used to promote formation of the peptide bond. Blocking groups can be used to protect functional groups, for example the side chain residues and some of the amino or carboxyl groups, against unwanted side reactions.

For example, you can use the method described in U.S. Patent No. 3,849,550, in which the N-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate and N-trifluoroacetyl-lysine are polymerized at room temperature in anhydrous dioxane with diethylamine as initiator. The γ-carboxyl group in glutamic acid can be unblocked with hydrogen bromide in glacial acetic acid. The trifluoroacetyl groups can be removed from lysine with 1 molar piperidine. A person skilled in the art will easily understand that the method can be adjusted for the production of peptides and polypeptides that contain the desired amino acids, for example only three of the four amino acids in copolymer 1, by selectively removing the reactions that apply to either glutamic acid, alanine, tyrosine or lysine. For the purposes of the present application, the terms "ambient temperature" and "room temperature" mean a temperature in the range from approximately 20 to approximately 26 degrees Celsius (°C).

The average molecular weight of the terpolymers can be adjusted during the polypeptide synthesis or after the terpolymers are prepared. To adjust the average molecular weight during polypeptide synthesis, the synthesis conditions or the amount of the amino acids are adjusted so that the synthesis stops when the polypeptide has approximately reached the desired length. After the synthesis, polypeptides with the desired average molecular weight can be obtained by any available size selection method, for example chromatography of the polypeptides on a column or gel that fractionates based on molecular weight and collection of the desired molecular weight ranges. The polypeptides according to the invention can also be partially hydrolysed to remove molecules with a high molecular weight, for example by acid hydrolysis or enzymatic hydrolysis, and then purified to remove acid or enzymes.

The following examples are to be considered illustrative examples only. More specifically, the invention is not intended to be limited by the methods, materials, conditions, process parameters, apparatus and the like specifically described herein.

EXAMPLE 1

PRODUCTION OF POLYPEPTIDES

Preparation of protected polypeptides

Protected polypeptides are prepared as described by Teitelbaum et al. using the N-carboxyanhydride (NCA)-blocked amino acids tyrosine, alanine, glutamic acid and trifluoroacetyl lysine dissolved in dioxane. 1. EUR. J. IMMUN. 242 (1971). The carboxylate group in glutamic acid is blocked with a benzyl (BZ) group.

The polymerization process is initiated by the addition of 0.01-0.2% diethylamine. The reaction mixture is stirred at room temperature for 20 hours and then poured into water. The protected polypeptide product is filtered off, washed with water and dried. Removal of the blocking γ-benzyl groups from the glutamate residue is carried out by treating the protected polypeptide with 33% hydrogen bromide in glacial acetic acid at room temperature for 6-12 hours with stirring. The product is extracted in an excess of water, filtered off, washed and dried to obtain the protected polypeptide.

Production of polypeptides with a molecular weight of 7,000 ± 2,000 Da

Treatment of γ-benzyl-protected polypeptides with 33% HBr in acetic acid not only ensures removal of the protecting benzyl group from the γ-carboxylate of the glutamate residue, but also cleaves the polypeptides into smaller polypeptides.

The time required to obtain a polypeptide with a molecular weight of 7,000 ± 2,000 daltons depends on the reaction temperature and the size of the protected polypeptide. At temperatures of between 20 °C and 28 °C, a test reaction is carried out for each portion over different periods of time, for example from 10 to 50 hours. A curve of the average molecular weight is kept as a function of time is drawn. The time required to obtain a molecular weight of 7,000 ± 2,000 Da is calculated from the curve, and the reaction is carried out on a larger scale. At 26 °C, the average time for obtaining a mixture of polypeptides with a molecular weight of 7,000 ± 2,000 Da is 17 hours. The product is extracted in an excess of water, filtered off, washed and dried to obtain a polypeptide with a desired average molecular weight range.

Production of lysine-containing polypeptides with low toxicity

Protected trifluoroacetyl polypeptide (20 g) is finely divided in 1 liter of water, and 100 g of piperidine is added. The mixture is stirred for 24 hours at room temperature and filtered. The solution of impure polypeptide is distributed on dialysis bags and dialyzed at 10-20 °C in water until pH 8 is achieved. The polypeptide solution is dialysed against 0.3% acetic acid and then again against water until pH 5.5-6.0 is obtained. This solution is then concentrated and freeze-dried to dryness.

Synthesis of: TGA poly[L-Tyr<0136>, L-Glu<0>,<21>, L-Ala<0>'<648>] with molecular weight 7,600

2) Procedure:

L-Tyr-NCA (6.5 g) is placed in dioxane (211 ml) and heated to 60 °C for 10 minutes, then filtered. 5-BZ-L-Glu-NCA (13 g) is then placed in dioxane (226 ml) and stirred at 20-25 °C for 10 minutes, then filtered. Place L-Ala-NCA (18.74 g) in dioxane (350 mL) and stir at 20-25 °C for 10 minutes, then filter.

A 2L Erlenmeyer flask equipped with magnetic stirrers is filled with the solution of L-Tyr-NCA, the solution of 5-BZ-L-Glu-NCA and the solution of L-Ala-NCA. Diethylamine (0.165 g) in dioxane (2.5 ml) is added to the reaction mixture and it is stirred at 20-25 °C for 20 hours. The reaction mixture is added to deionized water (IL), filtered and dried at 60 °C under vacuum. Yield: 25.8 g.

A solution of phenol (4.8 g) in HBr/AcOH (33%, 480 ml) is stirred for 20 hours. A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with a solution of HBr/AcOH, poly[5-BZ-L-Glu,L-Ala, L-L Tyr] which is stirred at 26 ± 1 °C for 16 hours. The reaction mixture is added to deionized water (2L) and stirred for 1 hour. Precipitated material is filtered off and washed with deionized water until the pH is 6. The product is dried in an oven with circulating air at 40 °C for approximately 40 hours. Yield: 24 g.

A 2 L Erlenmeyer flask equipped with a magnetic stirrer is filled with piperidine (13.2 ml), deionized water (1200 ml) and poly(Glu, L-Ala, L-L Tyr), and then stirred at 20-25 °C for 24 hours. The resulting solution is ultrafiltered through polyethersulfone membranes with a molecular weight limit of 5,000 Dalton until two-thirds of the original volume has been removed. Freshly deionized water is added to the original volume. The process is repeated 5 times until the content of impurities is less than 1%, assessed by HPLC. The resulting solution (in full volume) is acidified to pH 4.4 with acetic acid. The solution is ultrafiltered to pH 5.5-6, and the volume is reduced to a third. The resulting solution is freeze-dried to dryness.

Synthesis of GAL, poly[Glu<0153>, L-Ala<0>'<479>, L-Lys<0>,<365>] with molecular weight 8850

2) Procedure:

N6-TFA-L-Lys-NCA (22.9) in dioxane (420 ml) is stirred at 20-25 °C for 10 minutes and filtered. 5-BZ-L-Glu-NCA (9.8 g) in dioxane (170 ml) is stirred at 20-25 °C for 10 minutes and filtered. L-Ala-NCA (14 g) in dioxane (260 ml) is stirred at 20-25 °C for 10 minutes and filtered. A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with the solution of N6-TFA-L-Lys-NCA, the solution of 5-BZ-L-Glu-NCA and the solution of L-Ala-NCA. Diethylamine (0.12 g) in dioxane (2.5 ml) is added and the reaction mixture is stirred at 20-25 °C for 20 hours. The reaction mixture is added to deionized water (IL), filtered and dried at 60 °C under vacuum.

A solution of phenol (4.8 g) in HBr/AcOH (33%; 480 ml) is stirred for 20 hours. A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with a solution of HBr/AcOH, poly[5-BZ-L-Glu, L-Ala, N6-TFA-L-Lys] (24 g) which is stirred at 26 ±1°C in 16 hours. The reaction mixture is added to deionized water (2L) and stirred for 1 hour. Precipitated material is filtered off and washed with deionized water until pH = 6. The product is dried in an oven with circulating air at 40 °C for approximately 40 hours.

A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with piperidine (13.2 mL), deionized water (1200 mL) and poly[L-Glu, L-Ala, N6-TFA-L-Lys] (24 g) which is stirred at 20- 25 °C for 24 hours. The solution is ultrafiltered through polyethersulfone membranes with a limit value of 5,000 Dalton until two-thirds of the original volume has been removed. The volume is adjusted to the original volume by adding freshly deionized water. The process is repeated 5 times, until the content of impurities is less than 1% (assessed by HPLC). The resulting solution (in full volume) is acidified to pH 4.4 with acetic acid. The solution is ultrafiltered until the pH is 5.5-6 and the volume reduced to one third. The resulting solution is freeze-dried to dryness.

Synthesis of TGL, poly[L-Tyr<0>,<162>, L-Glu<0>,<259>, L-Lys<0>,<579>] with molecular weight 11,050

2) Procedure

L-Tyr-NCA (5.2 g) in dioxane (180 ml) is heated to 60 °C for 10 minutes and filtered. N6-TFA-L-Lys-NCA (24.16 g) in dioxane (450 ml) is stirred at 20-25°C for 10 minutes and filtered. 5-BZ-L-Glu_NCA (10.35 g) in dioxane (180 ml) is stirred at 20-25°C for 10 minutes and filtered.

A 2L Erlenmeyer flask equipped with magnetic stirrers is filled with the solution of N6-TFA-L-Lys-NCA, the solution of L-Tyr-NCA and the solution of 5-BZ-L-Glu-NCA. Diethylamine (0.095 g) in dioxane (2.5 ml) is added and the reaction mixture is stirred at 20-25 °C for 20 hours. Deionized water (0.7 L) is added to the reaction mixture, filtered and dried at 60 °C under vacuum.

A solution of phenol (4.6 g) in HBr/AcOH (33%; 460 mL) is stirred for 20 hours. A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with the solution of HB/r/AcOH and poly]5-BZ-L-Glu, L-Tyr, N6-TFA-L-Lys]. The mixture is stirred at 26 pp]. The mixture is stirred at 26 ± 1°C for 16 hours, after which it is added to deionized water (1.5L) and stirred for Vi hour. Precipitated material is filtered off and washed with deionized water until the pH is 5.5.

A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with piperidine (150 ml), deionized water (1400 ml) and poly]L-Glu, L-Tyr, N6-TFA-L-Lys] (50 g) which is stirred at 20-25 ° C for 24 hours. The resulting solution of poly[L-Glu, L-Tyr, L-Lys] is ultrafiltered through polyethersulfone membranes with a limit value of 5,000 Dalton until two-thirds of the original volume has been removed. The volume is adjusted to the initial volume by adding freshly deionized water. The process is repeated five times until the content of impurities is less than 1% (assessed by HPLC). The resulting solution (in full volume) is acidified to pH 4.2 with acetic acid. The solution is ultrafiltered until the pH is 5.5-6 and the volume is reduced to one third. The resulting solution is freeze-dried to dryness.

Synthesis of TAL, poly[L-Tyr<0>,<102>, L-Ala<0>'<542>, L-Lys<0>,<353>], with molecular weight 20,000

2) Procedure

L-Tyr-NCA (4.9 g) in dioxane (170 ml) is heated to 60 °C for 10 minutes and filtered. N 6 TFA-L-Lys-NCA (22.9 g) in dioxane (417 ml) is stirred at 20-25 °C for 10 minutes and filtered. L-Ala-NCA (14 g) in dioxane (260 ml) is stirred at 20-25 °C for 10 minutes and filtered.

A 2L Erlenmeyer flask equipped with magnetic stirrers is filled with the solution of N6-TFA-L-Lys-NCA, the solution of Tyr-NCA in dioxane and the solution of L-Ala-NCA. Diethylamine (0.1 g) in dioxane (2 ml) is added and the mixture is stirred at 20-25 °C for 20 hours. The reaction mixture is then added to deionized water (IL), filtered and dried at 60 °C under vacuum.

A solution of phenol (5 g) HBr/AcOH (33%; 500 ml) is stirred for 20 hours. A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with the solution of HB/r/AcOH and poly[L-Ala, L-Tyr, N6-TFA-L-Lys] and stirred at 62 ± 1°C for 16 hours. The reaction mixture is then added to deionized water (2L) and stirred for Vi hour. The starting material is filtered off and washed with deionized water until the pH is 5.5.

A 2L Erlenmeyer flask equipped with a magnetic stirrer is filled with piperidine (162 ml), water (1500 ml) and poly[L-Ala, L-Tyr, N6-TFA-L-Lys] (30 g) which is stirred at 20-25 °C for 24 hours. The resulting solution of poly[L-Ala, L-Tyr, L-Lys] is ultrafiltered through polyethersulfone membranes with a cut-off value of 5,000 daltons until two-thirds of the starting volume has been removed. The volume is adjusted to the original volume by adding freshly deionized water. The process is repeated five times until the content of impurities is less than 1% (assessed by HPLC). The resulting solution (in full volume) is acidified to pH 4.4 with acetic acid. The solution is ultrafiltered until the pH is 5.5-6 and the volume reduced to one third. The resulting solution is freeze-dried to dryness.

EXAMPLE 2

LOW MOLECULAR WEIGHT POLYPEPTIDES

In vivo assays

Three portions of copolymer-1 with an average molecular weight of 7.3 KDa and 8.4 KDa (less than 2.5% copolymer-1 molecules with a molecular weight above 40 KDa) and 22 KDa

(more than 5% copolymer-1 molecules with molecular weight above 40 KDa) were analyzed by the toxicity assay described below. Five mice were used in each experimental group.

Approach

Copolymer-1 is dissolved in distilled water to form a solution with 2.5 g of the active ingredient. Each mouse is injected with 0.5 ml of the assay solution into the lateral tail vein. The mice were observed for mortality and relevant clinical signs over a period of 48 hours. The observations were made 10 minutes, 24 hours and 48 hours after the injection. If all animals were alive after 48 hours and no adverse signs had been observed, the portion was designated as "non-toxic". However, if one or more of the mice had died or had shown undesirable signs, the portion was designated as "toxic".

Results

Three out of five mice died after 48 hours of treatment with the copolymer 1 polypeptides with an average molecular weight of 22 KDa. Accordingly, the high average molecular weight portion is termed "toxic". The copolymer 1 portions with average molecular weights of 7.3 and 8.4 KDa were both designated "non-toxic".

Rat basophilic leukemia cell degranulation assay in vitro

Histamine (or serotonin) release from basophil cells is an in vitro model of immediate hypersensitivity. The rat basophilic leukemia cell line RBL-2Ho is uniform and easy to maintain in culture, but a highly sensitive and reproducible system for the analysis of degranulation. E.L. Basumian, et al., 11 EUR. J. IMMUNOL. 317 (1981). The physiological stimulus for histamine release involves binding of the antigen to membrane-bound IgE molecules, which triggers an intricate biochemical cascade. Degranulation can be induced by non-IgE-mediated stimuli, including various peptides and synthetic polymers, such as polylysine. R. P. Sirganian, TRENDS IN PHARMACOLOGICAL SCIENCES 432 (Oet. 1983). RBL degranulation analysis is therefore used to identify portions of COP-1 which trigger extensive degranulation and which can thus be thought to trigger unwanted local and/or systemic side effects.

Approach

Rat basophilic leukemia cells (RBL-2Ho) are labeled with [H<3>]-serotonin, followed by incubation with a 100 µg of the COP-1 preparation to be analyzed. Portions of COP-1 that induce nonspecific degranulation release [H<3>]-serotonin into the medium. The radioactivity in the medium is measured in a scintillation counter, and the total amount of radioactively labeled serotonin in the cells is measured in centrifuged cells. Percent degranulation is calculated as percent serotonin released, relative to the total incorporated.

Results

Four portions of copolymer 1 with an average molecular weight between 8,250-14,500 were analyzed, both for percent molecules with a molecular weight above 40 KDa and for degranulation of RBL. The results are summarized in table 1.

It can be seen from the table that if the percentage of molecules with a high average molecular weight is low (less than 2.5%), the percentage release of serotonin is also low, and vice versa. These results suggest that copolymer 1 polypeptides with a low average molecular weight are preferable to copolymer 1 polypeptides with a higher average molecular weight.

EXAMPLE 3

SUPPRESSION OF EAE BY THE POLYPEPTIDES

Injection of copolymer 1 in incomplete Freund's adjuvant before disease induction can suppress experimental allergic encephalomyelitis (EAE). This suppression appears to be mediated via copolymer 1-specific TH2-type suppressor T cells that cross-react with myelin basic protein. Lando et al., 123 J. IMMUNOL. 2156

(1979); Aharoni et al., 17 EUR. J. IMMUNOL. 23 (1993); Aharoni et al., 94 PROC. NATL. AC AD. SCI. US 10821 (1997). Other researchers have observed that the therapeutic effect of copolymer 1 in patients with multiple sclerosis is also associated with the induction of TH2 cells. Lahat et al., 244 J. NEUROL. 129 (1997). In the present example, EAE is suppressed by different polypeptides according to the present invention.

METHODS OF PROCESSING

Copolymer 1

Copolymer 1, lots 02095 and 55495, with average molecular weight 6,000 Da and 5,800 Da, respectively, was obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel).

Terpolymers

Four terpolymers were obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel). The properties of these terpolymers are given below: 1) GAL terpolymer (SD-1689) is a mixture of polypeptides containing the following mole fraction of the amino acids glutamic acid (0.153), alanine (0.479) and lysine (0.365). The range of average molecular weight for GAL is from approximately 4,650 daltons to approximately 20,050 daltons. The average molecular weight of this GAL preparation is

8,850 daltons.

2) TGA terpolymer (SD-1690) is a mixture of polypeptides containing the following mole fraction of the amino acids tyrosine (0.136), glutamic acid (0.210) and alanine (0.648). The molecular weight range for TGA is from approximately 1,000 daltons to approximately 40,000 daltons. The average molecular weight of this TGA preparation is

7,600 daltons.

3) TAL terpolymer (SD-1691) is a mixture of polypeptides containing the following mole fraction of the amino acids tyrosine (0.102), alanine (0.542) and lysine (0.353). The molecular weight range for TAL is from approximately 5,700 daltons to approximately 34,400 daltons. The average molecular weight of this TAL preparation is approximately 20,000

dalton.

4) GTL terpolymers (SC-1697) are a mixture of polypeptides containing the following mole fraction of the amino acids glutamic acid (0.259), tyrosine (0.162) and lysine

(0.579). The molecular weight range for TGL is from approximately 4,000 daltons to approximately 23,500 daltons. The average molecular weight of this GTL preparation is approximately 11,050 daltons.

A control polypeptide is used which consists of a mixture of polypeptides containing a 1:1 mixture of the amino acids alanine, glutamic acid and tyrosine, with an average molecular weight of 26,700 daltons. This polypeptide was obtained from Sigma Chemical Company (St. Louis, MO).

Induction of EAE

Two- to three-month-old (SJL/JxBALB/c) Fl female mice are injected in all four footpads with mouse spinal cord homogenate (3.5 mg/mouse) emulsified in a 1:1 ratio in complete Freund's adjuvant (CFA) supplemented with 4 mg/ ml H37Ra. Pertussis toxin (0.25 ml, 250ng, Sigma) is injected intravenously immediately after and 48 hours later. The mice are examined daily from day 10 after induction for clinical signs of EAE which are scored on a scale of 0-5 as described in Lando et al., 123 J. IMMUNOL. 2157 (1979)..

EAE suppression by injection with incomplete adjuvant

The analyzed polypeptides (10 mg/mouse) are injected subcutaneously in incomplete Freund's adjuvant (ICFA) in 2-3 points in a neck area. EAE is induced 3 weeks later as described above.

EAE suppression by oral administration

In a second assay, female Lewis rats are fed 5 mg/kg guinea pig BP or copolymer 1 dissolved in phosphate buffered saline (PBS) at 2-3 day intervals before induction of EAE. EAE is induced two days after the last feeding by injection of 25 µg MBP from guinea pigs emulsified in 1:1 CFA to which is added 4 mg/ml mycobacterium tuberculosis (H37Ra) (Difco Lag, Detroit, Mich.). A total volume of 0.1 ml is injected into each of the two hind paws. Control rats are sham-fed with phosphate-buffered saline.

The efficacy of orally administered copolymer 1 in preventing EAE in rats is compared with that of rats fed guinea pig MBP by the method of Higgins et al., 140 J. IMMUNOL. 440 (1988).

The animals are examined daily from day 10 after the induction for signs of disease. EAE is scored as follows: 0 = no disease, 1 = flaccid tail, 2 = paralysis of the hind limbs, 3 = paralysis of all four limbs, 4 = moribund state and 5 = death.

RESULTS

EAE suppression by injection with incomplete adjuvant

Table 2 shows the effects of the polypeptides according to the invention when injected into the ICFA before the induction of EAE. Of the four polypeptides analyzed, copolymer 1 and TAL produced the strongest EAE suppression. GTL also showed suppressive activity. GAL and TGA were somewhat less effective. D-copolymer 1 is a copolymer of d-lysine, d-tyrosine, d-glutamic acid and d-alanine in the same molar ratio as in copolymer 1.

Table 3 shows the effect of orally administered copolymer 1 in preventing the clinical manifestations of EAE in Lewis rats, compared to rats given only phosphate buffered saline (PBS) or guinea pig basic protein.

Each number represents the cumulative results of 3-5 independent experiments. The P values represent the statistical significance of the difference from the control group (PBS group). The average maximum score is calculated for the entire group.

These results show that the present polypeptides are therapeutically effective for preventing the onset and extent of EAE when administered either orally or by injection.

EXAMPLE 4

Binding to antigen-presenting cells

Several of the polypeptides according to the invention bind effectively to living antigen-presenting cells.

METHODS OF PROCESSING

Copolymer 1

Copolymer 1 from lots 02095 and 55495, with average molecular weight of 6,000 daltons and 5,800 daltons, respectively, was obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel).

Terpolymers

GAL, TGA, TAL, GTA and the control polypeptides are as described above under example 3.

Biotinylation of antigens

Biotinylation of copolymer 1 and terpolymers is carried out at 0 °C with biotin-Af-hydroxysuccinimide (Sigma) according to Fridkis-Hareli et al. 91 PROC. NATL. ACAD. SCI. U.S. 4872 (1994).

Binding of biotinylated antigens to antigen-presenting cells

The biotinylated polypeptides were analyzed for binding to live mouse and human antigen-presenting cells using fluorescently labeled streptavidin and FACS analysis. Adherent spleen cells from (SJL/JxBALB/c)Fl mice or EBV-transformed human B cells of the DR7, s 11 haplotype (1 x 106/100 µl) were incubated at 37 °C for 20 h with 50 µg of biotinylated copolymer 1 or terpolymers dissolved in 100 µl PBS supplemented with 0.1% BSA). The cells were then incubated at 4°C for 30 minutes with phycoerythrin-conjugated streptavidin (Jackson Immuno Research, West Grove, PA) at a concentration of 0.5 µg/100 µl cell suspension. After each incubation, the cells were washed three times with PBS supplemented with 0.1% BSA. The cells were then analyzed by flow cytometry with a FACScan (Becton-Dickinson, Mountain View, CA). For each analysis, 5,000 cells were examined. Dead cells were excluded based on forward and lateral light scattering.

Epstein-Barr virus (EBV)-transformed B-cell lines

EBV-transformed B-cell lines were initiated according to Teitelbaum et al., 89 PROC. NATL. ACAD. SCI. US 137 (1992). Approximately 20 x 10<6> peripheral blood mononuclear cells were cultured with B95.8 cell line supernatant for 1 hour at 37°C. The cells were then washed and cultured in RPMI medium with 10% FCS and cyclosporin A (10 µg/ml) to remove T cells.

RESULTS

Table 4 shows binding of terpolymers and copolymer 1 polypeptides to living antigen-presenting cells, including macrophages from mouse spleen and human EBV-transformed B-cell lines. Results are given which show both percent binding and the intensity of the cell staining (table 41 + II). TAL was bound most efficiently - better than even copolymer 1. GAL and GTL were also very well bound - as well or even slightly better than copolymer 1. TGA was well bound, but somewhat less than copolymer 1.

TAL bound most efficiently, both to splenic macrophages from (SJL/JxBALB/c) Fi mice and to EBV-transformed B cells from a normal DR7.wl 1 donor, as shown by the binding intensity (Table 4).

EXAMPLE 5

BINDING TO PURIFIED HUMAN LEUCOCYTE ANTIGENS (HLA)

This example shows polypeptides that bind to human B cells and to purified human lymphocyte antigens, including HLA-DR1, HLA-DR2 and HLA-DR4 molecules, with high affinity.

METHODS OF PROCESSING

Cell lines and antibodies

The homozygous EBV-transformed human B-lymphocyte lines used for immunoaffinity purification of HLA-DR1, -DR2 and -DR4 molecules were LG-2 (DBR1-), MGAR (DRB1 ± 1501) and Pressis (DRB-0401/ DRB4-0101).

The cells were grown in RPMI1640 supplemented with 10% FCS, 2 mM glutamine, 50 U/ml penicillin G and 50 µg/ml streptomycin in roller bottles and stored with centrifuge at -80 °C. Anti-DR hybridoma LB3.1 (lgG2b) is grown in serum-free medium (Macrophage-SFM, Gibco BRL). See Gorga et al., 103 CELL. IMMUNOL. 160 (1986).

Protein purification

Immunoafflinite purification of HLA-DR1, -DR" and -DR4 molecules is performed as previously reported by Gorga et al., 262 J. BIOL. CHEm. 16087 (1987) with minor modifications. Briefly, detergent-soluble membrane preparations were obtained from LG-2 -, MGAR and Priess cells are passed at a flow rate of approximately 11 ml/hr through a series of columns in the following order: Sepharose CL-6B (30 ml), normal mouse serum Aff gel 10 (10 ml), protein A -Sepharose CL-4B (5 ml) and LB3.1 protein A-Sepharose CL-4B (5 ml).DR2a (DRB5*0101) and Drw53)

(DRB4<*>0101), the products of DR genes linked to the DRBl alleles were not removed from MGAR and Priess lysates before passage through the LB3.1 immunoaffinity column and contaminate the DR2 and DR4 preparation at an amount of 5- 10%. All subsequent steps were as described previously by Gorga et al., 262 J. BIOL. Chem. 16087 (1987). The eluate is dialyzed against 0.1% dioxycholate, 10 mM tris-HCl, pH 8.0 and concentrated on a Centriprep 30 membrane (Amicon). Protein concentration was determined by bicinchonitrilic acid assay (Pierce Chemical Co.).

Polypeptides and control antigens

Copolymer 1

Copolymer 1, lots 55495 and 52596, with an average molecular weight of 5,800 daltons and 8,150 daltons, respectively, was obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel). Copolymer 1, lot 52596, had a molar ratio of 1 tyrosine: 1.5 glutamic acid: 4.3 alanine: 3.1 lysine.

Myelin basic protein and hemagglutinin control peptides

MBP peptides were synthesized in an Applied Biosystems peptide synthesis instrument using solid phase techniques. Barany et al., THE PEPTIDES 1 (1979). The peptides were purified by reverse phase HPLC. The peptides used were HA 306-318, with the sequence PKYVKQNTLKLAT (molecular weight 1718) (SEQ ID NO: 2) and MBP 84 102, with the frequency DENPWHFFKNIVTPRTPP (molecular weight 2529) (SEQ ID NO: 1).

Terpolymers

GAL, TGA, TAL, GTL and the control polypeptides are as described above under example 3.

Polypeptide labeling

Biotinylation of the various polypeptides is carried out as in example 4. Unreacted biotin is removed by dialysis (Spectra/Por® membrane MWCO 500, Spectrum Medical Industries).

Analysis of polypeptide binding to class II MHC proteins

Solutions: the solutions used in this assay have the following: binding buffer is 20 mM 2-[N-morpholino]ethanesulfonic acid (MES), 1% n-octyl 6-D-glycopyranoside, 140 mM NaCl, 0.05% NaN3, pH 5.0, unless otherwise stated. PBS is 150 mM sodium chloride, 7.5 mM dibasic sodium phosphate, 2.5 mM monobasic sodium phosphate, pH 7.2. TBS is 137 mM sodium chloride, 25 mM Tris pH 8.0, 2.7 mM potassium chloride. TTBS is TBS plus 0.05% Tween-20.

Preparation of microtiter assay plates: Ninety-six-well microtiter immunoassay plates (PRO_BIND™, Falcon) were coated with 1 µg/well of affinity-purified monoclonal antibody LB3.1 in PBS (100 µl total) for 18 h at 4 °C. The wells were then blocked with TBS supplemented with 3% BSA for 1 hour at 37 °C and washed 3 times with TTBS. Before adding sample, 50 ul of TBS/1% BSA is added to each well.

Binding reactions: Detergent-resolved HLA-DR1, -DR2, and -DR4 molecules (0.5 µg/sample) were incubated with biotinylated peptides at various concentrations for 40 h at 37 °C in 50 µl binding buffer and transferred to prepared microtiter assay plates and incubated for 1 hour at 37°C to capture polypeptide-class II complexes.

Inhibition reactions: Biotinylated polypeptides at a final concentration of 1.5 µM in 50 µl binding buffer co-incubate with unlabeled polypeptides as well as the peptides HA 306-318 (SEQ ID NO: 2) or MBP 84-102 (SEQ ID NO : 1), used as inhibitors, and HLA-DR molecules for 40 hours at 37 °C.

Detection of class II/polypeptide complexes: Bound polypeptide-biotin is detected using streptavidin-conjugated alkaline phosphatase as follows: Plates were washed three times with TTBS and incubated with 100 µl streptavidin-conjugated alkaline phosphatase (1:3000, BioRad) for 1 hour at 37°C , followed by the addition of p-nitrophenyl phosphate in triethanolamine buffer (BioRad). The absorbance at 410 nm is measured in a microplate reader.

RESULTS

Binding of terpolymers to class II MHC proteins

Detergent-free HLA-DR1, HLA-DR2 and HLA-DR4 proteins were purified from the homozygous EBV-transformed B cell lines LG-2(DRB1<*>0101), MGAR (DRB1<*>1501) and Priess (DRB 1*0401), as described previously by Fridkis-Hareli et al, 160 J. IMMUNOL. 4386 (1998). Three different preparations of copolymer 1 bound these molecules with high affinity. To determine the affinity of the terpolymers for HLA-DR proteins, binding assays were performed with biotinylated terpolymers and compared to copolymer 1. The polypeptides were incubated at a range of concentrations with purified HLA-DR1, HLA-DR2 and HLA-DR4 molecules at pH 5 ,0, followed by capture with class H-specific mAb and detection with alkaline phosphatase-streptavidin.

Binding of TAL and copolymer 1 to detergent-free HLA-DR1 and HLA-DR4 molecules is better than the binding of GAL, TGA or GTL. However, GTL and copolymer 1 bind better than the other polypeptides to HLA-DR2, based on the saturation binding curves (Fig. 2A) and Ka values, calculated from double reciprocal plots of the binding results (Fig. 2B, Table 5).

Competitive binding assays were performed with biotinylated copolymer 1 and the following unlabeled inhibitors: Copolymer 1, TAL, TGA, GAL, TGL, MBP 84-102 and HA 306-318 polypeptides (Fig. 3). MBP 84-102 (SEQ ID NO: 1) peptides are a poor inhibitor of the binding of copolymer 1 to HLA-DR2. Binding of biotinylated copolymer 1 to detergent-free HLA-DR1 and DR4 molecules is effectively inhibited by unlabeled TGA, TAL, HA 306-318 (SEQ ID NO: 2) and copolymer 1. Binding of biotinylated copolymer 1 to detergent-free HLA-DR1 and -DR4 molecules are, however, more than 10 times less inhibited by TGL based on the dose for 50% inhibition (IC50) (Fig. 3, Table 5). Similarly, TAL is also a poor inhibitor of binding of copolymer 1 to HLA-DR1 and -DR4 molecules. In general, the binding pattern of HLA-DR2 is similar to the pattern observed for HLA-DR1 (Table 5). These results show that the three-amino acid polypeptides, particularly TAL and TGA, bind to class II MHC molecules with an affinity range corresponding to that of antigenic peptides and to copolymer 1. Consequently, TAL and TGA are effective competitors for the class II MHC molecules that copolymer 1 is bound to. Based on the binding capacity, the polypeptides can be arranged in the following order: (A) binding to HLA-DR1: copolymer 1 > TAL > GTL > TGA » GAL; (B) binding to HLA-DR2: copolymer 1 > GTL > TAL > GAL > TGA;

(C) binding to HLA-DR4: TAL > copolymer 1 » TGA > GTL > GAL.

<1> Copolymer 1 polypeptides with average molecular weight 5,800, TAL: molecular weight 20,000 GAL: molecular weight 8,850, TGA: molecular weight 7,600 and TGL: molecular weight 11,050 were incubated in a series of concentrations with purified HLA-DR1, -DR2 and -DR4 molecules at pH 5.0, followed by capture with class II-specific mAb and detection of peptide with alkaline phosphatase-streptavidin. 2 Detergent-resolved HLA-DR1, -DR2 and DR4 molecules were purified as described in materials and methods.

Ka, the dissociation constant at equilibrium, is calculated from the slope angle in the double reciprocal plot (Fig. 2B), and is expressed as x 10-<8> M.

<4>IC50, the inhibitor concentration that gives 50% inhibition, is calculated based on the competitive binding assays (Fig. 3) and is plotted as x 10_6M.

<5>The IC50 values for GAL could not be determined precisely, but lower than 1000 uM (see Fig. 3).

Effect of superantigens on binding of polypeptides to HLA-DR molecules

The bacterial superantigens SEA, SEG and TSST-1 have been shown to inhibit copolymer 1 binding to purified HLA-DR molecules only at very high concentrations. Fridkis-Hareli et al., 160 J. IMMUNOL. 4386 (1998). To investigate the effect of these superantigens on the binding of terpolymers to purified HLA-DR1, HLA-DR2 and HLA-DR4 molecules, competitive binding assays were performed with unlabelled SEA, SEG and TSST-1.

Binding of TAL to HLA-DR1-, HLA-DR2- and HLA-DR4 is only inhibited by the superantigens at a high molar ratio of superantigen, for example a fifty-fold higher amount of superantigen is required for inhibition of TAL binding (Fig. 4A) . However, binding of TGA and GAL is inhibited to a greater extent by the superantigens (Fig. 4B and C), which shows that these polypeptides bind to the HLA antigens with lower affinity.

EXAMPLE 6

INHIBITION OF MBP-INDUCED T- CELL PROLIFERATION

This example shows that the proliferation of T cells which are normally activated by myelin basic protein (MBP) can be inhibited by simultaneous treatment with certain polypeptides.

METHODS OF PROCESSING

Copolymer 1

Copolymer 1, portions 02095 and 55495 with average molecular weight 6,000 da and 5,800 da, respectively, were obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel).

Myelin basic protein peptides were synthesized in an Applied Biosystems peptide synthesis instrument using solid phase techniques. Barany et al, THE PEPTIDES 1

(1979). The peptides were purified by reverse phase HPLC. The peptides used were HA 306-318, with the sequence PKYVKQNTLKLAT (molecular weight 1718) (SEQ ID NO: 2) and MBP 84 102, with the sequence DENPVVHFFKNTTVTPRPTP (molecular weight 2529)

(SEQ ID NO: 1).

Terpolymers

GAL, TGA, TAL, GTL and the control polypeptides are as described above under example 3.

T cell lines and clones

Myelin basic protein (MBP)-specific T cell lines were derived from the spleens of mice that had been immunized ten days previously with 20 µg of the 84-102 peptide from MBP emulsified in complete Freund's adjuvant supplemented with 4 mg/ml Mycobacterium tuberculosis H37Ra. The cells were cultured and selected in vitro using the immunizing antigen (0.2-1 mg/plate) in culture medium (RPMI, 2 mM glutamine, 1 mM sodium pyruvate, non-essential amino acids, 5 x 10"<5> M 2 -mercaptoethanol, 100 ug/ml penicillin, 100 ug/ml streptomycin), supplemented with 2% autologous serum. After 4 days, the cells were transferred to culture medium containing 10% FCS and supplemented with 10% supernatant from Con A-activated, normal spleen cells which T cell growth factor (TCGF) Every fourteenth to twenty-first day, cells were stimulated by treatment with the immunizing antigen, presented on syngeneic, irradiated (3,000 rad) spleen cells (50 x 10<6>/plate) for 3 days, followed by culture in TCGF medium Cloning of T-cell lines is performed by limiting dilution to 0.3 cells/well in microtiter plates in the presence of the antigen (2-10 µg/well) and irradiated syngeneic spleen cells (5 x 106/well).

Human T cell lines were derived from peripheral blood mononuclear cells according to Teitelbaum et al., 89 PROC. NATL. ACAD. SCI. US 137 (1992). Approximately 5 x 10<6> cells were incubated in each well of a 24-well culture plate with copolymer 1 or MBP (50 µg/ml) in culture medium supplemented with 10% heat-inactivated autologous serum. After 7 days of culture, the cells were transferred to culture medium containing 10% fetal calf serum and recombinant human interleukin 2 (20 units/ml). The cells were grown continuously in this medium by periodic treatment with antigen presented on irradiated (3,000 rad), autologous mononuclear cells every 14-18 days.

Proliferation assay

Cells from T lines or clones were analyzed for specific proliferative response 10-21 days after antigen stimulation. The T cells (1.5 x 104) were cultured in triplicate with 5 x 10<5 >irradiated spleen cells and with the indicated antigens in a final volume of 0.2 ml in culture medium with 10% FCS. After 48 hours of incubation, the culture was pulse labeled with 1 uCi[<3>H]-thymidine (standard deviation <20% of mean cpm) and harvested 6-12 hours later.

Inhibition studies

Inhibition of the t-cell proliferative response is investigated by adding different concentrations of copolymer 1 and terpolymers as well as the stimulating MBP antigen to the proliferation assay system. The inhibition calculated as percent inhibition using equation 2: % inhibition = [1 - (cpm with inhibitor / cpm without inhibitor)] x 100. 2

RESULTS

Figure 1 shows how copolymer 1 and terpolymers affect the proliferation of t cells specific for certain myelin basic protein (MBP) peptides. In general, t cells proliferate when exposed to the antigen to which they have been sensitized. Thus, MBP, and in particular these antigenic peptides from MBP, stimulate proliferation of MBP-specific t cells.

Copolymer 1 and terpolymers did not stimulate proliferation of t cells specific for the 84-102 peptide from MBP. In contrast, they significantly inhibited the proliferation of t cells treated with this MBP antigen.

TAL showed the most effective inhibition of proliferation of t cells specific for MBP 84-102 antigen. The inhibition by TAL is even greater than that achieved by copolymer 1. TGA induced lower inhibition of t-cell proliferation. GAL and GTL inhibited T-cell proliferation in a dose-dependent manner, similarly to copolymer 1.

EXAMPLE 7

TERPOLYMERS ARE RECOGNIZED BY CERTAIN COPOLYMERS 1

1- SPECIFIC CELLS

Terpolymers can stimulate certain copolymer 1-specific t-cell lines to proliferate and secrete the cytokine IL-4.

METHODS OF PROCESSING

Copolymer 1

Copolymer 1, portions 02095 and 55495 with average molecular weight 6,000 da and 5800 da respectively were obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel.

Myelin basic protein peptides were synthesized in an Applied Biosystems peptide synthesis instrument using solid phase techniques. Barany et al, THE PEPTIDES 1 (1979). The peptides were purified by reverse phase HPLC. The peptides used were HA 306-318 with the sequence PKYVKQNTLKLAT (mol weight 1718) (SEQ ID NO: 2), and MBP 84 102, with the sequence DENPVVHFFKNIVTPRTPP (mol weight 2529) (SEQ ID NO: 1).

Terpolymers

GAL, TGA, TAL, GTL and the control polypeptides are as described above under example 3.

T cell lines and clones

Mouse T-cell lenses and clones were established according to Aharoni et al., 23 EUR. J. IMMUNOL. 17 (1993); Aharoni et al., 94 PROC. NATL. ACAD. SCI. US 10821 (1997). Copolymer 1-specific cell lines originated in the spleens of mice that 15-35 days earlier had been rendered non-responsive to EAE by subcutaneous injection into each mouse of 5-10 mg of copolymer 1 emulsified in incomplete Freund's adjuvant (Difco). Alternatively, copolymer 1-specific cell lines were obtained from the lymph nodes of mice that had been immunized ten days earlier with 200 µg of copolymer 1 emulsified in complete Freund's adjuvant (Difco) supplemented with 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco).

Copolymer 1-specific t-cell lines were used, as well as the t-cell clones LN-1, LN-2, S-3, 5-22-5, C-14 and C-52. The LN-2 antibodies were derived from lymph nodes of (SJL/JxBALB/c)Fi mice injected with copolymer 1 in CRA. The 5-22-5 antibodies were obtained from the spleens of (SJL/JxBALB/C)Fi mice injected with copolymer 1 in CFA according to Aharoni et al., 23 EUR. J. IMMUNOL. 17 (1993); Aharoni et al., PROC. NATL. ACAD. SCI. US 10821 (1997).

Human T-cell clones were derived from peripheral blood mononuclear cells according to Teitelbaum et al., 89 PROC. NATL. ACAD. SCI. US 137 (1992). Approximately 5 x 10<6> cells were incubated in each well of a 24-well culture plate with copolymer 1 or MBP (50 µg/ml) in culture medium supplemented with 10% heat-inactivated autologous serum. After 7 days of culture, the cells were transferred to culture medium containing 10% fetal calf serum and recombinant human interleukin 2 (20 units/ml). The cells were cultured continuously in this medium with periodic exposure to antigen presented on irradiated (3,000 rad), autologous mononuclear cells every 14-18 days. The T-cell clone C-52 was derived from a DR7, wl 1 donor, also according to Teiltelbaum et al., 89 PROC. NATL. ACAD. SCI. US 137 (1992).

Proliferation assay

Cells from t-cell lines or clones were analyzed for specific proliferative response 10-21 days after antigen stimulation. The T cells (1.5 x 104) were cultured in triplicate with 5 x 10<5 >irradiated spleen cells or with human EBV-transformed B cells (5 x 10<4>), and with the indicated antigens in a final volume of 0.2 ml in 10% FCS culture medium. After 48 hours of incubation, the cultures were pulse labeled with 1 uCi[<3>H]-thymidine and harvested 6-12 hours later. The triplicate samples' variation from the mean value was below 20%.

The cell line cross-reacted only with TAL. No cell line cross-reacted with GTL or with AGT (1:1:1), which has the same amino acids as TGA but in a molar ratio different from that in TGA or copolymer 1.

EXAMPLE 8

TERPOLYMERS CROSS-REACT WITH ANTI-COPOLYMER1 ANTIBODIES

Antibodies directed against copolymer 1 cross-react with terpolymers lacking either tyrosine, vitamin acid or alanine. However, terpolymers lacking lysine are not efficiently recognized by anti-copolymer 1 antibodies.

Antibodies

Mouse monoclonal anti-MBP and anti-copolymer 1 antibodies were obtained by fusion of MBP- or copolymer 1-immunized spleen cells from SJL/J mice with the mouse plasmacytoma cell line NSO/1. Teitelbaum et al., Proe. Nati. Acad. Pollock. USA 9528

(1991).

Radioimmunoassay

Flexible plastic microtiter plates were coated with copolymer 1 or terpolymers (2 µg/ml). After 16 hours of incubation at room temperature, the plates were washed three times and saturated for 2 hours with PBS supplemented with 2% bovine serum albumin, 0.05% Tween 20, 0.1% sodium azide, 10 mM EDTA and heparin at 5 units/ml ('TBS buffer"). Monoclonal antibody supernatants (50 µl) were added to the wells for incubation for 2 toms, and the wells were again washed with PBS buffer. <125>I-labeled goat anti-mouse Fab antibodies (1 x 10<5> cpm/well) was added for overnight incubation at 4° C. After extensive washing, the radioactivity is measured in a gamma counter.

Procedures

ELISA assay for antibody cross-reactivity

A standard ELISA assay using polyclonal anti-copolymer 1 antibodies and microtiter plates coated with 2 ug/ml terpolymer preparation is used.

Copolymer 1

Copolymer 1 with the following amino acid composition is obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel).

Terpolymers

The four terpolymers from example 1 were used.

Results

Table 7 shows that polyclonal anti-copolymer 1 antibodies cross-react with terpolymers lacking either tyrosine, glutamic acid or alanine. The relatively high percentage binding of terpolymers lacking glutamic acid (TAL) can be explained by their high average molecular weight. Terpolymers lacking lysine are not efficiently recognized by anti-polymer 1 antibodies. These results suggest that charged amino acids such as lysine may play a role in the recognition and binding of copolymer 1 and terpolymers.

Cross-reactivity of terpolymers with monoclonal copolymer 1-reactive antibodies

The cross-reactivity of the terpolymers with copolymer 1 at the B-cell response level is analyzed using monoclonal antibodies (mAb), which either react with both copolymer 1 and MBP (mAb 2-2-18 and 3-1-45) or only with copolymer 1 (mAb 3-3-9 and 5-7-2). Teitelbaum et al., 88 Proe. Nati. Acad. Pollock. U.S. 9528 (1991).

Table 8 shows that the terpolymers differed in their ability to bind to these mAbs. TGA and GTL were not recognized by any of the copolymer 1-specific mAbs. In contrast, TAL and GAL bound to copolymer 1- and MBP-specific mAb with an affinity similar to that of copolymer 1. GAL and TAL differed only in binding to a mAb, i.e. 5-7-2, which bound TAL and not CRAZY.

2- 2-18 is a monoclonal anti-mouse MBP antibody that cross-reacts with copolymer 1. 3- 1-45 is a monoclonal anti-copolymer 1 antibody that cross-reacts with MBP. 3-3-9 is a monoclonal anti-copolymer 1 antibody that does not cross-react with MBP. 5-7-2 is a monoclonal anti-copolymer 1 antibody that does not cross-react with MBP.

EXAMPLE 9

COPOLYMER 1 AND TERPOLYMERS COMPETE WITH COLLAGEN FOR BINDING TO HUMAN LEUCOCYTE ANTIGENS AND INHIBIT

COLLAGEN-SPECIFIC T-CELL RESPONSE

This example shows that copolymer 1, TAL, GAL, GTL and TGA compete for binding to MHC proteins with the rheumatoid arthritis-associated immunodominant collagen antigen CII 261-273 (SEQ ID NO: 3).

METHODS OF PROCESSING

Protein expression and purification

Recombinant HLA-DR1 and HLA-DR4 molecules (encoded by DRA/DRB 1 <*>0101 and <*>0401, respectively) were expressed in Drosophila S2 cells as described in Stern, L. et al. 68 CELL 465 (1992) and Dessen, A. et al. 7IMMUNITY 473 (1997). The cells were grown in roller bottles at 26°C in Excell 401 medium (Sigma, St. Louis, MO) supplemented with 0-5% fetal bovine serum (Sigma). The cells were induced by the addition of CUSO4 to a final concentration of 1 mM and then incubated for a further 4-5 days. Immunoaffinity purification of recombinant HLA-DR1 and HLA-DR4 is performed as previously reported by Stern, L. et al. 68 CELL 465 (1992) and Dessen, A. et al. 7 IMMUNITY 473 (1997). Supernatant from harvested cells is passed in this order through protein A, protein G and protein A-LB3.1 columns, followed by elution of bound HLA-DR with 50 mM 3-cyclohexylamino-l-propanesulfonic acid (CAPS), pH 11.5, and neutralization with 20 mM phosphate (pH 6.0). The eluate is concentrated on a Centriprep 10 membrane (Amicon). Protein concentration concentrations were determined with the bicinchoninic acid assay (Pierce Chemical Co.).

Labeling of polypeptide

The present polypeptides and HA 306-318 peptide were biotinylated as in Examples 4 and 7.

Class II MHC protein binding assay.

In this assay, water-soluble, recombinantly produced proteins were incubated with biotinylated polypeptides of the present invention and varying amounts of unlabeled competitor polypeptides, collagen CII peptides or HA peptides from influenza virus. The analyzes were performed in 96-well microtiter immunoassay plates (PRO-BOND™, Falcon) which were coated with affinity-purified monoclonal LB3.1 antibodies. Antibody coating is performed by placing 10 µl of 10.0 µg/ml LB3.1 monoclonal antibodies in each well and incubating at 4°C for 18 hours. The microtiter wells were then blocked with Tris-buffered saline (TBS) supplemented with 3% bovine serum albumin (BSA) for 1 hour at 37 °C and washed three times with TTBS. Before sample addition, 50 ul of TBS with 1% BSA is added to each well. Phosphate buffered saline (TBS) is 150 mM sodium chloride, 7.5 mM dibasic sodium phosphate, 2.5 mM monobasic sodium phosphate, pH 7.2. Tris-buffered saline (TBS) is 137 mM sodium chloride, 25 mM Tris pH 8.0, 2.7 mM potassium chloride. TTBS is TBS with 0.05% Tween-2G. Other solutions used in this analysis are described in Fridkis-Hareli, M. et al., 160 J. IMMUNOL. 4386 (1998).

The binding analysis is performed by incubating the water-soluble DR molecules with biotinylated polypeptides according to the present invention and varying concentrations of unlabeled inhibitors (copolymer 1, TAL, GAL, GTL, TGA, collagen CII 261-273 peptide or HA 306-318 peptide). Collagen type CII peptide 261-273 has SEQ. ID NO: 3 (AGFKGEQGPKGEP) and a molecular weight of 1516. The concentration of DR used is 0.15 µM. The final concentration of biotinylated copolymer 1 or terpolymers is 1.5 µM. The incubation takes place for 40 hours at 37 °C in 50 ul binding buffer at pH 5.0.

Bound labeled compound is detected using streptavidin-conjugated alkaline phosphatase as follows. The plates were washed three times with TTBS and incubated with 100 μl of streptavidin-conjugated alkaline phosphatase (1:3000, BioRad, Richmond, VA) for 1 h at 37 °C, followed by the addition of p-nitrophenyl phosphate in triethanolamine buffer (BioRad). The absorbance at 410 nm is measured in a microplate reader (model MR4000, Dynatech, Chantilly, VA).

Binding assays for T-cell hybridomas and antigen-presenting cells (APCs)

Copolymer 1 and terpolymers were analyzed to determine whether they could inhibit activation of t cells responding to collagen CII peptide. The IDR1-restricted t-cell hybridomas 3.19 and 19.3 from mice and the DR4-restricted t-cell hybridomas 3838 and D3 from mice were used. Rosloniec. E. F. et al., 185 J. EXP. WITH. 1113-1122

(1997); Andersson, E.C. et al., PROC. NATL. ACAD. SCI. United States (1998). Antigen presenting cells (APC) were L cells transfected with DR1 (the L57.23 cell lines obtained from Rosloniec, E. F., et al., 1997, 185 J. Exp. Med. 113-1122

(1997)), L cells transfected with DR4 and Priess cells (DRB1<*>0401/DRB4<*>0101). T cell crowding experiments were performed in 96-well microtiter plates in a total volume of 0.2 ml. Irradiated (3000-rad) APC (2.5 x 10<4>/well) were incubated with CII 261-273 (40 µg/ml) and varying concentrations of the present polypeptides for 2 h at 37 °C, after which t- cells (5 x 10<4>/well) were added and the incubation continued for 24 hours at 37 °C. Supernatants (30 µl) were removed and incubated with IL-2-dependent CTL-L (5 x 10<4>/well) for 12 hours, followed by labeling with <3>H-thymidine (1 µC/well) in 12 hours. Plates were harvested and radioactivity measured using a 1450 microbeta Plus liquid scintillation counter (Wallc, Gaithersburg, MD).

RESULTS

Class II MHC protein binding assay

The recombinant water-soluble HLA-DR1 and -DR4 proteins produced in insect cells were largely free of bound autoantigens or other peptides. Consequently, results obtained from proteins produced in insect cells may provide a more accurate picture of the actual binding affinities for polypeptides. Fridkis-Hareli et al., 160 J. IMMUNOL. 4386

(1998) (the entire contents of which are incorporated herein by reference).

Competitive binding of copolymer 1 and the terpolymers to HLA-DR1, HLA-DR2 and HLA-DR4 molecules is shown in Figure 5. Binding of copolymer 1 (YEAK, upper panel in Fig. 5 A) and test polymers is significantly higher than binding of CII 261-273 (SEQ ID NO: 3) peptide, estimated from the amount of CII 261-273 (SEQ ID NO: 3) peptide required for 50% inhibition. Also as observed above, TAL bound to HLA-DR1 and HLA-DR4 with higher affinity than copolymer 1. The kinetics of inhibition with unlabeled TAL (YAK, lower panel in Fig. 5A) polypeptides were also somewhat better than the kinetics of influenza virus peptide HA 306 -318 (SEQ ID NO: 2). However, influenza virus peptide HA 306-318 inhibited binding of copolymer 1 and terpolymers more effectively than CU 261-273 peptide.

Results from binding analysis for T-cell hybridomas and antigen-presenting cells

Copolymer 1 and terpolymers also inhibited DR 1-restricted t-cell activation by CII collagen peptide (Fig. 6). The T-cell activation was demonstrated by observing the new production from DR1-restricted CII-specific T-cell hybridomas. Collagen peptide CII 261-273 (SEQ ID NO: 3) at various concentrations was incubated with one of the present polypeptides, after which t cells (clone 3.19 or 19.3 as indicated) were added and the mixtures further incubated. Supernatants from these incubated cells were collected and analyzed for JL-2 by observing whether the supernatant induced proliferation of IL-2-dependent cytotoxic T-lymphocytes (CTL-L). The extent of inhibition by TAL is shown as filled circles (O), by TGA as filled triangles (A), by GTL as open triangles (A) and by copolymer 1 as filled squares ( ). Percent inhibition of CTL-L proliferation shown on the ordinate was calculated using Equation 1.

Again, TAL is the most potent inhibitor. However, GTL and copolymer 1 were also potent inhibitors of t-cell activation by Cll collagen peptide. TGA inhibited the activation less effectively. Similar results were obtained with other portions of copolymer 1 and terpolymers.

A corresponding inhibition of the activation of DR4-restricted t-cells was observed as shown in Figure 7. IL-2 production was used to estimate activation of DR4-restricted Cll-specific t-cell hybridomas (3838 and D3). The presence of different polypeptides inhibited IL-2 production, suggesting that they inhibited DR4-restricted t-cell activation. Fig. 7 A shows the effect of incubating irradiated 3838 or D3-Priess cells with collagen peptide CII 261-273 (SEQ ID NO: 3) at a fixed concentration of 40 µg/ml and with varying concentrations of polypeptides for 2 hours at 37 °C. Fig. 7B shows the effect of incubating L cells transfected with a gene encoding HLA-DR4 with collagen peptide CII 261-273 (SEQ ID NO: 3) at a fixed concentration of 40 µg/ml and with varying concentrations of GAL, TAL, TGL, TGA and copolymer 1, for 2 h at 37 °C. T cells were then added (clones 3838 or D3, as indicated), and the samples were further incubated for 24 hours at 37°C. The supernatants (30 µl) were then exposed and analyzed for active IL-2-induced proliferation of IL-2-dependent cytotoxic T-lymphocytes (CTL-L). Each polypeptide mixture was analyzed in duplicate. The concentration of the peptides present is indicated on the abscissa. The extent of inhibition by TAL is shown as filled circles (O), by TGA as filled triangles (A), by GTL as open triangles (A) and by copolymer 1 as filled squares ( ). Percent inhibition of CTL-L proliferation shown on the ordinate was calculated using Equation 1.

EXAMPLE 10

COPOLYMER 1 INHIBITS ACTIVATION OF T CELLS RESPONDING TO AN ANTIGENIC MYASTHENIA GRAVIS PEPTIDE

METHODS OF PROCESSING

Copolymer 1

Copolymer 1 was obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel).

Myasthenia Gravis-related peptides were synthesized in an Applied Biosystems peptide synthesis instrument using solid phase techniques. Barany et al., THE PEPTIDES 1 (1979). The peptides were purified by reverse phase HPLC. The peptides used were p259 peptide, see Zisman et al., Hum Immuol 1995 Nov; 44 (3): 121-30; Brocke et al., Immunology 1990 Apr; 69(4): 495-500.

IL-2 secretion

Secretion of IL-2 from the WCB AB cell line in response to the myasthenia gravis peptides and/or copolymer 1 was analyzed. The cells (1.5 x 10<4>) were incubated with the indicated antigen. Secretion of IL-2 from the WCB AB cell line was used as a measure of activation of the t-cell line in question.

RESULTS

Table 12 shows that the p259 peptide stimulates the secretion of the t cells. However, if copolymer 1 is incubated together with the p259 peptide, the secretion of IL-2 by the t cells is inhibited in a dose-dependent manner. At a concentration of 100 µM, copolymer 1 inhibits approximately 91% of IL-2 secretion (table 13), which shows that copolymer 1 is a powerful inhibitor of t-cell activation.

The absorbance is the average value for 3 samples. The reliability values were calculated from the absorbance of a blank sample %CV = SD/(average blank sample)<*>100.

Claims (24)

1. Use of a mixture of polypeptides having an average molecular weight of 2-40 kDa, where each polypeptide consists of glutamic acid, tyrosine, alanine and lysine, for the preparation of a pharmaceutical composition for the treatment of an autoimmune disease except multiple sclerosis, wherein said mixture of polypeptides is in an amount effective to treat a human suffering from an autoimmune disease, and wherein said pharmaceutical composition is suitable for administration to an individual suffering from an autoimmune disease. 2. Use according to claim 1, where said autoimmune disease is autoimmune hemolytic anemia, autoimmune oophoritis, autoimmune thyroiditis, autoimmune uveoretinitis, Crohn's disease, chronic immune thrombocytopenic purpura, colitis, contact sensitivity disease, diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease , idiopathic myxedema, myasthenia gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis, or systemic lupus erythematosus. 3. Use according to claim 1 or 2, wherein the autoimmune disease is rheumatoid arthritis. 4. Use according to claim 1 or 2, wherein the autoimmune disease is myasthenia gravis. 5. Use according to claim 1 or 2, wherein the autoimmune disease is Crohn's disease. 6. Use according to claim 1 or 2, wherein the autoimmune disease is colitis. 7. Use according to claim 1 or 2, wherein the autoimmune disease is autoimmune uveoretinitis. 8. Use according to any one of claims 1-7, wherein glutamic acid is present in the mixture of polypeptides in a mole fraction of approx. 0.14; tyrosine is present in the mixture of polypeptides in a mole fraction of approx. 0.10; alanine is present in the mixture of polypeptides in a mole fraction of approx. 0.43; and lysine is present in the mixture of polypeptides in a mole fraction of approx. 0.34. 9. Use according to any one of claims 1-8, where the mixture of polypeptides has an average molecular weight of approx. 4-12 kDa. 10. Use according to claim 9, where the mixture of polypeptides has an average molecular weight of approx. 4-9 kDa. 11. Use according to any one of claims 1-10, wherein glutamic acid is present in the mixture of polypeptides in a mole fraction of approx. 0.14; tyrosine is present in the mixture of polypeptides in a mole fraction of approx. 0.10; alanine is present in the mixture of polypeptides in a mole fraction of approx. 0.43; and lysine is present in the mixture of polypeptides in a mole fraction of approx. 0.34; and where the mixture of polypeptides has an average molecular weight of approx. 4-9 kDa. 12. Use according to any one of claims 1-11, wherein the amount of the mixture of polypeptides is at least 5 mg. 13. Use according to claim 12, wherein the amount of the mixture of polypeptides is at least 10 mg. 14. Use according to claim 13, where the amount of the mixture of polypeptides is at least 15 mg. 15. Use according to claim 14, wherein the amount of the mixture of polypeptides is at least 20 mg. 16. Use according to any one of claims 1-11, wherein the amount of the mixture of polypeptides is 25-400 µg per kg body weight per day. 17. Use according to any one of claims 1-16, wherein said pharmaceutical composition is administered orally, topically, by inhalation or by injection. 18. Use according to claim 17, where said pharmaceutical composition is administered orally. 19. Use according to any one of claims 1-18, wherein said pharmaceutical composition is lyophilized. 20. Use according to claim 1 or 2, where said autoimmune disease is a B-cell mediated autoimmune disease. 21. Use according to claim 1 or 2, where said autoimmune disease is a T-cell mediated autoimmune disease. 22. Use according to claim 1 or 2, where said autoimmune disease is an arthritic condition. 23. Use according to claim 1 or 2, where said autoimmune disease is a demyelination disease. 24. Use according to claim 1 or 2, where said autoimmune disease is an inflammatory disease.