JP2002521387A - Treatment of autoimmune diseases with Copolymer 1 and related copolymers and peptides - Google Patents

Abstract

  (57) [Summary] The present invention relates to polypeptides and peptides comprising at least three amino acids, randomly linked in a linear sequence, wherein at least one of the three amino acids is an aromatic amino acid and at least one of the three amino acids Are charged amino acids, and at least one is directed to polypeptides and peptides that are aliphatic amino acids. In a preferred embodiment, the polypeptide comprises 3-4 amino acids of the following amino acids: tyrosine, alanine, glutamic acid or lysine. According to the present invention, the polypeptide binds to antigen presenting cells, purified human lymphocyte antigen (HLA) and / or copolymer 1 specific T cells. Further in accordance with the invention, these polypeptides can be formulated into pharmaceutical compositions for treating autoimmune diseases. The present invention further contemplates a method of treating an autoimmune disease in a mammal, comprising administering to the mammal a therapeutically effective amount of any one of the polypeptides or peptides.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[Related application]

In this application, a provisional application, 60 / 093,859,199 filed on July 23, 1998
60 / 101,825 filed on September 25, 2008, and 60 / 101,825 filed on October 2, 1998
/ 102,960, filed October 30, 1998, 60 / 106,350,19.
Claim the benefits of 60 / 108,184 filed on November 12, 1998 and 60 / 123,675 filed on March 9, 1999, all of which are incorporated herein by reference. .

[0002]

[Government support]

This invention was sponsored in part by the government under CA47554 recognized by the National Institutes of Health.

[0003]

[Introduction]

The present invention provides compositions and methods for treating an autoimmune disease using a therapeutically effective amount of a peptide or polypeptide related to Copolymer 1. Copolymer 1 is a heterogeneous mixture of synthetic random linear copolymers of tyrosine, alanine, glutamic acid and lysine, used in the treatment of multiple sclerosis at an appropriate therapeutic dose and average molecular size. Such a mixture of synthetic random linear copolymers is copolymer 1
Are essentially called ternary copolymers when they consist essentially of three of the four amino acids found in Table 1. The present invention relates, in part, to terpolymers. Preferably, the terpolymer comprises tyrosine, alanine and lysine, or from glutamic acid, tyrosine and lysine, or from glutamic acid, alanine and lysine. Surprisingly, terpolymers have efficacy in the treatment of various autoimmune diseases,
Binds major histocompatibility complex (MHC) molecules, as well as antigen presenting cells.

[0004] The present invention also encompasses certain peptides comprising the four amino acids of Copolymer 1, which have efficacy in the treatment of various autoimmune diseases, and which comprise the class II major histocompatibility complex (MHC) It has also been found to bind molecules, as well as antigen presenting cells. Such a peptide is hereinafter referred to as a “copeptide” with emphasis on the relationship with the copolymer 1.

[0005]

BACKGROUND OF THE INVENTION

Autoimmune diseases occur when an organism's immune system does not recognize some of its own tissues as "self" and attacks them as "foreign". Usually, self-tolerance occurs early in the immune system due to developmental events, preventing the organism's own T and B cells from reacting with the organism's own tissues. MHC cell surface proteins bind to T cells and help regulate these early immune responses by providing processed peptides to T cells.

When an autoimmune disease develops, this self-tolerant process is disrupted. Soon the organism's own tissues and proteins are recognized as "self-antigens" 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 sheath, which covers and protects nerves. It is a progressive disease characterized by loss of neurons and motor function, second to demyelination. Rheumatoid arthritis ("RA") is also an autoimmune disease involving chronic inflammation of synovial cell joints and invasion by activated T cells, macrophages and plasma cells, thought to lead to progressive destruction of articular cartilage. I have. It is the most severe arthropathy. Collagen II is a self-antigen attacked by rheumatoid arthritis
Although the type is a candidate, its properties are not well understood.

[0007] The tendency to develop multiple sclerosis and rheumatoid arthritis is inherited-these
Disease occurs more frequently in individuals with one or more unique MHC class II alleles.
This. For example, the inherited rheumatoid arthritis susceptibility is MHC class II DRB1 ^* 0401, DRB1^*0404, or DRB1^*0405, or DRB1 ^* It is strongly associated with the 0101 allele. Histocompatibility locus antigen (HLA)
It is found on the cell surface and helps determine the individuality of tissues of various people. Organization
The gene for the matching locus antigen is the same chromosome as the major histocompatibility complex (MHC)
It is located in the sixth area. MHC region has many unique features in various cells of the body
Of the class I molecules, whose genes are arranged in chromosomal order in the class I
, II and III MHC genes. Class I gene is HLA gene
Which are further subdivided into A, B and C subdivisions. Class II inheritance
The child is subdivided into DR, DQ and DP subdivisions. MHC-DR molecule is the best
Well known, these are macrophages, dendritic cells of lymphoid tissues and epidermis
Appears on the surface of antigen presenting cells such as cells. Class III MHC products look like the complement system
It is expressed in various components, as well as in some non-immune related cells.

[0008] Steroidal and non-steroidal anti-inflammatory drugs (eg, methotrexate)
Numerous therapeutic agents have been developed to treat autoimmune diseases, including various interferons, and inhibitors of prostaglandin synthesis. However, these drugs are toxic except for short-term use, and can cause unwanted side effects. Other therapeutic agents bind and / or tumor necrosis factor (TNF)
, Which inhibit the inflammatory activity of, for example, an anti-TNF-specific antibody or antibody fragment, or a soluble form of the TNF receptor, these agents target proteins on the surface of T cells and are generally associated with antigen presenting cells (APC Hinder interaction. However, therapeutic compositions containing natural folded proteins are often produced, formulated,
Difficult to store and deliver. In addition, the innate heterogeneity of the immune system limits the efficacy of the drug, complicating long-term treatment of autoimmune diseases.

Thus, there is a need for new drugs that do not have the side effects of the present therapeutics and that fully address the innate heterogeneity of the immune system in order to effectively treat autoimmune and other immune disorders.

[0010]

[References]

[0011]

Summary of the Invention

Surprisingly, Copolymer 1 and the terpolymers and copeptides of the invention can be used to treat various autoimmune diseases in heterogeneous patient groups. These terpolymers and copeptides can inhibit some physiological responses of T cells that attack self antigens as these diseases progress.
In addition, Copolymer 1 and the terpolymers and copeptides of the present invention are highly active with antigen presenting cells of various genetic backgrounds and with some class II MHC molecules that interfere with and attack immune cell recognition. Bind with affinity. In addition, terpolymers and copeptides can stimulate the proliferation and function of copolymer 1-specific T cells, and can also treat and prevent various autoimmune diseases.

Therefore, the present invention relates to a group of amino acids comprising copolymer 1, ie, copolymer 1
A pharmaceutical composition having a polypeptide comprising three different amino acids selected from glutamic acid, alanine, lysine, and tyrosine in an appropriate relative molar ratio found in the above.

The present invention is also directed to a peptide comprising three different amino acids selected from the group of amino acids containing copolymer 1.

The invention also consists essentially of the amino acids tyrosine, alanine and lysine,
About 0.005 to about 0.250 tyrosine, about 0.3 to about 0.6 alanine, and about 0
. It is also directed to a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 1 to about 0.5 lysine and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of glutamic acid.

[0015] The present invention further provides about 0.005 to about 0.300 glutamic acid, about 0.005 to about 0.250 tyrosine, and about 0.3 to about 0.3 g of glutamic acid, tyrosine and lysine. Provided is a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 7 lysines and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of alanine.

[0016] The present invention also relates to a composition comprising about 0.005 to about 0.25 tyrosine, about 0.005 to about 0.3 glutamic acid, and about 0.005 to about 0 It is also directed to a pharmaceutical composition comprising a therapeutically effective amount of a tertiary copolymer in a molar ratio of .8 lysine and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of lysine.

[0017] The present invention also relates to a compound comprising about glutamic acid, alanine and lysine, comprising about 0
. 005 to about 0.3 glutamic acid, about 0.005 to about 0.6 alanine, and about 0
. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 2 to about 0.7 lysine and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of tyrosine.

The present invention also provides a pharmaceutical composition for treating an autoimmune disease, comprising a therapeutically effective amount of copolymer 1 or polypeptide consisting essentially of glutamic acid, alanine, tyrosine and lysine, and Also provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

The present invention is further a method for treating and preventing an autoimmune disease in a mammal, comprising administering a therapeutically effective amount of a composition comprising Copolymer 1, terpolymer or copeptide. A method comprising: In another aspect, the method for treating an autoimmune disease in a mammal further comprises inhibiting T cell proliferation associated with an immune attack. In another aspect, a method for treating an autoimmune disease in a mammal comprises combining the terpolymer or copeptide with an antigen presenting cell. In yet another aspect, a method for treating an autoimmune disease in a mammal comprises combining a terpolymer or copeptide with a major histocompatibility complex class II protein associated with the autoimmune disease. .

The autoimmune diseases contemplated by the present invention include arthritis diseases, demyelination diseases and inflammatory diseases. For example, autoimmune diseases that can be treated with the present compositions include:
Multiple sclerosis, autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmune uveal retinitis, Crohn's disease, chronic autoimmune thrombocytopenic purpura, colitis, contact Hypersensitivity, diabetes mellitus, Graves' disease, Guillain-Barre syndrome,
If you have Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis, or lupus erythematosus. The compositions can be used to treat one or more of these diseases.

[0021]

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, polypeptides and peptides having at least three different amino acids, randomly polymerized in linear form, are useful for treating autoimmune diseases. Autoimmune diseases occur when the immune system is able to respond inappropriately to certain tissues or cells. The polypeptides and peptides of the present invention include, for example, T
Alternatively, the immune system can be prevented from attacking by inhibiting the growth or activity of B cells, or by protecting the tissue from attack by binding to MHC proteins on the surface of cells that make up the tissue.

The amino acids of the present invention include, but are not limited to, the 20 commonly occurring amino acids. Also included are naturally occurring and synthetic derivatives such as selenocysteine. In addition, amino acids include amino acid analogs. Amino acid "analogs" are defined as chemically similar forms of amino acids having different configurations, such as isomers, or D-configuration rather than L-configuration, or organic molecules containing amino acids of appropriate size and shape, or peptide bonds. Amino acids that have been modified at the relevant atom and are resistant to proteases during polymerization in peptides and polypeptides.

“Amino acid” and “amino acid sequence” as defined herein and in the claims refer to any or all of the one or more of the 20 naturally occurring amino acids represented by the sequence. One or more components that are amino acid derivatives and / or amino acid analogs may be included. For example, in amino acid sequences having one or more tyrosine residues, some of those one or more residues can be replaced with homotyrosine. Furthermore, amino acid sequences having one or more non-peptide or peptide-like bonds between two adjacent residues are included in this definition.

The one- and three-letter amino acid codes (and the amino acids indicated by each) are as follows: A means ala (alanine); C means cys (cysteine); D is asp (asparagine). E means glu (glutamic acid); F means phe (phenylalanine); G means gly (glycine); H means his (histidine); I means ile ( K means lys (lysine); L means leu (leucine); M means m
et means methionine; N means asn (asparagine); P means pr
Q means gln (glutamine); R means arg (arginine); S means ser (serine); T means thr (threonine); W means val (valine); W means trp (tryptophan); and Y means tyr (tyrosine).

A “hydrophobic” amino acid is defined herein and in the claims as an aliphatic amino acid, 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) (in brackets, one letter of each amino acid and 3
Letter standard code abbreviations), and aromatic amino acids, tryptophan (W or tr
p), phenylalanine (F or phe), and tyrosine (Y or tyr)
) Is defined. When found as residues in proteins, amino acids confer hydrophobicity as a function of aliphatic side chain length and aromatic side chain size.

“Charged” amino acids are defined herein and in the claims as an aqueous solution of a protein containing these residues at physiological pH values (his, lys and arg).
A) aspartic acid (D or asp), glutamic acid (E or glu), histidine (H or his), arginine (R or arg) and lysine (K or lys) which confer charge or negative (asp and gly) charges; Defined.

Polypeptide Compositions Contemplated by the Present Invention The polypeptides and peptides of the present invention comprise copolymer 1 and three of the four amino acids of copolymer 1, ie, tyrosine, glutamic acid, alanine and lysine.
Comprising a terpolymer consisting essentially of seeds. However, one skilled in the art can readily substitute structurally similar and / or charge related amino acids without departing from the spirit of the invention. Thus, the present invention further contemplates conservative amino acid substitutions for tyrosine, glutamic acid, alanine and lysine in the polypeptides. Such conservative alternatives are structurally related amino acid alternatives, such as those amino acids that are similar in charge, hydrophobicity and size to tyrosine, glutamic acid, alanine or lysine. For example, lysine is structurally related to arginine and histidine; glutamic acid is structurally related to aspartic acid; tyrosine is structurally related to serine, threonine, phenylalanine and tryptophan; and alanine is valine, leucine. And is structurally related to isoleucine. These and other such conservative alternatives and structurally related synthetic amino acids are contemplated by the present invention.

Further, the terpolymer and the copeptide may consist of d-amino acids,
And / or α-amino acids. As is known to those skilled in the art, l
-Amino acids are present in most natural proteins. However, d-amino acids are commercially available and may be used in place of some or all of the amino acids used to make terpolymers and copeptides. The present invention relates to terpolymers and copeptides formed from mixtures of d- and l-amino acids, and
Tertiary copolymers and copeptides consisting essentially of either-or d-amino acids are contemplated.

The average molecular weight and average mole fraction of amino acids in the terpolymer can vary. However, it is believed that the range of average molecular weight is from about 2,000 to about 40,000 daltons. The length of the copeptide may be about 7-100 amino acids, or about 7-50 amino acids, about 7-25 amino acids, about 7-15 amino acids. For a preferred average molecular weight range and terpolymer production process, see US Pat.
No. 800,808, which is incorporated herein by reference.

In one embodiment, the present invention provides a terpolymer comprising tyrosine, alanine and lysine. The average mole fraction of amino acids in these terpolymers can vary, for example, tyrosine is present in a mole fraction of about 0.005 to about 0.250; Present in a molar fraction of 0.6;
It may be present in a mole fraction of about 0.5. The average molecular weight is about 2,000 to about 40,0
00 daltons, preferably between about 3,000 and about 35,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,
000 daltons.

In another aspect, the invention provides a terpolymer comprising tyrosine, glutamic acid and lysine. The average mole fraction of amino acids in these polypeptides can vary, for example, glutamic acid is present in a mole fraction of about 0.005 to about 0.300; tyrosine is present in about 0.005 to about 0.250. And lysine may be present in a mole fraction of about 0.3 to about 0.7. Average molecular weight is about 2
Between about 3,000 and about 40,000 daltons, preferably between about 3,000 and about 3
Between 5,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,000 daltons.

In another aspect, the invention provides a terpolymer comprising glutamic acid, alanine and lysine. The average molar fraction of amino acids in these polypeptides can vary, for example, glutamic acid is present in a molar fraction of about 0.005 to about 0.300; alanine is present in about 0.005 to about 0.600. And lysine may be present in a mole fraction of about 0.2 to about 0.7. Average molecular weight is about 2
Between about 3,000 and about 40,000 daltons, preferably between about 3,000 and about 3
Between 5,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,000 daltons.

In another aspect, the invention provides a terpolymer comprising tyrosine, glutamic acid and alanine. The average mole fraction of amino acids in these polypeptides can vary, for example, tyrosine is present in a mole fraction of about 0.005 to about 0.250; glutamic acid is present in about 0.005 to about 0.300. Present in a mole fraction of
And alanine may be present in a mole fraction of about 0.005 to about 0.800. The average molecular weight is between about 2,000 and about 40,000 daltons, preferably about 3,
Between 000 and about 35,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,000 daltons.

In a further preferred embodiment, the molar fraction of amino acids in the terpolymer is also preferred for copolymer 1. The molar fractions of amino acids in copolymer 1 are glutamic acid (about 0.14), alanine (about 0.43), tyrosine (about 0.10) and lysine (about 0.34). The most preferred average molecular weight of Copolymer 1 is about 5,
Between 000 and about 9,000 daltons.

A more preferred terpolymer monomer, glutamic acid, alanine and tyrosine molar ratio is about 0.21: about 0.65: about 0.14.

A more preferred terpolymer has a molar ratio of monomers, glutamic acid, alanine and lysine of about 0.15: about 0.48: about 0.36.

A more preferred terpolymer has a molar ratio of monomer, glutamic acid, tyrosine and lysine of about 0.26: about 0.16: about 0.58.

A more preferred terpolymer has a molar ratio of monomers, tyrosine, alanine and lysine of about 0.10: about 0.54: about 0.35.

In one embodiment, the terpolymers and copeptides of the present invention are capable of binding to MHC class II proteins, preferably associated with autoimmune diseases. Using any available method, one can determine whether the terpolymer and copeptide bind to one or more MHC class II proteins. For example, after labeling a polypeptide with a reporter molecule (such as a radionuclide or biotin), mixing with a crude or pure MHC class II protein preparation and removing unbound polypeptide, the reporter molecule may be labeled with an MHC class II protein. If it is in contact with the protein, binding is detected.

[0040] In another embodiment, the terpolymers and copeptides of the invention are capable of binding to MHC class II proteins associated with multiple sclerosis. The polypeptide of this embodiment has a similar or higher affinity to the antigen binding groove of the MHC class II protein associated with multiple sclerosis than Copolymer 1. Thus, the intended polypeptide either inhibits the binding of myelin autoantigen or inhibits its binding to MH
It can be replaced from C class II proteins. 1 related to multiple sclerosis
One MHC class II protein is HLA-DR4 (DRB1 ^* 1501).

In another embodiment, the copolymer 1, terpolymer and co-peptide of the invention are capable of binding to MHC class II proteins associated with arthritis diseases such as rheumatoid arthritis or osteoarthritis. The copolymer 1, terpolymer or copeptide of this embodiment has a higher affinity than the type II collagen 261-273 (SEQ ID NO: 3) peptide for the antigen-binding groove of MHC class II proteins associated with autoimmune diseases. Have. Thus, the intended copolymer 1, terpolymer or copeptide is
Inhibit the binding of type II collagen 261-273 peptide or reduce it to MH
It can replace the antigen binding groove of the C class II protein. Class II M
HC proteins consist of approximately equal-sized α and β subunits, both of which are transmembrane proteins. The peptide bond cross-section is created by the amino terminal portion of both the α and β subunits. This peptide bond cross-section is the presentation of the antigen to T cells. At least three types of class II MHC molecules: HLA-DR, HL
There are A-DQ and HLA-DP molecules. Also, each type of these HL
There are a number of alleles that encode the A molecule. Class II MHC molecules have excellent expression on the surface of antigen presenting cells such as B lymphocytes and macrophages.

The copeptide of the present invention is a synthetic peptide having a length of at least 7 amino acid residues and capable of binding to an MHC class II protein associated with an autoimmune disease. The copeptide may include an amino-terminal alanine, the sequence of which is alanine-glutamic-lysine-tyrosine-alanine (AEKYA) (SEQ ID NO: 4); alanine-glutamic-lysine-valine-alanine (AEKVA) (SEQ ID NO: 5); -Glutamic acid-lysine-phenylalanine-alanine (AEKFA) (SEQ ID NO: 6); lysine-glutamic acid-tyrosine-alanine (KEYA) (SEQ ID NO: 7); lysine-tyrosine-alanine-glutamic acid (KYAE) (SEQ ID NO: 8)
Lysine-glutamic acid-valine-alanine (KEVA) (SEQ ID NO: 9); lysine-valine-alanine-glutamic acid (KVAE) (SEQ ID NO: 10);
Glutamic acid-phenylalanine-alanine (KEFA) (SEQ ID NO: 11); Lysine-phenylalanine-alanine-glutamic acid (KFAE) (SEQ ID NO: 12)
); Lysine-tyrosine-alanine-alanine (KYAA) (SEQ ID NO: 13); lysine-lysine-tyrosine-alanine (KKYA) (SEQ ID NO: 14); lysine-valine-alanine-alanine (KVAA) (SEQ ID NO: 15); Consists of lysine-lysine-valine-alanine (KKVA) (SEQ ID NO: 16); lysine-phenylalanine-alanine-alanine (KFAA) (SEQ ID NO: 17); and lysine-lysine-phenylalanine-alanine (KKFA) (SEQ ID NO: 18). It is selected from the group of amino acid sequences. In this embodiment, the peptide may further comprise two alanine residues, the sequence of which is alanine-lysine-tyrosine-alanine-glutamic acid (AKYAE) (SEQ ID NO: 19); glutamic acid-alanine-lysine-tyrosine-alanine (EAKYA). (SEQ ID NO: 20); alanine-lysine-valine-alanine-glutamic acid (AKVAE) (SEQ ID NO: 21); glutamic acid-alanine-lysine-valine-alanine (EAKVA) (SEQ ID NO: 22); alanine-lysine-phenylalanine-alanine- Glutamic acid (AKFAE) (SEQ ID NO: 23)
); And glutamic acid-alanine-lysine-phenylalanine-alanine (E
AKFA) (SEQ ID NO: 24).

In another embodiment, the polypeptide is: AKEYAAAAAAKAAAAA (SEQ ID NO: 25) AAEYAAAAAAAKAAAAA (SEQ ID NO: 26) AAKYAEAAAAAKAAAAA (SEQ ID NO: 27) : 29) KEYAAAAAAAKAAAAA (Sequence recognition number: 30) AEYAAAAAAAKAAAAA (Sequence recognition number: 31) EKAYAAAAAAAKAAAAA (Sequence recognition number: 32) AAKEAAAKAAAAAA (A) ) AKYEAAAAAAAAAAA (Distribution (Sequence recognition number: 36) AAEYKAAAAAAAAA (sequence recognition number: 37) AAKYEAAAAAAAAA (sequence recognition number: 38) AAKYAEAAAAAAAAAAA (sequence recognition number: 39) No. 42) and AKYAEAAAAAAAAAA (SEQ ID NO: 43) having a sequence selected from the group consisting of: MH
Has high affinity for C class II proteins.

Yet another aspect of the invention is a copeptide having any of the above sequences wherein tyrosine (Y) is replaced with valine (V) or phenylalanine (F).

In another aspect of the present invention, there is provided a copeptide having an amino acid sequence capable of inhibiting an immune response to a mammalian self-antigen, wherein the identity and position of at least one amino acid in the polypeptide sequence are MHC. A copeptide corresponding to the identity and position of at least one amino acid in the peptide binding groove of a Class II protein is provided (in this embodiment, the copeptide may include other amino acids shown below). To treat multiple sclerosis and rheumatoid arthritis, MHC class II proteins are converted to HLA-DR1 protein, HLA-D
It can be selected from the group consisting of R4 protein or HLA-DR2 protein. In a preferred embodiment, the identity and position of at least one amino acid in the polypeptide corresponds to the identity and position of at least one amino acid in the P1 pocket of the MHC class II peptide binding groove. More preferably, the copeptide has a tyrosine, valine, or phenylalanine corresponding to the position of the P1 pocket of the MHC class II peptide binding groove. The present embodiment further provides a copeptide composition wherein the amino acid residue corresponding to the first amino acid of the P1 pocket in the MHC class II peptide binding groove is alanine. The present embodiment further provides a peptide in which the amino acid corresponding to the eighth amino acid of the P1 pocket in the MHC class II peptide binding groove is lysine or alanine, and the amino acid residue corresponding to the P1 pocket is tyrosine, valine or phenylalanine. .

In another example of the invention, the first co-peptide and the second co-peptide, the first co-peptide with additional lysine and the eighth co-located beyond the P1 pocket in the MHC class II peptide binding groove. A composition comprising a second copeptide having an alanine at a position corresponding to an amino acid is provided.

The terpolymer and copeptide of the present invention can be formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein includes any or all of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweeteners, and the like. Pharmaceutically acceptable carriers include, but are not limited to, a wide variety of materials such as flavorings, sweeteners and buffers and absorbents required to prepare certain therapeutic compositions. May be prepared from a wide range of materials.

The combination of such materials with pharmaceutically active substances is well known in the art.
Except insofar as any conventional material or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

The compositions of the present invention may be formulated as injectable solutions or suspensions, spray solutions or suspensions.

Therapeutic Methods Contemplated by the Invention The present invention is further directed to a method for treating and preventing an autoimmune disease in a mammal, comprising Copolymer 1, an amino acid containing glutamic acid, tyrosine, lysine and alanine. Administering a therapeutically effective amount of a composition comprising at least three different amino acids, randomly polymerized in linear form, selected from the group consisting of: Providing a method comprising: In one embodiment, the polypeptide is Copolymer 1 or a terpolymer.

Autoimmune diseases contemplated by the present invention include either cell-mediated diseases (eg, T cells) or antibody-mediated (eg, B cell) disorders. Such disorders are, inter alia, arthritis, demyelination and inflammatory diseases. For example, autoimmune diseases that can be treated by the polypeptides include multiple sclerosis, autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmune uveo-retinitis, Crohn's disease, chronic Autoimmune thrombocytopenic purpura, colitis, contact hypersensitivity,
Diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic myxedema,
Examples include myasthenia gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis or systemic lupus erythematosus. The compositions can be used to treat one or more of these diseases.

As used herein, “arthritis disease” refers to a condition in which at least one symptom of rheumatoid arthritis affects at least one joint of a mammal, for example, the scapula, knee, hip, hip, spine or toes of the mammal. It is a disease seen. Examples of arthritis diseases are "polyarthritis", an arthritis disease affecting multiple joints, "juvenile arthritis", an arthritis disease in humans under the age of 21, and neutropenia, splenomegaly, weight loss, anemia It may include Felty's syndrome, which includes symptoms of sickness, lymph node damage as well as pigment spots on the skin.

[0053] In one embodiment, the intended polypeptide is an MH associated with an autoimmune disease
Any autoimmune disease can be treated with the polypeptide, as long as it binds to the C class II protein. One aspect of this embodiment is that antigenic peptides and MHC classes
A method comprising selecting a polypeptide that inhibits binding to a II protein, e.g., where step (a) is a class II for an MHC class II protein-peptide complex
Provided is a method further comprising selecting a heteropolymer that inhibits a specific T cell response, and wherein the antigenic peptide is associated with an autoimmune disease. In another aspect of the invention, the MHC class A method is provided wherein the II protein is associated with an autoimmune disease.

In another embodiment, the method for treating an autoimmune disease in a mammal further comprises inhibiting the proliferation or function of T cells that respond to the self antigen. RA is a T-cell mediated autoimmune disease that can be treated by the polypeptide. Autoimmune diseases and the pathological progression of the immune response are mediated by T cells. Upon binding and recognition of the antigen, the T cells proliferate, secrete cytokines, and additional inflammatory and cytotoxic cells increase at the site. The polypeptides inhibit T cell proliferation and T cell function, such as secretion of cytokines and proliferation to sites of inflammatory and cytotoxic cells. When the autoimmune disease is an arthritis disease, the self antigen is collagen, and the polypeptide can inhibit the proliferation and function of collagen-responsive T cells.

In another embodiment, the method for treating an autoimmune disease in a mammal comprises binding the polypeptide to an antigen presenting cell such as a macrophage, a dendritic cell of lymphoid tissue or an epidermal cell. included. When the appropriate antigen is presented, T cell proliferation and function is activated. It is believed that the polypeptide arrests or otherwise interferes with T cell activation by binding to antigen presenting cells.

[0056] In yet another embodiment, the method for treating an autoimmune disease in a mammal comprises combining the polypeptide with a major histocompatibility complex class II protein associated with the autoimmune disease. . Class II MHC proteins are well expressed on the surface of antigen presenting cells such as B lymphocytes and macrophages. These class II MHC proteins have a peptide binding cross-section where the antigenic peptide is presented to T cells. When the polypeptides bind to the major histocompatibility complex class II proteins, these polypeptides can stop or otherwise interfere with antigen presentation and / or T cell activation.

[0057] In another embodiment, the method for treating an autoimmune disease in a mammal comprises combining the polypeptide with a copolymer 1 reactive B cell antibody and / or a copolymer 1 reactive T cell. To be included. Copolymer 1 reactive _TH2 /
3T cells promote the therapeutic effect of Copolymer 1. By binding to Copolymer 1 reactive T cells, the polypeptides stimulate their T cell proliferation, secretion of anti-inflammatory cytokines, increasing the therapeutic benefit of treatment with the present methods. According to the present invention,
The polypeptides also bind to autoantigen-reactive antibodies and stop the antibodies from attacking the target tissue, thereby helping to prevent the development of an autoimmune disease. For example, if the polypeptide binds to an MBP-specific antibody, it will not bind to these antibodies MBP, thereby resulting in destruction of the myelin myelin MBP.

The polypeptide may be administered by any convenient route. In one embodiment, the polypeptides and peptides can be administered by injection to facilitate delivery to tissues affected by the autoimmune disease. Thus, the peptides and polypeptides may for example be injected, ingested, inhaled or topically applied. The polypeptide of interest may be mixed with a cream, solution or suspension for topical administration.
The polypeptide is preferably administered orally, topically or by injection without the addition of an adjuvant.

Useful Kits of the Present Invention Another aspect of the present invention is a kit for assaying the binding of an analyte to an MHC protein, comprising a water-soluble MHC recombinantly produced in a non-mammalian cell.
A kit comprising a protein, means for detecting an analyte bound on the MHC protein, and instructions for use is provided. The MHC protein used in the kit was M
An MHC class II protein selected from the group consisting of HC class II HLA-DR1 protein, MHC class II HLA-DR2 protein and MHC class II HLA-DR4 protein. The kit may further comprise an autoantigenic peptide.

In a preferred embodiment, the MHC class II protein is produced in an invertebrate or bacterial cell, such as an insect or yeast cell, and thus has no peptide bound at the antigen cross section. Means for detecting the binding of an analyte to an MHC protein include radiation, fluorescence measurements, and methods commonly known in the art.
It may be a chemiluminescent, enzymatic or colorimetric means. In a preferred embodiment, the MHC protein is a class II HLA-DR1 or HLA-DR4
It is a protein. Further, the kit may comprise an autoantigenic peptide, such as a collagen II peptide or a peptide from myelin basic protein, a myelin poor dendritic protein, or a peptide from some other protein involved in an autoimmune disease. .

Synthesis of Ternary Copolymers of the Invention Ternary copolymers can be prepared by any technique available to those skilled in the art. For example, the terpolymer can be prepared using dissolved amino acids at the desired mole fraction or under solid-state synthesis techniques under condensation conditions. The condensation conditions include a suitable temperature, pH and solvent conditions for condensing the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond. Condensing agents, such as dicyclohexylcarbodiimide, may be used to facilitate the formation of peptide bonds. Protecting groups may be used to protect the side-chain moieties and functional groups such as some amino or carboxyl groups to provide for unwanted side reactions.

For example, in the process disclosed in US Pat. No. 3,849,550, the N-carboxyanhydride of tyrosine, alanine, γ-benzylglutamate and N-trifluoroacetyl-lysine are dissolved in anhydrous dioxane. The addition of diethylamine as an initiator and polymerization at ambient temperature can be used. The γ-carboxyl group of glutamic acid can be deprotected with hydrogen bromide in glacial acetic acid. The trifluoroacetyl group can be removed from lysine with 1 molar piperidine. By selectively eliminating reactions related to any one of glutamic acid, alanine, tyrosine or lysine, the process can be carried out in Copolymer 1 with a desired amino acid, for example,
It can be readily appreciated by one of skill in the art that peptides and polypeptides containing only three of the four amino acids can be tailored to produce. For purposes of application, "ambient temperature" and "room temperature" are between about 20 to about 26 degrees Celsius (° C).
).

[0063] The average molecular weight of the terpolymer can be adjusted during polypeptide synthesis or after the terpolymer is prepared. To adjust the average molecular weight during polypeptide synthesis, the conditions or amounts of amino acid synthesis are adjusted so that synthesis stops when the polypeptide reaches the desired desired length. After synthesis, a polypeptide having the desired average molecular weight can be obtained by any available size selection procedure, for example, by chromatography of the polypeptide on a molecular weight sizing column or gel and collection of the desired average molecular weight range. The polypeptide may also be partially hydrolyzed, for example by hydrolysis with an acid or an enzyme, to eliminate high molecular weight species and later purified to eliminate the acid or enzyme.

In one embodiment, the invention is a method of making a terpolymer having a desired average molecular weight, comprising reacting a protected polypeptide with hydrobromic acid to obtain a desired average molecular weight profile. Methods comprising forming a polypeptide having the same. The reaction is performed at a time and temperature predetermined by one or more test reactions. During the test reaction, the time and temperature are varied to determine the average molecular weight range for a given batch of test polypeptide. The test conditions that give the optimal average molecular weight range for the batch of polypeptide are used for the batch. Thus, a polypeptide having the desired average molecular weight profile can be produced by a method comprising reacting the protected polypeptide with hydrobromic acid at a predetermined time and temperature in a test reaction.

In a preferred embodiment, a test sample of a given batch of protected polypeptide is reacted with hydrobromic acid at about 20-28 ° C. for about 10-50 hours. Perform the test reaction several times to determine the best conditions for the batch. For example, in one embodiment, the protected polypeptide is reacted with hydrobromic acid at about 26 ° C. for about 17 hours.

The following examples describe the invention in detail in terms of how to show certain typical embodiments thereof, but only with regard to materials, equipment and processing steps. It is understood that the examples are intended to be illustrative. In particular, the invention does not limit the methods, materials, conditions, processing parameters, equipment, etc. described herein.

In this application, various publications, patents and patent applications are referenced. The teachings and disclosures of these publications, patents and patent applications are all incorporated herein by reference and fully describe the state of the art to which this invention pertains.

It should be understood and assumed by those skilled in the art that variations on the principles of the invention disclosed herein may be made, and such modifications are intended to be included within the scope of the invention.

[0069]

【Example】

Preparation protective polypeptide for manufacturing protective polypeptide of Example 1 polypeptides as described by Teitelbaum et el., Was dissolved in dioxane, amino acids tyrosine and blocked with N- carboxyanhydride (NCA), alanine, glutamic acid and Prepared using trifluoroacetyl lysine. 1
EUR. J. IMMUN. 242 (1971). The carboxylation group of glutamic acid is replaced with benzyl (BZ).
Block with a group.

The polymerization process is started by adding 0.01-0.02% diethylamine. The reaction mixture is stirred at room temperature for 20 hours and then poured into water. The protected polypeptide product is filtered, washed with water and dried. Removal of the γ-benzyl blocking group from glutamic acid residues can be achieved by protecting the protected polypeptide with 33% hydrobromic acid in glacial acetic acid at room temperature for 6-1
This is performed by treating with stirring for 2 hours. The product is poured into excess water, filtered, washed and dried to give the protected polypeptide.

Preparation of a Polypeptide Having a Molecular Weight of 7,000 ± 2,000 Da Treatment of a γ-benzyl protected polypeptide with 33% HBr in acetic acid allows the removal of a benzyl protecting group from the γ-carboxylate of a glutamic acid residue. Not only is removal guaranteed, but the polypeptide is cleaved 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. The test reaction of each batch is carried out at a temperature between 20 and 28 ° C. for various times, for example 10 to 50 hours. Subtract the average molecular weight curve obtained over time. Molecular weight 7,000 ± 2,000
The time required to obtain ODa is calculated from the curve and the reaction is performed on a large scale.
On average, at 26 ° C., the time to obtain a mixture of polypeptides having a molecular weight of 7,000 ± 2,000 Da is 17 hours. The product is poured into excess water, filtered, washed and dried to yield a polypeptide having the desired average molecular weight range.

Preparation of Low Toxic Lysine Containing Polypeptide Protected trifluoroacetyl polypeptide (20 g) is dispersed in one liter of water and 100 g of piperidine is added. The mixture is stirred at room temperature for 24 hours and filtered. Dispense the unpurified polypeptide solution into dialysis bags and allow 1 until pH 8 is reached.
Dialyze against water at 0-20 ° C. The polypeptide solution is dialyzed against 0.3% acetic acid and then dialyzed again with water until the pH becomes 5.5 to 6.0. The solution is then concentrated and lyophilized.

Synthesis of poly (L-Tyr ^0.136 , L-Glu ^0.21 , L-Ala ^0.648 ) with molecular weight of 7,600 TGA 1) Raw material: 18.74 g of L-Ala-NCA L-Tyr-NCA 6.5 g 5-BZ-L-Glu-NCA 13 g diethylamine 0.165 g dioxane 790 ml HBr / AcOH (33%) 480 ml phenol 4.8 g piperidine 13.2 ml deionized water acetic acid 2) Procedure L-Tyr-NCA (6. 5 g) in dioxane (211 ml) at 60 ° C.
And then filtered. 5-BZ-L-Glu-NCA (13 g) is put in dioxane (226 ml), stirred at 20 to 25 ° C. for 10 minutes, and then filtered. L-Ala-NCA (18.74 g) is placed in dioxane (350 ml), stirred at 20 to 25 ° C. for 10 minutes, and filtered.

The L-Tyr-NCA solution, 5-L was added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
The BZ-L-Glu-NCA solution and the L-Ala-NCA solution are charged. Diethylamine (0.165 g) in dioxane (2.5 ml) is charged to the reaction mixture and stirred at 20-25 ° C. for 20 hours. Add the reaction mixture to deionized water (1 L), filter and dry at 60 ° C. in vacuo. Yield -25.8 g.

A solution of phenol (4.8 g) in HBr / AcOH (33%; 480 ml) is stirred for 20 hours. HBr / A in 2L Erlenmeyer equipped with magnetic stirrer
A solution of cOH and poly [5-BZ-L-Glu, L-Ala, LL-Tyr] is charged and stirred at 26 ± 1 ° C. for 16 hours. The reaction mixture is deionized water (2 L)
And stir for 1 hour. The precipitate is filtered and washed with deionized water until the pH is 6. The product is dried in a circulating air oven at 40 ° C. for about 40 hours. Yield-24
g.

Piperidine (13.2 ml) in a 2 L Erlenmeyer equipped with a magnetic stirrer,
Deionized water (1200 ml) and poly [L-Glu, L-Ala, LLT
After stirring, the mixture was stirred at 20 to 25 ° C for 24 hours. The resulting solution is ultrafiltered through a 5000 dalton polyethersulfone membrane until two thirds of the original volume has been eliminated. Add fresh deionized water and return to original volume. HPL
This step is repeated 5 times until the impurity content is less than 1% by C. The resulting solution (total) is acidified with acetic acid to pH 4.4. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

Synthesis of molecular weight 8850GAL, poly [L-Glu ^0.153 , L-Ala ^0.479 , L-Lys ^0.365 ] 1) Raw material: L-Ala-NCA 14g N6-TFA-L-Lys-NCA 22.9 g 5-BZ-L-Glu-NCA 9.8 g diethylamine 0.12 g dioxane 850 ml HBr / AcOH (33%) 480 ml phenol 4.8 g piperidine 13.2 ml deionized water acetic acid 2) Procedure in dioxane (420 ml) N6-TFA-L-Lys-NCA (22.9
g) is stirred at 20-25 <0> 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) was added to 2
Stir at 0-25 ° C for 10 minutes and filter. N6-TFA-L-Lys-NCA solution, 5-BZ-L-Glu-NC in a 2 L Erlenmeyer equipped with a magnetic stirrer
A solution and L-Ala-NCA solution are charged. Diethylamine (0.12 g) in dioxane (2.5 ml) is added and the reaction mixture is stirred at 20-25 ° C. for 20 hours. Add the reaction mixture to deionized water (1 L), filter and dry at 60 ° C. in vacuo.

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

Piperidine (13.2 ml) was placed in a 2 L Erlenmeyer equipped with a magnetic stirrer,
Deionized water (1200 ml) and poly [L-Glu, L-Ala, N6-TF
[A-L-Lys], and then stirred at 20 to 25 ° C for 24 hours. The solution is ultrafiltered on a 5000 dalton exclusion limit polyethersulfone membrane until two thirds of the original volume has been eliminated. Add fresh deionized water and return to original volume.
This process is repeated 5 times (by HPLC) until the impurity content is less than 1%.
The resulting solution (total) is acidified with acetic acid to pH 4.4. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

[0081] Molecular weight 11,050TGL, poly ^{[L-Tyr 0.162, L-} Glu 0.2 59, L-Lys 0.579] Synthesis 1) Raw material: 5-BZ-L-Glu -NCA 10.34g N6-TFA-L-Lys-NCA 24.16 g L-Tyr-NCA 5.2 g Diethylamine 0.095 g Dioxane 810 ml HBr / AcOH (33%) 460 ml Phenol 4.6 g Piperidine 150 ml Deionized water Acetic acid 2) Procedure Dioxane (180 ml) L-Tyr-NCA (5.2 g) in
Heat for 0 minutes and filter. N6-TFA-LL in dioxane (450 ml)
Stir ys-NCA (24.16 g) at 20-25 ° C. for 10 minutes and filter. 5-BZ-L-Glu-NCA (10.35 g) in dioxane (180 ml) is stirred at 20-25 ° C. for 10 minutes and filtered.

N2-TFA-L-Lys-N was added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
A CA solution, an L-Tyr-NCA solution and a 5-BZ-L-Glu-NCA solution are charged. Charge diethylamine (0.095 g) in dioxane (2.5 ml) and stir the reaction mixture at 20-25 <0> C for 20 hours. Add the reaction mixture to deionized water (0.7 L), filter and dry at 60 ° C. in vacuo.

A solution of phenol (4.6 g) in HBr / AcOH (33%; 460 ml) is stirred for 20 hours. HBr / A in 2L Erlenmeyer equipped with magnetic stirrer
cOH and poly [5-BZ-L-Glu, L-Tyr, N6-TFA-LL
ys]. The mixture is stirred at 26 ± 1 ° C. for 16 hours, then the mixture is added to deionized water (1.5 L) and stirred for １ ／ hour. Filter the precipitate, pH
Wash with deionized water until pH is 5.5.

In a 2 L Erlenmeyer equipped with a magnetic stirrer, piperidine (150 ml), deionized water (1400 ml) and poly [L-Glu, L-Tyr, N6-TFA
-L-Lys] (50 g) and stirred at 20 to 25 ° C for 24 hours. Poly [
[L-Glu, L-Tyr, L-Lys] was obtained.
Is ultrafiltered through a 5000 dalton polyethersulfone membrane until exclusion is achieved. Return to original volume by adding fresh deionized water. This process is repeated 5 times (by HPLC) until the impurity content is less than 1%. The resulting solution (total) is acidified with acetic acid to pH 4.2. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

[0085] Molecular weight 20,000TAL, the synthesis of poly ^{[L-Tyr 0.102, L-} Ala 0.5 42, L-Lys 0.353] 1) material: L-Ala-NCA 14g N6 -TFA-L- Lys-NCA 22.9 g L-Tyr-NCA 4.9 g Diethylamine 0.1 g Dioxane 850 ml HBr / AcOH (33%) 500 ml Phenol 5 g Piperidine 162 ml deionized water acetic acid 2) Procedure L-Tyr-NCA in dioxane (170 ml) (4.9 g) at 60 ° C.
Heat for 0 minutes and filter. N6-TFA-LL in dioxane (417 ml)
Stir ys-NCA (22.9 g) at 20-25 ° C. for 10 minutes and filter. L-Ala-NCA (14 g) in dioxane (260 ml) was added at 20-25 ° C for 10
Stir for minutes and filter.

N2-TFA-L-Lys-N was added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
Charge CA solution, L-Tyr-NCA solution in dioxane and L-Ala-NCA solution. Charge diethylamine (0.1 g) in dioxane (2 ml),
The mixture is stirred at 20-25 ° C for 20 hours. The reaction mixture is deionized water (1 L)
), Filter and dry in vacuo at 60 ° C.

A solution of phenol (5 g) in HBr / AcOH (33%; 500 ml) was treated with 2
Stir for 0 hours. HBr / AcO in 2L Erlenmeyer equipped with magnetic stirrer
A solution of H and poly [L-Ala, L-Tyr, N6-TFA-L-Lys] is charged and stirred at 26 ± 1 ° C. for 16 hours. The reaction mixture was then washed with deionized water (2 L).
) And stir for 1/2 hour. The precipitate is filtered and washed with deionized water until the pH is 5.5.

Piperidine (162 ml), water (1500 ml) and poly [L-Ala, L-Tyr, N6-TFA-LL were added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
ys] (30 g) and stirred at 20 to 25 ° C for 24 hours. Poly [L-Al
a, L-Tyr, L-Lys] is ultrafiltered through a polyethersulfone membrane with an exclusion limit of 5000 daltons until two thirds of the original volume has been eliminated. Return to original volume by adding fresh deionized water. Impurity content is 1 (by HPLC)
This step is repeated 5 times until it is less than%. PH of the resulting solution (total) with acetic acid
Acidify to 4.4. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

Example 2 In Vivo Testing of Low Molecular Weight Polypeptides Average molecular weights of 7.3 and 8.4 KDa (one copolymer above 40 KDa
. Three batches of Copolymer 1 having less than 5%) and 22 KDa (more than 5% of one copolymer greater than 40 KDa) were subjected to the toxicity tests described below. Five mice were used in each test group.

Method Copolymer 1 is dissolved in distilled water to obtain a solution of 2 mg / ml of the active ingredient. Each mouse is injected with 0.5 ml of the test solution into the lateral tail vein. The number of deaths and associated clinical signs of the mice for 48 hours were observed. 10 minutes, 24 hours and 48 after injection
Observations were recorded at time. At the end of 48 hours, if all animals are alive and show no bad signs, the batch is considered "non-toxic." However, if one or more mice die or show bad symptoms, the batch is considered "toxic."

Results When treated with Copolymer 1 polypeptide having an average molecular weight of 22 KDa, three of the five mice died after 48 hours. Therefore, this batch with a high average molecular weight is referred to as
Toxicity. One batch of copolymer having an average molecular weight of 7.3 and 8.4 KDa was both "non-toxic".

In Vitro Rat Basophilic Leukemia Cell Degranulation Assay Histamine (or serotonin) release from basophils is an in vitro model of immediate hypersensitivity. Rat basophilic leukemia cell line, RBL-2H ₀ is homogeneous, but it is easy to maintain in culture, there is a high sensitivity to test for degranulation, and a reproducible certain systems. EL Basumian, et al., 11 EUR. J.
IMMUNOL. 317 (1981). Physiological stimuli for histamine release include the association of antigens with membrane-bound IgE molecules that trigger a complex biochemical cascade. Degranulation is induced by non-IgE-mediated stimuli including various polypeptides and synthetic polymers such as polylysine. RP Siraganian, TRENDS IN PHARMACO
LOGICAL SCIENCES 432 (Oct, 1983). Thus, using the RBL degranulation test, a batch of that COP-1 that can cause substantial degranulation, thereby inducing unwanted local and / or systemic side effects. To select.

Methods Rat basophil leukemia cells (RBL-2H ₀ ) are fed [H ³ ] -serotonin and then incubated with 100 μg of COP-1 to be tested. The COP-1 batch to induce non-specific degranulation in medium [H ^3] - releasing serotonin. Radioactivity in the medium is measured with a scintillation counter, and total radiolabeled serotonin incorporated into the cells is determined in the pellet cells. % Degranulation is calculated as the percentage of serotonin released from all incorporated serotonin.

Results Four batches of Copolymer 1 with an average molecular weight between 8,250 and 14,500 were analyzed for both% of species above 40 kDa and RBL degranulation. The results are summarized in Table 1.

Table 1

[Table 1] As can be seen, when the percentage of the high average molecular weight species is low (less than 2.5%), the% serotonin release is low, and vice versa. These data indicate that a copolymer 1 polypeptide with a lower average molecular weight is preferred over a copolymer 1 polypeptide with a higher average molecular weight.

Example 3 Inhibition of EAE by Polypeptide Experimental Allergic Encephalomyelitis (EAE) can be inhibited by injecting Copolymer 1 in Freund's incomplete adjuvant before disease induction. This inhibition is believed to T _{H 2} type of copolymer 1-specific suppressor T cells that cross-react with myelin basic protein is mediated. Lando et al., 123 J. IMMUNOL.
2156 (1979); Aharoni et al., 17 EUR.J. IMMUNOL. 23 (1993); Aharoni et a
l., also 94 PROC. NATL. ACAD. SCI . USA 10821 (1997). that other researchers are associated with the therapeutic effect of Copolymer 1 also T _{H 2} cell induction in patients with multiple sclerosis Admit. Lahat et al., 244 J. Neuro. 129 (1997). In this example, EAE is inhibited by various polypeptides of the present invention.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

Ternary Copolymers Four terpolymers are available from Teva Pharmaceutical Industries (Petach Tikva, Israe).
Obtained from l). The properties of these terpolymers are as follows.

1) GAL terpolymer (SD-1689) is a polypeptide mixture containing the following molar fractions of the amino acids glutamic acid (0.153), alanine (0.479) and lysine (0.365) is there. The GAL average molecular weight ranges from about 4650 daltons to about 20,050 daltons. The average molecular weight of this GAL preparation is 8850 daltons.

2) The TGA terpolymer (SD-1690) contains the following molar fractions of the amino acids tyrosine (0.136), glutamic acid (0.210) and alanine (0.648)
And a polypeptide mixture comprising: The TGA average molecular weight ranges from about 1000 Daltons to about 40,000 Daltons. The average molecular weight of this TGA preparation is 7600
Dalton.

3) TAL terpolymer (SD-1691) is a polypeptide mixture containing the following molar fractions of the amino acids tyrosine (0.102), alanine (0.542) and lysine (0.353) is there. The TAL average molecular weight ranges from about 5700 daltons to about 34,400 daltons. The average molecular weight of this TAL preparation is 20,000 daltons.

4) GTL terpolymer (SD-1697) is a polypeptide mixture containing the following mole fractions of the amino acids glutamic acid (0.259), tyrosine (0.162) and lysine (0.579) is there. The GTL average molecular weight ranges from about 4,000 daltons to about 23,500 daltons. The average molecular weight of this GTL preparation is 1105
0 Daltons.

A control polypeptide consisting of a polypeptide mixture comprising a 1: 1: 1 mixture of the amino acids alanine, glutamic acid and tyrosine and having an average molecular weight of 26,700 Da is used. This polypeptide was obtained from Sigma Chemical Company (St. Louis, MO).
Obtain from

Induction of EAE 4 mg / ml H in 2-3 month old female (SJL / J × BALB / c) FI mice
Mouse spinal cord homogenate (3.5 mg / mouse) emulsified at a 1: 1 ratio with Freund's complete adjuvant (CFA) supplemented with 37Ra is injected into all four footpads. Immediately after and 48 hours later, pertussis toxin (0.25 ml, 250 ng, Sigma)
Is injected intravenously. Mice are examined daily for clinical signs of EAE from day 10 post-challenge and evaluated on a scale of 0-5 as described by Lando et al., 123 J. IMMUNOL. 2156 (1979).

Inhibition of EAE by Injection of Incomplete Adjuvant The polypeptide to be tested (10 mg / mouse) is injected subcutaneously in Freund's incomplete adjuvant (ICFA) into a few of the nodal regions. EAE is induced after 3 weeks as described above.

EAE Inhibition by Oral Administration In a second study, female Lewis rats were injected with 5 mg / kg guinea pig BP or co-administered in phosphate buffered saline (PBS) at 2-3 day intervals before EAE induction. Polymer 1 is provided. EAE is 4 mg / ml Mycobacterium tuberculosis (H37Ra) (Difco, Lab, Det.
(Roit, Mich.) and induced 2 days after final feeding by injection of 25 μg guinea pig MBP emulsified 1: 1 with CFA. 0 total capacity in each of the two hind footpads
. Inject 1 ml. Control rats are mock receiving phosphate buffered saline.

The effect of oral administration of Copolymer 1 on the prevention of EAE in rats was determined by Higgins et al.
al., 140 J. IMMUNOL. 440 (1988) and compared to rats given guinea pig MBP.

Mice were examined daily for symptoms from day 10 post-challenge. EAE is evaluated as follows: 0 = no symptoms; 1 = fragile tail; 2 = hind limb paralysis; 3 = all limbs paralyzed;
= Moribund condition; and 5 = dead.

Results Inhibition of EAE by Injection of Incomplete Adjuvant Table 2 shows the effect of injecting the polypeptide of the present invention by ICFA before EAE induction. Of the four polypeptides tested, Copolymer 1 and TAL resulted in maximum EAE suppression. GTL also showed excellent inhibitory activity. GAL
And TGA were somewhat less potent.

Table 2 EAE suppression in mice by injection of the polypeptide of the invention

[Table 2] * MMS = average highest point D-copolymer 1 is d-lysine, d-tyrosine, d- in the molar ratio of copolymer 1.
It is a copolymer of glutamic acid and d-alanine.

Table 3 compares the efficacy of orally administered Copolymer 1 in preventing clinical signs of EAE in Lewis rats compared to rats receiving phosphate buffered saline (PBS) alone or guinea pig basic protein (GPBP). Is shown.

Table 3 EAE suppression in rats by oral administration of the polypeptide of the present invention

[Table 3] Each number is the cumulative result of three to five independent tests. The p value is the control (P
BS) shows the statistical significance of the difference from the group. The average highest score is calculated for all groups.

These data indicate that the polypeptides of the invention are therapeutically effective when administered either orally or by infusion to reduce the signs and severity of EAE.

Example 4 Binding to Antigen Presenting Cells Some of the polypeptides efficiently bind live antigen presenting cells.

Methods Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

Biotinylation of Antigen Biotinylation of Copolymer 1 and the terpolymer are described in Fridkis-Hareli et al. 91 PRO.
Perform at 0 ° C. with biotin-N-hydroxysuccinimide (Sigma) according to C. NATL. ACAD. SCI. USA 4872 (1994).

Binding of Biotinylated Antigen to Antigen Presenting Cells The binding of the biotinylated polypeptide to fluorescently labeled streptavidin and FACS analysis was examined for binding of live antigen presenting cells of mouse and human origin. (SJ
L / J × BALB / c) adherent spleen cells from FI mice, or DR7. wII
EBV-transformed human B cells of haplotype ^{(1 × 10 8 / 100μl)} 0.1
The mixture was incubated at 37 ° C. for 20 hours with 50 μg of biotinylated copolymer 1 or ternary copolymer dissolved in 100 μl of PBS containing% BSA. Cells were then incubated with phycoerythrin-conjugated streptavidin (Jackson Immuno Research, West Grove, PA) at a cell suspension concentration of 0.5 μg / 100 μl at 4 ° C. for 3 hours.
Incubated for 0 minutes. After each incubation, the cells are
The plate was washed three times with PBS containing% BSA. The cells were then analyzed by flow cytometry using a FACS scan (Becton-Dickinson, Mountain View, CA). For each analysis, 5000 cells were tested. Dead cells show forward and side light scatter (f
orward and side-angle light scatter).

Epstein-Barr Virus (EBV) Transformed B Cell Line The EBV transformed B cell line was isolated from Teitelbaum et al. 89 PROC. NATL. ACAD. SCI.
Induced according to USA 137 (1992). About 20 × 10 ⁶ peripheral blood mononuclear cells were B95.8
The cells were cultured with the cell line supernatant at 37 ° C. for 1 hour. The cells are then washed and
The cells were cultured in RPMI medium containing% FCS and cyclosporin A (10 μg / ml) to deplete T cells.

Results Table 4 shows the binding of the terpolymer and Copolymer 1 polypeptide to live antigen-presenting cells, including mouse spleen macrophages and human EBV-transformed B cell lines. Data showing both% binding and cell staining intensity (Table 4 I + II)
. TAL was even better than Copolymer 1 and bound with maximum efficiency. GAL and GTL also bound very successfully, and were the same or somewhat better than Copolymer 1. TGA bound well, but was somewhat inferior to Copolymer 1.

As represented by the bond strength (Table 4), TAL is (SJL / J × BALB)
/ C) _{F 1} mice of splenic macrophages and normal DR7. It bound with maximum efficiency to EBV transformed B cells from w11 donor.

Table 4 Binding of terpolymers to antigen presenting cells Antigen-presenting cells derived from mouse spleen macrophages

[0123]

[Table 4-1] ^* MFI = average fluorescence intensity II. Antigen presenting cells from human EBV transformed B cell line

[0124]

[Table 4-2] ^* MFI = average fluorescence intensity Example 5 This polypeptide binds to purified human leukocyte antigen (HLA) In this example, the polypeptide of the present invention binds to human B cells and HLA-DR1, HL
It shows binding to purified human lymphocyte antigen (HLA) with high affinity, such as A-DR2 and HLA-DR4 molecules.

Methods Cell Lines and Antibodies The homozygous EBV-transformed human B lymphocyte line used for immunoaffinity purification of HLA-DR1, -DR2 and -DR4 molecules was LG-2 (D
RB1), MGAR (DRB1-1501) and Preiss (DRB-04
01 / DRB4-0101).

Cells were treated with 10% FCS, 2 mM glutamine, 50 U / ml penicillin G and 5
The cells were grown in roller bottles containing RPMI 1640 supplemented with 0 μg / ml streptomycin and stored at −80 ° C. as pellets. Anti-DR hybridoma LB3.1 (IgG2b) is grown in serum-free medium (Macrophage-SFM, Gibco BRL). See Gorga et al., 103 CELL. IMMUNOL. 160 (1986).

Protein Purification The immunoaffinity purification of HLA-DR1, -DR2 and -DR4 molecules was
Performed with slight modifications as previously reported by Gorga et al., 262 J. BIOL. CHEM. 16087 (1987). In short, LG-2, MGAR and Pries
S-cell-derived detergent-soluble membrane preparation was applied to a series of columns at a flow rate of about 11 ml / hr in the following order: Sepharose CL-6B (30 ml), normal mouse serum-Af
f-gel 10 (10 ml), protein A-Sepharose CL-4B (5 ml)
And LB3.1-protein A-Sepharose CL-4B (5 ml). Before passage through the LB3.1 immunoaffinity column, DR2a (DRB5 ^* 0
101) and Drw53 (DRB4 ^* 0101), the product of the DR gene associated with the DRB1 allele, were not removed from the MGAR and Priess lysates, and the DR2 and DR4 preparations were contaminated in amounts of 5-10%. Was. All subsequent steps were as previously described by Gorga et al., 262 J. BIOL. CHEM. 16087 (1987). The eluate was washed with 0.1% deoxycholate, 10 mM Tris.
-Dialyzed against HCl, pH 8.0, Centriprep 30 membrane (amicon)
And concentrate. Protein concentration was determined using the bicinchonitric acid assay (Pi
erce Chemical Co.).

Peptides and Polypeptides Method Copolymer 1 One batch of copolymer having an average molecular weight of 5800 daltons and 8,150 daltons, # 55495 and 52596, respectively, is Teva Pharmaceutical Industri
es (Petach Tikva, Israel). One batch 52596 of the copolymer had a molar ratio of 1 tyrosine: 1.5 glutamic acid: 4.3 alanine: 3.1 lysine.

Myelin basic protein peptide MBP protein was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (1979). The peptides were purified by reverse phase HPLC. The peptide used was the sequence PKYVKQNTLKL
HA306-318 with AT (MW1718) (SEQ ID NO: 2) and sequence D
MBP84-102 with ENPVVHFFKNIVTPRTPP (MW2529) (SEQ ID NO: 1).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

Polypeptide Labeling Biotinylation of various polypeptides is performed as in Example 4. Unreacted biotin is dialyzed (Spectra / Por® membrane MWCO500, Spectrum Medic
al Industries).

Assay for Polypeptides that Bind to Class II MHC Proteins Solutions: The solutions used for this assay are as follows. The binding buffer was 20 mM 2- [N-morpholino] ethanesulfonic acid (ME unless otherwise noted).
S) 1% n-octyl β-D-glucopyranoside, 140 mM NaCl,
0.05% NaN ₃ , pH 5.0. PBS is 150 mM sodium chloride, 7.5 mM sodium phosphate, dibasic, 2.5 mM sodium phosphate, monobasic, pH 7.2. TBS was 137 mM sodium chloride, 25 mM Tr.
is pH 8.0, 2.7 mM potassium chloride. TTBS is 0.0 for TBS
5% Tween-20 was added.

Microtiter assay plate preparation: 96-well microtiter immunoassay plates (PRO-BIND ™, Falcon) were loaded with PBS (100 total).
1 μg / well of affinity purified LB3.1 monoclonal antibody in μl) at 4 ° C. for 18 hours. The wells were then sealed with TBS containing 3% BSA at 37 ° C. for 1 hour and washed three times with TTBS. 50 μl of TBS / 1% BSA was added to each well before adding samples.

Binding reaction: detergent soluble HLA-DR1, -DR2 and -DR4 molecules (0.
5 μg / sample) in 50 μl of binding buffer at various concentrations with biotinylated peptide at 37 ° C. for 40 h, transfer to prepared microtiter assay plates and 37 ° C. to capture polypeptide-class II complex Incubated for 1 hour.

Inhibition reaction: 1.5 μM final concentration of biotinylated polypeptide in 50 μl binding buffer, unlabeled polypeptide and peptide HA30 used as inhibitor
6-318 (SEQ ID NO: 2) or MBP84-102 (SEQ ID NO: 1), and H
Co-incubated with LA-DR molecule at 37 ° C for 40 hours.

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 of streptavidin-conjugated alkaline phosphatase (1: 3000, BioRad) for 1 hour at 37 ° C., then p-nitrophenyl phosphate in triethanolamine buffer (BioRad) was added. The absorbance at 410 nm is monitored by a microplate reader (Dynatech MR4000).

Results Terpolymer Coupling with Class II MHC Proteins The detergent soluble HLA-DR1, HLA-DR2 and HLA-DR4 proteins were each homozygous EBV transformed B cell line LG-2 (DRB1 ^* 010
1), MGAR (DRB1 ^* 1501) and Priess (DRB1 ^* 040)
Purified from 1) as previously described by Fridkis-Hareli et al. 160 J. IMMUNOL. 4386 (1998). Three different preparations of Copolymer 1 bound to these molecules with high affinity. Id. To determine the affinity of the terpolymer for the HLA-DR protein, a binding assay of the biotinylated terpolymer was performed and compared to Copolymer 1. HLA-DR1, wherein the polypeptide is purified at a range of concentrations,
Incubate with HLA-DR2 and HLA-DR4 molecules at pH 5.0, then capture by class II specific mAbs and alkaline phosphatase-
Detection with streptavidin was performed.

Detergent Soluble HLA-DR1 and HLA-DR4 of TAL and Copolymer 1
The binding to the molecule is better than that of GAL, TGA or GTL. However, GTL and Copolymer 1 were based on the saturation binding curve (FIG. 2A) and the _Kα value calculated from the double reciprocal plot of the binding data (FIG. 2B, Table 5), indicating that other polypeptides were Better binding.

Biotinylated copolymer 1 and the following unlabeled inhibitors: copolymer 1, TAL, T
Competitive binding assays were performed on GA, GAL, TGL, MBP84-102 and HA306-318 polypeptides (FIG. 3). The MBP84-102 (SEQ ID NO: 1) peptide is a poor inhibitor of the binding between Copolymer 1 and HLA-DR2. Binding of biotinylated copolymer 1 to detergent soluble HLA-DR1 and -DR4 molecules is efficiently inhibited by unlabeled TGA, TAL, HA306-318 (SEQ ID NO: 2) and copolymer 1. However, the inhibition by TGL of the binding between biotinylated copolymer 1 and detergent soluble HLA-DR1 and -DR4 molecules is 10-fold lower as indicated by the amount of inhibition (IC ₅₀ ) (FIG. 3, Table 5). Similarly, G
AL is also a poor inhibitor of the binding of Copolymer 1 to HLA-DR1 and -DR4 molecules. Generally, the binding pattern with HLA-DR2 is the same as that seen with HLA-DR1 (Table 5). These results indicate that the three amino acid polypeptides, particularly TAL and TGA, bind to class II MHC molecules having the same affinity range as that of antigenic peptide and copolymer 1. Therefore, T
AL and TGA are effective competitors for Class II MHC molecules that bind Copolymer 1. Based on these binding capacities, the polypeptides are ordered 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> T
GA; (C) Binding to HLA-DR4: TAL> Copolymer 1 >>TGA>GTL>
GAL;

Table 5 Purified human HLA-DR1, -DR2 and -DR of the polypeptide
Affinity for 4 molecules

[Table 5] Copolymer 1 polypeptide having ^one average molecular weight 5,800; molecular weight 20,0
GAL having a molecular weight of 8,850; TGA having a molecular weight of 7,600; and HLA-DR1, -DR obtained by purifying TGL having a molecular weight of 11,050 in a certain concentration range.
2 and -DR4 molecules at pH 5.0, then class II
Capture with a specific mAb and detection with alkaline phosphatase-streptavidin were performed.

^Two detergent soluble HLA-DR1, -DR2 and -DR4 molecules were purified as described in Materials and Methods.

³ K _α , the dissociation constant at equilibrium, was calculated from the slope of the double reciprocal plot (FIG. 2B) and expressed as × 10 ⁻⁸ M.

^{4 The} IC ₅₀ , the inhibitory concentration giving 50% inhibition, was calculated based on the competitive binding assay (FIG. 3) and expressed as × 10 ⁻⁶ M.

[0144] _{The IC 50} values of ⁵ GAL is less than 1000μM could not be accurately determined (see FIG. 3).

Effect of Superantigen on Binding Polypeptide to HLA-DR Molecule Bacterial superantigens SEA, SEB and TSST-1 inhibit Copolymer 1 which binds only very high concentrations to purified HLA-DR molecule Has been shown to do so. Frid
kis-Hareli et al. 160 J. IMMUNOL. 4386 (1998). Terpolymer and purified HL
To test the effect of these superantigens on binding to A-DR1, HLA-DR2 and HLA-DR4 molecules, competitive binding assays were performed on unlabeled SEA, SEB and TSST-1.

The binding of SEA, SEB and TSST-1 TAL to HLA-DR1, HLA-DR2 and HLA-DR4 is only inhibited by a high molar ratio of superantigen, eg 50-fold to inhibit TAL binding Amounts of superantigen are required (FIG. 4A). However, TGA and G
Binding to AL was more differentially inhibited by the superantigen (FIGS. 4B and C), indicating that these polypeptides bind to the lower affinity HLA antigen.

Example 6 Inhibition of T Cell Proliferation Induced by MBP This example demonstrates that the proliferation of T cells activated by myelin basic protein (MBP) can usually be inhibited by co-exposure to the polypeptide. Is shown.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

The myelin basic protein peptide was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (1
979). The peptide was purified by reverse phase HPLC. The peptide used was the sequence PKY
HA306-3 having VKQNTLKLAT (MW1718) (SEQ ID NO: 2)
18 and the sequence DENPVVHFFKNIVTPRTPP (MW2529) (SEQ ID NO: 1).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

T cell line and clones Myelin basic protein (MBP) specific T cells were 4 mg / ml 10 days before
Emulsified with Freund's complete adjuvant to which Mycobacterium tuberculosis H37Ra was added
Originated from the spleen of a mouse immunized with 20 μg of the 84-102 peptide. The cells are cultured and a medium (RPMI, 2 mM glutamine, 1 mM sodium pyruvate, non-essential amino acids, 5 × 10 ⁻⁵ M 2) supplemented with 1% autoserum using the immunizing antigen (0.2 to 1 mg / plate) -In vitro selection in (mercaptoethanol, 100 / ml penicillin, 100 μg / ml streptomycin). Four days later,
Cells were transferred to a medium containing 10% FCS, and 10% supernatant of ConA-activated normal spleen cells and T cell growth factor (TCGF) were added. Every 14-21 days cells were stimulated by 3 days of exposure to immunizing antigen appearing in syngeneic irradiation (3000 rad) spleen cells (50 × 10 ⁶ / plate) and then grown in TCGF medium. Cloning of T cells was performed in a microtiter plate in the presence of antigen (2-10 μg / well) and irradiated syngeneic spleen cells (50 × 10 ⁶ / well).
Performed by limiting dilution in 3 cells / well.

The human T cell line is Teitelbaum et al. 89 PROC. NATL. ACAD. SCI. USA 137 (1
992) derived from peripheral blood mononuclear cells. Approximately 5 × 10 ⁶ cells were incubated in each well of a 24-well culture plate with medium supplemented with 10% heat-inactivated autoserum with Copolymer 1 or MBP (50 μg / ml). After 7 days in culture, cells were harvested with 10% fetal calf serum and recombinant human interleukin-2 (20 units / m2).
l). Cells were grown continuously in this medium with regular exposure every 14 to 18 days to the antigen appearing on irradiated (3000 rad) autologous mononuclear cells.

Proliferation Assay Cells or clones of the T line were tested for their specific proliferative response 10 to 21 days after challenge. T cells (1.5 × 10 ⁴ ) were triplicated in 10% FCS medium with 5 × 10 ⁵ irradiated spleen cells and a final volume of 0.2 ml of the indicated antigen. At the end of the 48 hour incubation, 1 μCi [ ³ H] -thymidine (standard deviation <20% of mean cpm) was applied to the medium and harvested after 6-12 hours.

Inhibition Studies Inhibition of the T cell proliferative response was added to varying concentrations of Copolymer 1 and terpolymer,
The proliferation assay system was examined by adding a stimulating MBP antigen. Inhibition is calculated as% inhibition using equation 2:% inhibition = [1− (cpm with inhibitor / cpm without inhibitor)] × 100.

Results FIG. 1 shows how Copolymer 1 and the terpolymer result in T cell proliferation specific for certain myelin basic protein (MBP) peptides. Generally, T cells proliferate when exposed to the antigen they sensitize. Therefore M
BP and, in particular, certain antigenic peptides from MBP stimulate the proliferation of MBP-specific T cells.

Copolymer 1 and the terpolymer did not stimulate the proliferation of T cells specific for the 84-102 peptide of MBP. Instead, they significantly inhibited the proliferation of T cells exposed to this MBP antigen.

TAL was most effective at inhibiting proliferation of T cells specific for the MBP84-102 antigen. The inhibition by TAL was even greater than that by Copolymer 1. TGA-induced T cell proliferation was lower. GAL and GTL inhibited T cell proliferation with a similar dose response as Copolymer 1.

Example 7 Terpolymer Recognized by Some Copolymer 1 Specific T Cells Ternary Copolymer Stimulates Some Copolymer 1 Specific T Cell Lines Let
It can secrete a cytokine, IL-4.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

[0160] Myelin basic protein peptide was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (197
9). The peptide was purified by reverse phase HPLC. The peptide used was the sequence PKYV
HA306-31 having KQNTLKLAT (MW1718) (SEQ ID NO: 2)
8 and MBP84-102 having the sequence DENPVVHFFKNIVTPRTPP (MW2529) (SEQ ID NO: 1).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

T Cell Lines and Clones Mouse T cell lines and clones were obtained from Aharoni et al., 23 Eur. J. Immunol. 17
(1993); Aharoni et al., 94 PROC. NATL. ACAD. SCI. USA 10821 (1997). The strain specific for copolymer 1 originated from the mouse spleen,
Copolymer 1 emulsified with Freund's incomplete adjuvant (Difco) 35 days before
5-10 mg was injected subcutaneously into each mouse to reduce sensitivity to EAE. The strain specific to copolymer 1 was 4 mg / ml of M. tuberculosis H37 10 days ago.
Obtained from lymph nodes of mice immunized with 1200 μg of copolymer emulsified with Freund's complete adjuvant (Difco) supplemented with Ra (Difco).

Using a copolymer 1 specific T cell line, the T cell clones were LN-1, LN-2,
S-3, 5-22-5, C-14 and C-52 were used. LN-2 antibody is CF
Derived from injected copolymer 1 in A (SJL / BALB / c) F 1 mice lymph node. The 5-22-5 antibody was obtained from Aharoni et al., 23 EUR. J. IMMUNOL. 17 (19
93); Aharoni et al., PROC. NATL. ACAD. SCI. USA 10821 (1997).
It was obtained from the injected copolymer 1 in A (SJL / BALB / c) F 1 mice spleen.

Human T cell clones were obtained from Teitelbaum et al. 89 PROC. NATL. ACAD. SCI. USA 13
7 (1992) derived from peripheral blood mononuclear cells. Approximately 5 × 10 ⁶ cells were used for copolymer 1
Alternatively, the cells were incubated in each well of a 24-well culture plate containing a medium supplemented with 10% heat-inactivated autologous serum together with MBP (50 μg / ml). After 7 days in culture, cells were transferred to a medium containing 10% fetal calf serum and recombinant human interleukin-2 (20 units / ml). Cells were grown continuously in this medium with regular exposure every 14 to 18 days to the antigen appearing on irradiated (3000 rad) autologous mononuclear cells. The C-52 T cell clone is also available from Teitelbaum et al. 89 PROC. NATL. ACA.
D. according to D. SCI. USA 137 (1992). Derived from w11 donor.

Proliferation Assay Cells or clones of the T line were tested for their specific proliferative response 10 to 21 days after challenge. T cells (1.5 × 10 ⁴ ) in triplicate in 10% FCS medium with 5 × 10 ⁵ irradiated spleen cells or human EBV transformed B cells (5 × 10 ⁴ ) and a final volume of 0.2 ml of the indicated antigen did. At the end of the 48 hour incubation, 1 μCi [ ³ H] -thymidine was applied to the medium and harvested after 6-12 hours. The average deviation of the triplicate culture was less than 20%.

<< P51 >> The cell line cross-reacted only with TAL. None of the cell lines cross-reacted with GTL or cross-reacted with AGT (1: 1: 1) that had the same amino acids as TGA but in a different mole fraction than TGA or Copolymer 1.

Table 6 Cross-reactivity of terpolymer with Copolymer 1 on Copolymer 1 specific T cell lines and clones Murine T cell lines and clones

[0168]

[Table 6-1] ^* Prol = measurement by proliferation. ^‡ IL-4 = measurement by interleukin-4 II. Human T cell clone

[0169]

[Table 6-2] EXAMPLE 8 Terpolymer Cross-Reacts with Anti-Copolymer 1 Antibody Antibody directed against Copolymer 1 crosses terpolymer lacking either tyrosine, glutamic acid or alanine React. However, terpolymers lacking lysine have not been effectively recognized by anti-copolymer 1 antibody.

Antibodies Mouse anti-MBP and anti-copolymer 1 monoclonal antibodies were obtained by fusing SJL / J mouse MBP- or copolymer 1 immunized spleen cells with the NSO / 1 murine plasmacytoma cell line. Teitelbaum et al. Proc. Natl. Acad. Sci. USA 9528
(1991).

Radioimmunoassay Soft plastic microtiter plates were coated with Copolymer 1 or terpolymer (2 μg / ml). After incubation at room temperature for 16 hours, the plates were washed three times, 2% bovine serum albumin, 0.05% Tween 20, 0.1%
Saturated with PBS containing sodium azide, 10 mM EDTA and 5 units / ml heparin ("PBS buffer") for 2 hours. Monoclonal antibody supernatant (50 μl) was added to the wells during the 2 hour incubation and the wells were washed again with PBS buffer. ¹²⁵ I-labeled goat anti-mouse Fab antibody (1 ×
10 ⁵ cpm / well) was added during the overnight incubation at 4 ° C. After extensive washing, the radioactivity is measured with a gamma counter.

Methods ELISA Assay for Antibody Cross-Reactivity A standard ELISA assay is performed using microtiter plates coated with anti-copolymer 1 polyclonal antibody and a 2 μg / ml terpolymer preparation.

Copolymer 1 Copolymer 1 having the following amino acid composition was prepared using Teva Pharmaceutical Industries (P
etach Tikva, Israel).

Amino acid molar fraction L-glutamic acid 0.141 L-alanine 0.427 L-tyrosine 0.095 L-lysine 0.338 Terpolymer The four terpolymers of Example 1 were used. did.

Results Table 7 shows that anti-copolymer 1 polyclonal antibody cross-reacts with terpolymers lacking either tyrosine, glutamic acid or alanine. The binding to terpolymers lacking glutamic acid (TAL) is relatively high, as will be evident from its high average molecular weight. The tertiary copolymer lacking lysine has not been effectively recognized by the anti-copolymer 1 antibody. These data suggest that charged amino acids such as lysine play a role in the recognition and binding of Copolymer 1 and terpolymer.

Table 7

[Table 7] ^* Dalton ^‡ nd-not measurable Cross-reactivity between terpolymer and copolymer 1-reactive monoclonal antibody Cross-reactivity between terpolymer and copolymer 1 at B-cell response level Check using antibodies (mAbs). The monoclonal antibody is reactive with both Copolymer 1 and MBP (mAbs 2-2-18 and 3-1-45), or Copolymer 1 (mAbs 3-3-9 and 5-7-2). ) Is only reactive with Teitelbaum et al. 88 Proc. Natl. Acad. Sci.
USA 9528 (1991).

Table 8 shows that the terpolymers differed in their ability to bind to mAbs. TGA and GTL were not recognized by any of the copolymer 1 specific mAbs. On the other hand, TAL and GAL bound to copolymer 1 and MBP-specific mAbs having the same affinity as copolymer 1. GAL and T
AL differed only in binding to one mAbs, 5-7-2, which bound TAL but not GAL.

Table 8 Cross-reactivity of terpolymers with MBP- and copolymer 1-reactive B cell antibodies

[Table 8] 2-2-18 is an anti-mouse MBP monoclonal antibody cross-reactive with Copolymer 1 3-1-45 is an anti-copolymer 1 monoclonal antibody cross-reactive with MBP 3 -3-9 is an anti-copolymer 1 monoclonal antibody having no cross-reactivity with MBP 5-7-2 is an anti-copolymer 1 monoclonal antibody having no cross-reactivity with MBP Example 9 Copolymer 1 and terpolymer are combined with collagen in binding to human leukocyte antigen.
In this example, which competes and inhibits collagen-specific T cell responses, Copolymer 1, TAL, GAL, GTL and TGA bind rheumatoid arthritis-associated immune dominant collagen antigen in binding to MHC proteins;
CII 261-273 (SEQ ID NO: 3).

Methods Protein Expression and Purification Recombinant HLA-DR1 and HLA-DR4 molecules (DRA / DRB1 respectively) ^* 0101 and^*0401) is Stern, L. et al. 68 CELL
 465 (1992) and Dessen, A. et al. 7 IMMUNITY 473 (1997).
Was expressed in Drosophila S2 cells. Cells are 0-5%
Excell 401 medium (Sigma, St. Louis, MO) supplemented with fetal calf serum (Sigma)
) Were grown at 26 ° C. in roller bottles. CuSO₄To a final concentration of 1 mM
To induce cells, then incubate for another 4-5 days.
I did it. Immunoaffinity analysis of recombinant HLA-DR1 and HLA-DR4
Manufactured by Stern, L. et al. 68 CELL 465 (1992) and Dessen, A. et al. 7 IMMUNIT
Perform as previously reported by Y 473 (1997). Continuously collect the collected cell supernatant.
Protein A, Protein G and Protein A-LB3
. One column, followed by binding of HLA-DR to 50 mM 3-cyclohexyl
Elution with amino-1-propanesulfonic acid (CAPS), pH 11.5, 200
Neutralize with mm phosphate (pH 6.0). This eluate is centripre
Concentrate on a p10 membrane (Amicon). Protein concentration is bicinchoninic acid (bicin
choninic acid) assay (Pierce Chemical Co.).

Polypeptide Labeling The polypeptide and HA306-318 peptide were biotinylated as in Examples 4 and 7.

Class II MHC Protein Binding Assay In this assay, a recombinantly produced water soluble protein biotinylated polypeptide of the invention and varying amounts of unlabeled competitor polypeptide, collagen CII peptide or influenza virus HA Incubated with peptide. Assays were performed in 96-well microtiter immunoassay plates (PRO-BIN) coated with affinity-purified LB3.1 monoclonal antibody.
D ™, Falcon). Antibody coating was performed by adding 100 μl of 10.0 μg / ml LB3.1 monoclonal antibody to each well and incubating at 4 ° C. for 18 hours. The microtiter wells were then blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) at 37 ° C. for 1 hour,
Washed three times with TTBS. Add 1% BSA to each well before adding sample
Add 50 μl of TBS containing. 150 mM phosphate buffered saline (PBS)
Sodium chloride, 7.5 mM sodium phosphate, dibasic, 2.5 mM sodium phosphate, monobasic, 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 obtained by adding 0.05% Tween-2G to TBS. Other solutions used in this assay are described in Fridkis-Hareli et al. 160 J.
IMMUNOL. 4386 (1998).

[0182] Binding analysis was performed using the polypeptide of the present invention obtained by biotinylating a water-soluble DR molecule, and various concentrations of an unlabeled inhibitor (copolymer 1, TAL, GAL, GTL, TGA, collagen CII 261-273 peptide or HA306). -318 peptide). Collagen type CII peptide 261-2
73 has SEQ ID NO: 3 (AGFKGEQGPKGEP) and has a molecular weight of 1516. The DR concentration used is 0.15 μM. The final concentration of biotinylated copolymer 1 or terpolymer is 1.5 μM. Incubation is for 40 hours at 37 ° C. in 50 μl of binding buffer pH 5.0.

The bound label is detected using streptavidin-conjugated alkaline phosphatase as follows. The plate was washed three times with TTBS and 100 μl of streptavidin-conjugated alkaline phosphatase (1: 3000, BioRad, Richmond, VA).
for 1 hour at 37 ° C., then p-nitrophenyl phosphate in triethanolamine buffer (BioRad) was added. The absorbance at 410 nm was measured using a microplate reader (MR4000, Dynatech, Chantil).
ly, VA).

T Cell Hybridoma and Antigen Presenting Cell (APC) Binding Assay To determine whether Copolymer 1 and terpolymer can inhibit the activation of T cells in response to collagen CII peptide, Examined. Mouse DR
One restriction 3.19 and 19.3 T cell hybridomas and mouse DR4 restriction 3838 and D3 T cell hybridomas were used. Rosloniec. EF, et
al., 185 J. EXP. MED. 1113-1122 (1997); Andersson, EC, et al., PROC.
NATL. ACAD. SCI. USA (1998). Antigen presenting cells (APCs) were L cells transfected with DR1 (Rosloniec. EF, et al., 185 J. EXP. MED. 1113-1122 (
1997), L4 cells transfected with DR4, and Price (DRB1 ^* 0401 / DRB4 ^* 0101). T cell stimulation tests were performed in 96-well microtiter plates in a total volume of 0.2 ml. Irradiated (3000-rad) APCs (2.5 × 10 ⁴ / well) were co-incubated with CII261-273 (40 μg / ml) and various concentrations of the polypeptide at 37 ° C. for 2 hours and then T cells ( 5 × 10 ⁴ / well) and incubation is continued at 37 ° C. for 24 hours. The supernatant (30
μl) and remove 1 with IL-2-dependent CTL-L (5 × 10 ⁴ / well).
After incubation for 2 hours, the cells were labeled with ³ H-thymidine (1 μCi / well) for 12 hours. Plates are collected and radioactivity is monitored using a 1450 microbeta plus liquid scintillation counter (Wallac, Gaithersburg, MD).

Results Class II MHC Protein Binding Assay Recombinant, water-soluble HLA-DR1 and -DR4 proteins produced in insect cells were largely free of bound autoantigens or other peptides. Thus, the data obtained from the insect cells that produced the protein more accurately indicates the actual binding affinity for the polypeptide. Fridkis-Hareli et al. 160 J. IMMUNOL. 43
86 (1998), the entire contents of which are incorporated herein by reference.

HLA-DR1 and HLA-DR2 with each copolymer 1 and terpolymer
And competitive binding to HLA-DR4 molecules is shown in FIG. Each copolymer 1
(YEAK, FIG. 5A, top panel) and binding to the terpolymer was determined by the amount of CII 261-273 (SEQ ID NO: 3) peptide required for 50% inhibition, as indicated by C
Stronger than that of the II261-273 (SEQ ID NO: 3) peptide. Also, as can be seen from the above, TAL has a higher affinity than HLA-
It bound to DR1 and HLA-DR4 molecules. Unlabeled TAL (YAK, FIG. 5A,
(Bottom panel) The kinetics of inhibition by the polypeptide was also somewhat better than that of the influenza virus peptide HA306-318 (SEQ ID NO: 2). However, the influenza virus peptide HA306-318 has CII261-2.
The binding to copolymer 1 and the binding to the terpolymer were inhibited more efficiently than the 73 peptide.

T Cell Hybridoma and Antigen Presenting Cell Binding Assay Results Copolymer 1 and terpolymer are DR1 restricted T by CII collagen peptide
It also inhibited cell activation (FIG. 6). T cell activation was detected by observing IL-2 production by DR1-restricted CII-specific T cell hybridomas. Various concentrations of the collagen peptide CII 261-273 (SEQ ID NO: 3) were co-incubated with one of the polypeptides and then T cells (indicated clone 3.1)
9 or 19.3) was added and the mixture was further incubated. The supernatant of the incubated cells is removed and the supernatant is treated with IL-2-dependent cytotoxic T.
By observing whether proliferation of lymphocytes (CTL-L) was induced,
Assayed for L-2. Circle filled with the degree of inhibition by TAL (●)
, A triangle (▲) filled with TGA, an open triangle (△) with GTL, and a square (■) filled with copolymer 1.
). The percent inhibition of CTL-L proliferation indicated on the vertical axis was calculated using Equation 1.

TAL is also a leading inhibitor. However, GTL and Copolymer 1 were also potent inhibitors of T cell activation by CII collagen peptide.
Inhibition of TGA activation was not very effective. Similar results were obtained with other batches of Copolymer 1 and terpolymer.

As shown in FIG. 7, similar inhibition of activation of DR4 restricted T cells was seen. IL
-2 production was used to assess the activation of DR4 restricted CII-specific T cell hybridomas (3838 and D3). It was found that the presence of various polypeptides inhibited IL-2 production, which inhibited DR4 restricted T cell activation. FIG. 7A
Shows the effect of co-incubating irradiated 3838 or D3 Price cells with a constant concentration of 40 μg / ml of collagen peptide CII261-273 (SEQ ID NO: 3) and various concentrations of the polypeptide at 37 ° C. for 2 hours. FIG. 7B
L-cells transfected with the gene encoding HLA-DR4 were treated with a constant concentration of 40 μg / ml of collagen peptide CII261-273 (SEQ ID NO: 3) and various concentrations of GAL, TAL, GTL, TGA and copolymer 1, 37
It shows the effect of incubating at 2 ° C for 2 hours. Then the T cells (indicated clone 3
838 or D3) was added and the samples were further incubated at 37 ° C. for 24 hours. The supernatant (30 μl) was then removed and the IL- induced by IL-2 production was removed.
Assayed for activation by proliferation of 2-dependent cytotoxic T lymphocytes (CTL-L). Each polypeptide mixture was tested in duplicate. The concentration of the polypeptide is shown on the horizontal axis. The degree of inhibition by TAL is indicated by filled circles (●), triangles filled by TGA (▲), triangles opened by GTL (△), and squares filled by copolymer 1 Shown by (■). The percent inhibition of CTL-L proliferation indicated on the vertical axis was calculated using Equation 1.

Embodiment 10Copolymer binding to HLA-DR1, HLA-DR2 and HLA-DR4 molecules
One sequence motif Methods Protein Expression and Purification Recombinant HLA-DR1 and HLA-DR4 molecules (DRA / DRB1 respectively) ^* 0101 and^*0401) is Stern, L. et al. 68 CELL
 465 (1992) and Dessen, A. et al. 7 IMMUNITY 473 (1997).
Was expressed in Drosophila S2 cells. Cells are 0-5%
Excell 401 medium (Sigma, St. Louis, MO) supplemented with fetal calf serum (Sigma)
) Were grown at 26 ° C. in roller bottles. CuSO₄To a final concentration of 1 mM
To induce cells, then incubate for another 4-5 days.
I did it. Immunoaffinity analysis of recombinant HLA-DR1 and HLA-DR4
Manufactured by Stern, L. et al. 68 CELL 465 (1992) and Dessen, A. et al. 7 IMMUNIT
Perform as previously reported by Y 473 (1997). Continuously collect the collected cell supernatant.
Protein A, Protein G and Protein A-LB3
. One column, followed by binding of HLA-DR to 50 mM 3-cyclohexyl
Elution with amino-1-propanesulfonic acid (CAPS), pH 11.5, 200
Neutralize with mm phosphate (pH 6.0). This eluate is centripre
Concentrate on a p10 membrane (Amicon). Protein concentration is bicinchoninic acid
(Pierce Chemical Co.).

Bound and Quantification of Bound Polypeptide Copolymer 1 was dissolved in a 1: 1 molar ratio of water-soluble HLA-DR1, -DR2 or -DR.
Incubate with 4 molecules at 37 ° C. for 40 hours. The unbound copolymer 1 and the bound copolymer 1 are separated by Centricon ultrafiltration. The bound copolymer 1 from the HLA-DR complex was then extracted by acid treatment (Chicz,
R. et al. 178 J. EXP. MED. 27 (1993)) and amino acid analysis.

After elution, 5-10% of Copolymer 1 mixture for HPLC separation and microsequencing
Is fractionated by microballs. HPLC was performed on a Hewlett-Packard 1090 HPLC equipped with a 1040 diode array detector with Zorbax C ₁₈ 1
. Use a 0 mm reverse phase column. At a flow rate of 54 μl / min Copolymer 1 is eluted with a 0.055% trifluoroacetic acid (TFA) gradient in acetonitrile (0% at 0-10 minutes, 33% at 73 minutes and 60% at 105 minutes). The strategy of peak selection, reverse phase separation and Edman microsequencing are described in Chicz et al. 178 J.
EXP. MED. 27 (1993) and Lane, W. et al. 10 J. PROT. CHEM. 151 (1991).

Structural Properties of Bound Copolymer 1 Human HLA-DR1, HLA-DR from Homozygous EBV Transformed B Cell Line
By fractionating the copolymer 1 bound to 2 or HLA-DR4 molecules, almost all of the bound copolymer 1 is found in the high average molecular weight fraction and less than 10% of the bound copolymer 1 It was found that it was found in the fraction having a low average molecular weight.

To determine the hydrophobicity and size of Copolymer 1 binding to the isolated recombinant HLA molecule, a sample of bound and unbound Copolymer 1 was subjected to an acetonitrile gradient. Separation by reversed phase HPLC. Untreated control copolymer 1
Is eluted as a very broad peak with several small peaks, which spans an elution time of about 40-75 minutes. This elution profile is characteristic of a mixture of random polypeptides and is similar to the HPLC separation of Copolymer 1 in other batches. Copolymer 1 was converted to HLA-DR1, HLA-DR2 or HLA-DR4.
A similar profile was obtained upon elution from the molecule, indicating that the bound fraction was similar in chemical properties to the original Copolymer 1 preparation.

At least 95% of one molecule of the added copolymer is found in the fraction bound to the isolated HLA-DR1 protein, and similarly, at least 95% of one molecule of the added copolymer is isolated. It is found in the fraction bound to the HLA-DR4 protein. However, 80% of one molecule of the added copolymer binds to the HLA-DR2 protein. Copolymer 1 eluted from the HLA conjugate having the same amino acid tyrosine: glutamic acid: alanine: lysine ratio as that of the untreated control copolymer 1. These results indicate that the bound fraction of Copolymer 1 has an amino acid composition similar to that of the bound / unbound mixture, and that one molecule of the copolymer exhibits selective binding to different HLA-DR proteins. It can be seen that there was little or no indication.

Copolymer 1 bonded to each of HLA-DR1, -DR2 and -DR4 molecules
The HPLC fractions obtained above were pooled and analyzed by Hewlett-Packard
Automated Edman degradation was performed according to the manufacturer's routine 3.5 on a G1005A (Palo Alto, CA) protein sequencer.

For each HLA-DR protein, the bound copolymer had a random distribution of four amino acids corresponding to the input molar ratio of amino acids used for Copolymer 1 preparation. As inferred from the high initial molar ratio of alanine in Copolymer 1, alanine was found at much higher levels than glutamic acid, tyrosine and lysine. It can be seen that there is no sequence specificity of any amino acid of Copolymer 1, or no selective localization, whereby the bound fraction is also random and the same as the unfractionated Copolymer 1 preparation It was shown that.

Identification of Bound and Unbound Copolymer 1 Epitopes Using an anti-copolymer 1 polyclonal antibody, one fraction of the copolymer eluted from each of the HLA-DR molecules was added to the untreated control copolymer 1. It was determined whether the epitope found was included. The cross-reactivity of Copolymer 1 with the various eluted fractions was determined by a direct ELISA assay using a biotinylated anti-Copolymer 1 polyclonal antibody. 0.4 μg / ml of copolymer 1 or 1 fraction of copolymer
diluted to 100 μg / ml, duplicate at 100 μg / well, 96-well microtiter immunoassay plate (PRO-BIND ™, Falcon, Lincoln Park)
, NJ) and incubated at 37 ° C for 1 hour, 0.05% Tween-2.
Washed 3 times with 0-containing TBS. Then, the wells were sealed with TBS containing 3% BSA, and then biotinylated anti-copolymer 1 (1: 5000 dilution, 100 μl / well) was added. The antibody-ligand conjugate was composed of streptavidin-conjugated alkaline phosphatase (1: 3000 dilution, BioRad) and triethanolamine buffer (BioRad
Hercules, Calif.) Using p-nitrophenyl phosphate. 41
The absorbance at 0 nm was monitored by a microplate reader (Dynatech MR4000).

The antibody binding assay showed that all fractions were similarly recognized by the anti-copolymer antibody, and these bound heteropolymer fractions were similar to each other and to Control Copolymer 1, Or it was suggested that they share the same epitope.

Determination of Copolymer 1-HLA Binding Motif Upon binding, MHC class II proteins protect the Copolymer 1 binding site from peptidase digestion. In order to obtain the amino acid sequence of the binding site in the copolymer 1 of the HLA-DR molecule, 1 mM of the copolymer 1 was separately incubated with 100 μm of various HLA-DR molecules in a volume of 10 μl of PBS at 37 ° C. for 40 hours. The molar ratio of Copolymer 1 to -DR was 10: 1. During the last 18 hours of incubation, 2 units of aminopeptidase 1 were added to the reaction. Aminopeptidase 1 is a metalloprotein isolated from Streptomyces griseus (Spungin et al. 283 J. BIOCHEM 471 (1989)
)), Available from Sigma Chemicals, St. Louis, MO. Aminopeptidase 1 removes the amino terminus of Copolymer 1 protruding from the HLA-DR molecule and digests the remaining unbound Copolymer 1 (Mouritsen et al. 148 J. IMMUNOL. 1987 (1992); Lar
sen et al. 184 J. EXP. MED. 183 (1996)). 20- to 50-fold expanded volume of subsequent digestion of copolymer 1 with aminopeptidase, e.g.
Perform with 300 μl of heteropolymer digested with 1 l. Sample is Centricon
Spin-concentrate using a 10 ultrafiltration device to a final volume of about 100 μl.

[0201] Copolymer 1-HLA-DR conjugate and unbound Copolymer 1 were analyzed by SDS-PAGE using NOVEX small cell electrophoresis, 10% acrylamide separation gel and 5% acrylamide stacking gel. The copolymer 1-HLA-DR1 complex was run at 200 V for 1 hour under non-reducing conditions, stained with Coomassie brilliant blue, fixed with 10% methanol / 10% acetic acid for 3 hours, and on cellophane paper (BioRad). Dry at 25 ° C. The copolymer 1-HLA-DR complex withstands SDS-mediated dissociation, forms a high molecular weight complex with the HLA-DR1 αβ heterodimer, and has multiple molecules with a molecular weight greater than 50 kD on polyacrylamide gels. , Indicating that the copolymer 1-HLA-DR complex was protected.

The aminopeptidase 1 treatment revealed unbound Copolymer 1 as a stain on the lower part of the gel, indicating that it was completely digested by the enzyme.

Fractions containing the peak of the copolymer 1-HLA-DR complex were selected in the range of about 40 to 75 minutes of elution time of various HLA-DR complexes to obtain a binding motif sequence. Digested Conjugated Copolymer 1 polypeptide has no protruding amino termini and acetic acid (10%
) And incubated at 70 ° C. for 15 minutes, followed by ultrafiltration and Spe
Elute from HLA-DR by vacuum concentration on edVac (Savant Instruments, Farmingdale, NY). Fridkis-Hareli et al. 163 CELL. IMMUNOL. 229
(1995). The N-terminal 20-25 amino acids of Copolymer 1 protrude beyond the HLA-DR proteins and are not the binding motif portion of Copolymer 1 for these proteins. The sequence data (Table 9) shows that the copolymer 1 peptide bound to HLA-DR1 has very high levels of glutamic acid at the first and second positions of the copolymer 1 binding domain and at the second and third positions. It shows that it has high levels of lysine and high levels of tyrosine at positions 3-5. P1 site is HLA-DR
The third position (lysine or tyrosine) of the coupled copolymer 1 peptide is MHC class II because it is the third amino acid at the binding site of the other peptide that binds to 1.
It is thought to correspond to the P1 position of the peptide binding site in the groove. For example, tyrosine is found at the third (P1) position of the HA306-318 peptide, which corresponds to position Y308 of the HA protein. Stern et al. 368 NATURE (Lond.
215. These data show that the sequence specificity or lack of selective localization of any four amino acids in Copolymer 1 in the MHC class II groove found in untreated Copolymer 1. In contrast to the random pattern.

In HLA-DR2, both tyrosine and alanine are detected after the third cycle of Edman degradation (Table 9). The position corresponding to the anchor position following P1 (HLA-
Positions corresponding to P4, P6 or P9 of DR1 and HLA-DR4; and H
(Position corresponding to P4 or P7 of the LA-DR2b molecule) shows no sequence specificity or selective localization. In all samples, alanine levels were higher than glutamic acid, tyrosine and lysine, which was inferred from the higher molar ratio of alanine present in Copolymer 1. In each of the HLA-DR1 and HLA-DR4 molecules, tyrosine is found at the position corresponding to the first anchor position (the third residue in the sequence analysis), followed by alanine at the position corresponding to the next pocket. HLA
Even in Copolymer 1 bound to -DR2, a large amount of tyrosine was present at the position corresponding to P1. Glutamic acid is abundant in the first cycle position corresponding to P-2 position, and P-1
There was more lysine in the next adjacent position corresponding to the position. These residues are responsible for the interaction of Copolymer 1 with the HLA-DR molecule and the complex with the T cell receptor (TCR
) Can help stabilize the interaction.

These results indicate that Copolymer 1 contains a class II MHC binding motif. Copolymer 1 linked to the antigenic groove of the HLA-DR molecule can act either as a blocking peptide, or as an antagonist or semi-agonist, resulting in suppression of an autoimmune T cell response and / or allergy, or both. Is to gain. Applicants' invention does not rely on the theory based on the action of Copolymer 1 or terpolymer.

Table 9 Binding sequence of copolymer 1 peptide bound to HLA-DR1, HLA-DR2 and HLA-DR4 molecules

[Table 9] Example 11 Development of Peptide Binding to HLA-DR1 and HLA-DR4 Molecules In the above example, copolymer 1 and terpolymer were bound to purified human HLA-DR molecules in the peptide binding groove and It has been shown to inhibit binding to HA306-318, an influenza virus-derived peptide that binds with high affinity to both DR1 (DRB1 ^* 0101) and HLA-DR4 (DRB1 ^* 0401) molecules. Furthermore, a terpolymer composed of only three kinds of amino acids (
GAL, TGA, TAL and GTL) are purified HLA-DR1, HLA-D
Binds R2 and HLA-DR4 molecules and produces a collagen peptide, CII 261-2
73 and RA-associated HLA-DR1 (DRB1 ^* 0101) and -DR4 (DRB
1 ^* 0401) competed for binding to the protein molecule. The terpolymer also inhibited CII-reactive T cell clones.

In this example, a peptide having a defined sequence and length (15 residues) was synthesized using the binding motif sequences summarized in Table 9. These peptides are
The affinity and specificity of binding to class II HLA-DR protein molecules, the ability to inhibit binding to competitor molecules, and the ability to inhibit T cell responses were analyzed. According to the present invention, these properties are useful in therapeutic compositions for treating autoimmune diseases.

The peptides shown in Table 10 were prepared on an Applied Biosystems Peptide Synthesizer using solid phase techniques (Barany G. et al., 1979. The Peptides, E. Gross. Et al., Eds.
(New York, NY: Academic Press) and purified by reverse phase HPLC. The peptide sequence includes the sequence PKYVKQNTLKLAT (SEQ ID NO: 2) (MW17
HA) 306-318 with the sequence AGFKGEQGPKGEP (SEQ ID NO: 3) (MW1516); and HA306-318, APKYVKQNTLKLATA (SEQ ID NO: 44) with alanine at the N and C termini. Peptides are also available in the multipin peptide synthesis system (Chiron Tech
nologies, Raleigh, NC) on a 1 μmole scale. A peptide having a free amino group at the N-terminus and a free carboxyl group at the C-terminus, and a spacer SGS
G was linked to the N-terminus by G and synthesized as a 15-mer containing biotin having a free carboxyl group at the C-terminus. Peptide synthesis was monitored including two standard peptide sequences as controls, which were subjected to HPLC and mass spectrometry. HA306-
The 318 peptide was also used as a positive control in the binding test. The pin peptide was lyophilized and resuspended in dimethylsulfoxide (DMSO) to a concentration of 2 mg / ml. Most of the peptides were completely dissolved under these conditions. Biotinylated Fridkis-
Harid et al. 91 PROC. NAD. ACAD. SCI., USA 4872-4876 (1994), an excess of N-hydroxysuccinimide biotin in DMSO (Sigma.
Louis, MO). Unreacted biotin is dialyzed (SpectraPor® membrane MWCO500, Spectrum Medical Industries, Houston, TX)
Removed by

The peptides were made to have a test sequence motif for binding to the groove of the HLA-DR1 and -DR4 molecules (see Table 10). The peptides usually contained various combinations of glutamic acid, lysine and alanine at the N-terminus, followed by tyrosine at the position corresponding to P1 (shown in bold in Table 10), followed by alanine in the subsequent binding pocket. Test sequence motifs are classified into three different groups according to these common positions (Table 10). Group I peptides have a lysine at the position corresponding to P8 (
K) and tyrosine (Y) at the position corresponding to P1 (bold in Table 10). A reference peptide of this combination with lysine (K) at the position corresponding to P8 which enhances solubility and alanine at all other residues was previously synthesized. Ja
rdetzky et al., 9 EMBO J. 1797-1803 (1990). Peptides of group II had tyrosine (Y) at the position corresponding to P1, while alanine (A) was located at the position corresponding to P8.
). In the peptides of Group III, the amino acid tyrosine (T) at the P1 position of the HA306-318 peptide was shifted by one or two residues. All groups of peptides included one or more glutamic acid (E) and / or lysine (K) residues at various positions as found in the binding motif to enhance solubility.
Both N-terminal biotinylated and unlabeled assemblies of peptides were synthesized for these studies.

Table 10 Synthetic peptide groups and common positions

[Table 10] Using a competitive binding assay, the synthetic peptides were HLA-DR1 and HLA-D
In binding to the R4 protein, copolymer 1 or high affinity HA306-
Determine if it can successfully compete with the 318 peptide. In competitive binding assays, copolymer 1 or HA306-318 peptide with alanine (AP
KYVKQNTLKLATA (SEQ ID NO: 44) is biotinylated and an unlabeled copolymer 1 or peptide inhibitor is added. In kinetic studies, biotinylated copolymer 1 was found to be unlabeled copolymer 1 and HA306-318 (SEQ ID NO: 2) and recombinant H
It was shown that the binding to LA-DR1 was better inhibited than the peptides of Groups I to III. However, some of the peptides with lysine at position P8 (group I) are peptides with similar sequences but are better inhibitors than those with alanine at position P8 (groups II and III in Table 10). is there. On the other hand, the binding between the biotinylated copolymer 1 and the HLA-DR4 molecule was efficiently inhibited by many peptides in Groups I to III. Conversely, with regard to the binding between biotinylated HA306-318 and HLA-DR4, copolymer 1 inhibited better than HA306-318 or the peptides of Groups I to III.

To further investigate the relative affinities of the peptides of Groups I-III for binding to HLA-DR1, -DR2 and -DR4 molecules, a competitive binding assay was performed with a biotinylated multipin peptide and a non-inhibitor as an inhibitor. Labeled copolymer 1, HA306-3
18 or CII 261-273. The binding of most of the peptides of Groups I to III to HLA-DR1 and HLA-DR4 was unlabeled copolymer 1, HA306
-318 (SEQ ID NO: 2) and CII 261-273 (SEQ ID NO: 3), but were less effective than binding to HA306-318 (SEQ ID NO: 2). Some peptides are HLA-DR1 (Table 1A) or HLA-DR4
(Table 1B) For protein, Copolymer 1, HA306-318 or CII26
It showed higher affinity than 1-273.

Table 11A Affinity of Group I-III peptides for HLA-DR1 molecule

[0213]

[Table 11A] Table 11B Affinity of selected copeptides for HLA-DR4 (DRB1 ^* 04014)

[0214]

[Table 11B] Affinity was determined by competition with biotinylated competitors HA306-318 and copolymer 1.

All peptides were further examined for inhibition of CII-specific T cell responses using the methods described in the above examples. HA306-318 (peptide SEQ ID NO: 2)
Both R1 3.19 and 19.3 T cell clones were very efficiently (19.
3 and 3.19 cells inhibited more than 95% and 98%, respectively). However, the peptides having SEQ ID NOs: 27, 48, 36 and 53 were better or better inhibitors of the T cell response than the reference influenza virus hemagglutinin peptide HA306-318.

For HLA-DR4 restricted T cells, the following activity patterns were obtained using L fibroblasts transfected with HLA-DR4 as antigen presenting cells.

Peptide SEQ ID NOs: 29, 31, 34, 35, 50, 51, 54, 38 and 41
Was an excellent inhibitor of the DR4 3838 T cell clone, while the D3 clone was most inhibited by peptide SEQ ID NOs: 46, 27, 34, 28 and 54. These peptides resulted in over 80% inhibition levels on D3 and 3838 cells. Copolymer 1 had minimal effect on the CII-specific T cell response, which was worth giving less than 20% inhibition. HA306-318 (SEQ ID NO: 2) inhibited both DR4 3838 and D3 T cell clones, but was less efficient than the DR1, 3.19 and 19.3 clones (
<60% inhibition).

These data indicate that the peptides having SEQ ID NOs: 46, 27, 34, 28 and 41 are significantly better inhibitors of the T cell response than the reference influenza virus hemagglutinin peptide HA 306-318. . The peptides having SEQ ID NOs: 27 and 41 are HLA-DR-1- and HLA-DR-4 restricted CII
It is a high level inhibitor of both specific T cells.

The data in these examples are based on at least two sets of each peptide,
Of a specific synthetic peptide. Some of these are HA30
It inhibited binding of 6-318 and copolymer 1 to recombinant HLA-DR1 and -DR4 molecules. HA 306-318 or copolymer 1 and HLA-DR1 or-
The peptide that inhibited binding to the DR4 molecule contained tyrosine at the P1 position. P1
Glutamic acid, alanine and lysine, present in various combinations on the N-terminal side of, did not appear to affect binding affinity. Of the residues that follow, the lysine at P8 is not copolymer 1, which binds to HLA-DR1, but HA3.
06-318 was important. In contrast to HLA-DR1, a very large number of peptides inhibited the binding of HA306-318 and copolymer 1 to the HLA-DR4 molecule. These peptides contained a tyrosine at the position corresponding to P1 and a lysine or alanine at the position corresponding to P8, but lacked any significant amino acid preference at other positions.

The affinity of HA306-318 for recombinant HLA-DR4 was HLA-DR1
Lower than for the molecule, the affinity of Copolymer 1 for recombinant HLA-DR4 was higher than for the HLA-DR1 molecule. These affinities are similar to those found in HLA-DR1 and -DR4 molecules purified from human blood. The binding of some biotinylated peptides to either HLA-DR1 or -DR4 was via CII 261-273, and HA306-318 and copolymer 1
And that these peptides compete for presentation to CII-reactive T cells, as in all copolymer 1 mixtures. CII 261
-273, HA306-318 and peptides having an affinity close to or higher than that of the Copolymer 1 preparation are listed in Table 11A (for HLA-DR1) and Table 11B (for HLA-DR4). I will.

Some 15-mer peptides inhibited type II collagen-specific T cell clones. All of these peptides have tyrosine at the position corresponding to P1 and P8
Lysine or alanine at the position corresponding to, but there was no other specific sequence pattern. The examples herein show potent inhibition by several random heteropolymers of three amino acids selected from the group consisting of tyrosine, glutamic acid, alanine and lysine. These heteropolymers, especially TAL, are C
II261-273 competed with the RA-associated HLA-DR1 and -DR4 molecules for binding and inhibited CII-reactive T cell clones. In addition, peptide SEQ ID NO: 46 containing the direct sequence tyrosine-alanine-lysine has better inhibition of type II collagen-reactive T cells than copolymer 1 and has a simple tyrosine-alanine-lysine sequence of about 15 It has been found that peptides of amino acids can be used in place of the random polypeptide mixture found in TAL heteropolymers.

The results of the above examples show that embodiments of the present invention having each amino acid sequence containing at least three amino acids selected from tyrosine, glutamic acid, alanine and lysine have binding motifs at fixed positions that match a particular HLA-DR molecule. Which indicates that it can act as an effective therapeutic agent for autoimmune diseases. Pharmaceutical compositions comprising pure, short synthetic peptides of simple sequence are likely to have fewer side effects than mixtures of polypeptides of random sequence and have higher specificity for a particular binding site or autoimmune disease. On the other hand, the polypeptide mixtures contemplated by the present invention are believed to have broad efficacy in many types of autoimmune diseases. Thus, both simple sequence short peptide and polypeptide mixtures readily provided by the present invention are valuable therapeutic agents.

Example 12 Copolymer 1 Inhibits Activation of T Cells in Response to Myasthenia Gravis Antigenic Peptide
That method copolymer 1 copolymer 1 was obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel ).

The myasthenia gravis-related peptide was synthesized using a solid phase approach on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (1979).
Peptides were purified by reverse phase HPLC. The peptide used was the p259 peptide, Zisman et al., Hum Immunol 1995 Nov; 44 (3): 121-30; Brocke et
al., Immunology 1990 Apr, 69 (4): 495-500.

IL-2 Secretion IL-2 secretion by cell line WCB AB in response to myasthenia gravis peptide and / or copolymer 1 was evaluated. Cells (1.5 × 10 ⁴ ) were incubated with the indicated antigen. IL-2 secretion by the cell line WCB AB was used as a measure of activation of that T cell line.

Results Table 12 shows that the p259 peptide stimulates T cell secretion. However, when Copolymer 1 was incubated with the p259 peptide, IL-2
T cell secretion is inhibited in a dose-related manner. Copolymer 1 IL- at 100 μM
About 91% of 2 secretion was inhibited (Table 13), indicating that Copolymer 1 is a potent inhibitor of T cell activation.

Table 12 IL-2 secretion from WCB AB strain in response to p259

[Table 12] Table 13 WCB in response to Cop1 alone or 2 μM p259 + Cop1
IL-2 secretion from AB strain

[Table 13] Absorbance is the average of three samples. Confidence values were calculated for the absorbance of the blank (% CV = SD / (mean-blank) ^* 100).

[Brief description of the drawings]

Used as follows. “GAL” is a random terpolymer of glutamic acid, alanine and lysine; “TGA” or “YEA” is a random terpolymer of tyrosine, glutamic acid and alanine; “GTL” or “YEK” And "YEAK" is Copolymer 1; a random terpolymer of glutamic acid, tyrosine and lysine;

[Detailed description of drawings]

FIG. 1 shows the effect of Copolymer 1 or terpolymer on the proliferation of T cells specific for certain myelin basic protein (MBP) peptides. Some of the terpolymers of the present invention comprise a myelin basic protein antigen, MBP 84-10.
2 (SEQ ID NO: 1) inhibits the growth of T cell lines. The following symbols: Copolymer 1 (●); GAL (□); TGA (△); TAL (○); GTL (×) were used. FIG. 1A shows growth inhibition of mouse T cell clone MBP-sp-1 specific for MBP 84-102 peptide (0.5 g / well). As a control, the growth of mouse T cell clone MBP-sp-1 stimulated with MBP 84-102 (SEQ ID NO: 1) without inhibitor was 21,145 cpm. FIG. 1B shows MBP 8
Human T cell clone GP against 4-102 peptide (0.125 g / well)
2 shows inhibition of the -25 response. MBP 84-102 without inhibitor (SEQ ID NO: 1)
The proliferation of the human T cell clone GP-25 stimulated by was 4442 cpm.

FIG. 2A compares the binding of the polypeptide to the Class II major histocompatibility molecules DR1 (top), DR2 (middle) and DR4 (bottom) and of Copolymer 1. Coupling with copolymer 1 (■) and the following biotinylated terpolymer: GAL
(□); TGA (▲); GTL (△); and binding by TAL (結合) were compared. The amount of Class II major histocompatibility molecule was kept constant at 0.5 μ / sample and the peptide concentration was varied between 0 and 8 μM as shown on the y-axis. Binding is 37 ° C,
pH 5.0 for 40 hours. Binding was detected by capturing the polypeptide-class II complex with the LB3.1 antibody and reacting with streptavidin-conjugated alkaline phosphatase followed by monitoring the absorbance at 410 nm to detect the amount of biotinylated polypeptide bound. .

FIG. 2B is a Lineweaver-Burk plot of the binding data obtained in FIG. 2A.

FIG. 3 shows the antagonistic inhibition of binding between Copolymer 1 and class II major histocompatibility molecules by the present polypeptide. Purified HLA-DR1 (top), HLA-DR2 (middle) and HLA-DR4 (bottom) molecules alone or with the following unlabeled polypeptide: copolymer 1 (■); GAL (□); TGA (▲); in the presence of one of GTL (△); and TAL (◆), a fixed amount (1.5 μM
) Was incubated with the biotinylated copolymer 1). The inhibition by these polypeptides was compared to the inhibition by the myelin basic protein antigen HA 306-318 (O) (SEQ ID NO: 2). Binding was at 37 ° C., pH 5.0 for 40 hours. The amount of unlabeled polypeptide was varied between 0.1 and 1000 μM as shown on the y-axis. The binding rate was calculated by the formula 1:% inhibition = 100− (signal in the presence of competitor−background) × 100 / (
(% Signal without competitor-background).

FIG. 4A: TAL with bacterial superantigens SEA, SEB and TSST-1
Figure 5 shows competitive inhibition of binding of to class II major histocompatibility molecules. Purified HLA-DR
1 (top), HLA-DR2 (middle) and HLA-DR4 (bottom) molecules at increasing concentrations of unlabeled bacterial superantigen SEA (□); SEB (■); or TSST-1 (●)
Was incubated with a fixed amount of biotinylated TAL in the presence of Binding is 37
C., pH 5.0 for 40 hours. As shown on the y-axis, the amount of superantigen was 0.1 to 10
It was varied between 00 μM. The binding rate is shown as% inhibition according to equation 1.

FIG. 4B. TGA with bacterial superantigens SEA, SEB and TSST-1.
Figure 5 shows competitive inhibition of binding of to class II major histocompatibility molecules. Purified HLA-DR
1 (top), HLA-DR2 (middle) and HLA-DR4 (bottom) molecules at increasing concentrations of unlabeled bacterial superantigen SEA (□); SEB (■); or TSST-1 (●)
Was incubated with a fixed amount of the biotinylated polypeptide TGA in the presence of Binding was at 37 ° C., pH 5.0 for 40 hours. The amount of superantigen was varied between 0.1 and 1000 μM as shown on the y-axis. The binding rate is the inhibition% calculated by the above equation 1.
As shown.

FIG. 4C. GAL with bacterial superantigens SEA, SEB and TSST-1.
Figure 5 shows competitive inhibition of binding of to class II major histocompatibility molecules. Purified HLA-DR
1 (top) and HLA-DR2 (bottom) molecules at increasing concentrations of unlabeled bacterial superantigen SE
A (□); SEB (■); or TSST-1 (●) were incubated with a fixed amount of biotinylated polypeptide GAL. Binding is 37 ° C, pH5.
0 was 40 hours. The amount of superantigen was varied between 0.1 and 1000 μM as shown on the y-axis. The binding rate is shown as% inhibition calculated by the above equation 1.

FIG. 5A shows inhibition of binding between a labeled molecule and a purified MHC class II protein by the terpolymer of the present invention. FIG. 5A shows inhibition of binding to MHC HLA-DR1 protein.

FIG. 5B shows inhibition of binding between a labeled molecule and a purified MHC class II protein by the terpolymer of the present invention. FIG. 5B shows inhibition of binding to MHC HLA-DR4 protein. As unlabeled competitors, the terpolymer of the present invention, 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 shown on the horizontal axis. In each panel, CII 261-27
3 (SEQ ID NO: 3) is indicated by open circles (○) and HA 306-318 (
Inhibition by SEQ ID NO: 2) is indicated by filled circles (•), and inhibition by each of the terpolymers of the present invention is indicated by open triangles or squares, or filled triangles or squares. The degree of inhibition by GTL is indicated using an open triangle (△), the one with TAL is filled with a triangle (▲), the one with TGA is opened with a square (□), and the one with copolymer 1 is filled. Are indicated by squares (■). The observed and indicated binding rate on the vertical axis was calculated as% inhibition using Equation 1 above.

FIG. 6 shows inhibition of IL-2 production by DR1-restricted CII-specific T cell hybridomas in the presence of various polypeptides of the invention. Irradiated L57.
23 cells (fibroblasts transfected with the gene encoding HLA-DR1) were incubated with collagen peptide CII261-273 (40 μg / ml) and various concentrations of the polypeptide shown on the horizontal axis in duplicate, at 37 ° C. Co-incubated for 2 hours, then T cells (indicated clones 3.19 or 19.3) were added and the mixture was further incubated at 37 ° C. for 24 hours. Supernatant (30 μl
) Was then removed and assayed for IL-2-induced activation by proliferation of IL-2-dependent cytotoxic T lymphocytes (CTL-L). The degree of inhibition by TAL is indicated by filled circles (●), triangles filled by TGA (▲), triangles opened by GTL (△), and squares filled by copolymer 1 Shown by (■). CT shown on vertical axis
The% inhibition of LL proliferation was calculated according to equation 1.

FIG. 7A shows DR4 restricted CII in the presence of various polypeptides of the invention.
FIG. 9 shows inhibition of IL-2 production by specific T cell hybridomas (3838 and D3). FIG. 7A shows that irradiated 3838 or D3 Price cells were treated at a constant concentration of 4;
The effect of co-incubating with 0 μg / ml of collagen peptide CII 261-273 (SEQ ID NO: 3) and various concentrations of each of the present polypeptides at 37 ° C. for 2 hours is shown.

FIG. 7B. DR4 restricted CII in the presence of various polypeptides of the invention.
FIG. 9 shows inhibition of IL-2 production by specific T cell hybridomas (3838 and D3). FIG. 7B shows L transfected with the gene encoding HLA-DR4.
The effect of incubating cells with a constant concentration of 40 μg / ml of collagen peptide CII 261-273 (SEQ ID NO: 3) and various concentrations of each of the polypeptides at 37 ° C. for 2 hours is shown. The T cells (indicated clone 3838 or D
3) was added, the samples were further incubated at 37 ° C. for 24 hours, and the supernatant was assayed as described in FIG. Each polypeptide was tested in duplicate. The concentration of the polypeptide is shown on the horizontal axis. In this drawing, the same reference numerals as those in FIG. 6 are used.

[Sequence list]

[Procedure amendment]

[Submission date] October 9, 2001 (2001.1.10.9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

Title: Treatment of autoimmune diseases with Copolymer 1 and related copolymers and peptides

[Claims]

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[Related Application] In the present application, a provisional application, 60 / 093,859, 19 filed on July 23, 1998
60 / 101,825 filed on September 25, 1998, 6/101 filed on October 2, 1998.
0 / 102,960, 1998 October 30 filed 60 / 106,350, and claims the 60 / 108,184 benefit of 1998 November 12 filed 1999
Claims of PCT / US99 / 16747, filed on July 23, all of which are incorporated herein by reference.

[0002]

[Government Support] This invention was partially supported by the government under CA47554 approved by the National Institutes of Health.

[0003]

Introduction The present invention provides compositions and methods for treating an autoimmune disease using a therapeutically effective amount of a peptide or polypeptide related to Copolymer 1. Copolymer 1 is a heterogeneous mixture of synthetic random linear copolymers of tyrosine, alanine, glutamic acid and lysine, used in the treatment of multiple sclerosis at an appropriate therapeutic dose and average molecular size. Such a mixture of synthetic random linear copolymers is copolymer 1
Are essentially called ternary copolymers when they consist essentially of three of the four amino acids found in Table 1. The present invention relates, in part, to terpolymers. Preferably, the terpolymer comprises tyrosine, alanine and lysine, or from glutamic acid, tyrosine and lysine, or from glutamic acid, alanine and lysine. Surprisingly, terpolymers have efficacy in the treatment of various autoimmune diseases,
Binds major histocompatibility complex (MHC) molecules, as well as antigen presenting cells.

[0004]

BACKGROUND OF THE INVENTION Autoimmune diseases occur when an organism's immune system does not recognize some of its own tissues as "self" and attacks them as "foreign". Usually, self-tolerance occurs early in the immune system due to developmental events, preventing the organism's own T and B cells from reacting with the organism's own tissues. MHC cell surface proteins bind to T cells and help regulate these early immune responses by providing processed peptides to T cells.

When an autoimmune disease develops, this self-tolerance process is disrupted. Soon the organism's own tissues and proteins are recognized as "self-antigens" 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 sheath, which covers and protects nerves. It is a progressive disease characterized by loss of neurons and motor function, second to demyelination. Rheumatoid arthritis ("RA") is also an autoimmune disease involving chronic inflammation of synovial cell joints and invasion by activated T cells, macrophages and plasma cells, and is thought to lead to progressive destruction of articular cartilage ing. It is the most severe arthropathy. Collagen II is a self-antigen attacked by rheumatoid arthritis
Although the type is a candidate, its properties are not well understood.

[0006] The tendency to develop multiple sclerosis and rheumatoid arthritis is inherited-these
Disease occurs more frequently in individuals with one or more unique MHC class II alleles.
This. For example, the inherited rheumatoid arthritis susceptibility is MHC class II DRB1 ^* 0401, DRB1^*0404, or DRB1^*0405, or DRB1 ^* It is strongly associated with the 0101 allele. Histocompatibility locus antigen (HLA)
It is found on the cell surface and helps determine the individuality of tissues of various people. Organization
The gene for the matching locus antigen is the same chromosome as the major histocompatibility complex (MHC)
It is located in the sixth area. MHC region has many unique features in various cells of the body
Of the class I molecules, whose genes are arranged in chromosomal order in the class I
, II and III MHC genes. Class I gene is HLA gene
Which are further subdivided into A, B and C subdivisions. Class II inheritance
The child is subdivided into DR, DQ and DP subdivisions. MHC-DR molecule is the best
Well known, these are macrophages, dendritic cells of lymphoid tissues and epidermis
Appears on the surface of antigen presenting cells such as cells. Class III MHC products look like the complement system
It is expressed in various components, as well as in some non-immune related cells.

[0007] Steroidal and non-steroidal anti-inflammatory drugs (eg, methotrexate)
Numerous therapeutic agents have been developed to treat autoimmune diseases, including various interferons, and inhibitors of prostaglandin synthesis. However, these drugs are toxic except for short-term use, and can cause unwanted side effects. Other therapeutic agents bind and / or tumor necrosis factor (TNF)
, Which inhibit the inflammatory activity of, for example, an anti-TNF-specific antibody or antibody fragment, or a soluble form of the TNF receptor, these agents target proteins on the surface of T cells and are generally associated with antigen presenting cells (APC Hinder interaction. However, therapeutic compositions containing natural folded proteins are often produced, formulated,
Difficult to store and deliver. In addition, the innate heterogeneity of the immune system limits the efficacy of the drug, complicating long-term treatment of autoimmune diseases.

[0008] Thus, there is a need for new drugs that do not have the side effects of the present therapeutics and that fully address the innate heterogeneity of the immune system in order to effectively treat autoimmune and other immune disorders.

[0009]

[References]

[0010]

SUMMARY OF THE INVENTION Surprisingly, Copolymer 1 and the terpolymers of the present invention can be used to treat various autoimmune diseases in heterogeneous patient groups. These terpolymers can inhibit some physiological responses of T cells that attack self-antigens as these diseases progress. In addition, Copolymer 1 and the terpolymers and co-peptides of the present invention have various genetic backgrounds with antigen presenting cells, and some class II that interfere with and attack immune cell recognition.
Binds with high affinity to MHC molecules. In addition, terpolymers and copeptides can stimulate the proliferation and function of copolymer 1-specific T cells, and can also treat and prevent various autoimmune diseases.

Accordingly, the present invention relates to a group of amino acids comprising copolymer 1, ie, copolymer 1
A pharmaceutical composition having a polypeptide comprising three different amino acids selected from glutamic acid, alanine, lysine, and tyrosine in an appropriate relative molar ratio found in the above.

The invention also consists essentially of the amino acids tyrosine, alanine and lysine,
About 0.005 to about 0.250 tyrosine, about 0.3 to about 0.6 alanine, and about 0
. It is also directed to a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 1 to about 0.5 lysine and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of glutamic acid.

[0013] The invention further relates to about 0.005 to about 0.300 glutamic acid, about 0.005 to about 0.250 tyrosine, and about 0.3 to about 0.3 g of glutamic acid, tyrosine and lysine. Provided is a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 7 lysines and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of alanine.

[0014] The present invention also relates to a composition comprising about 0.005 to about 0.25 tyrosine, about 0.005 to about 0.3 glutamic acid, and about 0.005 to about 0 It is also directed to a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 0.8 alanine and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of lysine.

[0015] The present invention also relates to a compound comprising about glutamic acid, alanine and lysine, comprising about 0
. 005 to about 0.3 glutamic acid, about 0.005 to about 0.6 alanine, and about 0
. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of the terpolymer in a molar ratio of 2 to about 0.7 lysine and a pharmaceutically acceptable carrier. Preferably, the terpolymer is substantially free of tyrosine.

The present invention also provides a pharmaceutical composition for treating an autoimmune disease, comprising a therapeutically effective amount of copolymer 1 or polypeptide consisting essentially of glutamic acid, alanine, tyrosine and lysine, and Also provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

The invention further provides a method for treating and preventing an autoimmune disease in a mammal, comprising administering a therapeutically effective amount of a composition comprising Copolymer 1 or a terpolymer. A method comprising: In another aspect, the method for treating an autoimmune disease in a mammal further comprises inhibiting T cell proliferation associated with an immune attack. In another aspect, a method for treating an autoimmune disease in a mammal comprises combining the terpolymer or copeptide with an antigen presenting cell. In yet another embodiment, a method for treating an autoimmune disease in a mammal comprises combining the terpolymer with a major histocompatibility complex class II protein associated with the autoimmune disease.

[0018] Autoimmune diseases contemplated by the present invention include arthritic diseases, demyelinating diseases and inflammatory diseases. For example, autoimmune diseases that can be treated with the present compositions include:
Multiple sclerosis, autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmune uveal retinitis, Crohn's disease, chronic autoimmune thrombocytopenic purpura, colitis, contact Hypersensitivity, diabetes mellitus, Graves' disease, Guillain-Barre syndrome,
If you have Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis, or lupus erythematosus. The compositions can be used to treat one or more of these diseases.

[0019]

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, polypeptides having at least three different amino acids, randomly polymerized in linear form, are useful for treating autoimmune diseases. Autoimmune diseases occur when the immune system is able to respond inappropriately to certain tissues or cells. The polypeptides and peptides of the present invention protect tissues from attack, for example, by inhibiting the growth or activity of T or B cells responsible for the attack, or by binding to MHC proteins on the surface of cells that make up the tissue. This can prevent the immune system from attacking.

The amino acids of the present invention include, but are not limited to, the 20 commonly occurring amino acids. Also included are naturally occurring and synthetic derivatives such as selenocysteine. In addition, amino acids include amino acid analogs. Amino acid "analogs" are defined as chemically similar forms of amino acids having different configurations, such as isomers, or D-configuration rather than L-configuration, or organic molecules containing amino acids of appropriate size and shape, or peptide bonds. An amino acid that has been modified at the relevant atom and is resistant to proteases during polymerization in the polypeptide.

“Amino acid” and “amino acid sequence” as defined herein and in the claims refer to any or all of the one or more of the 20 naturally occurring amino acids represented by the sequence. One or more components that are amino acid derivatives and / or amino acid analogs may be included. For example, in amino acid sequences having one or more tyrosine residues, some of those one or more residues can be replaced with homotyrosine. Furthermore, amino acid sequences having one or more non-peptide or peptide-like bonds between two adjacent residues are included in this definition.

The one- and three-letter amino acid codes (and the amino acids indicated by each) are as follows: A means ala (alanine); C means cys (cysteine); D is asp (asparagine). E means glu (glutamic acid); F means phe (phenylalanine); G means gly (glycine); H means his (histidine); I means ile ( K means lys (lysine); L means leu (leucine); M means m
et means methionine; N means asn (asparagine); P means pr
Q means gln (glutamine); R means arg (arginine); S means ser (serine); T means thr (threonine); W means val (valine); W means trp (tryptophan); and Y means tyr (tyrosine).

A “hydrophobic” amino acid is defined herein as an aliphatic amino acid, 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) (in brackets, one letter of each amino acid and 3
Letter standard code abbreviations), and aromatic amino acids, tryptophan (W or tr
p), phenylalanine (F or phe), and tyrosine (Y or tyr)
) Is defined. When found as residues in proteins, amino acids confer hydrophobicity as a function of aliphatic side chain length and aromatic side chain size.

“Charged” amino acids are defined herein and in the claims as an aqueous solution of a protein containing these residues at physiological pH values (his, lys and arg).
A) aspartic acid (D or asp), glutamic acid (E or glu), histidine (H or his), arginine (R or arg) and lysine (K or lys) which confer charge or negative (asp and gly) charges; Defined.

Polypeptide Compositions Contemplated by the Invention The polypeptides of the invention consist essentially of Copolymer 1 and three of the four amino acids of Copolymer 1, ie, tyrosine, glutamic acid, alanine and lysine. Comprising a terpolymer. However, one skilled in the art can readily substitute structurally similar and / or charge related amino acids without departing from the spirit of the invention. Thus, the present invention further contemplates conservative amino acid substitutions for tyrosine, glutamic acid, alanine and lysine in the polypeptides. Such conservative alternatives are structurally related amino acid alternatives, such as those amino acids that are similar in charge, hydrophobicity and size to tyrosine, glutamic acid, alanine or lysine. For example, lysine is structurally related to arginine and histidine; glutamic acid is structurally related to aspartic acid; tyrosine is structurally related to serine, threonine, phenylalanine and tryptophan; and alanine is valine, leucine. And is structurally related to isoleucine. These and other such conservative alternatives and structurally related synthetic amino acids are contemplated by the present invention.

Further, the terpolymer may be composed of 1- or d-amino acids. As known to those skilled in the art, 1-amino acids are present in most natural proteins. However, d-amino acids are commercially available and may be used in place of some or all of the amino acids used to make the terpolymer. The present invention contemplates terpolymers formed from a mixture of d- and l-amino acids, as well as terpolymers consisting essentially of either l- or d-amino acids.

The average molecular weight and average mole fraction of amino acids in the terpolymer can vary. However, it is believed that the range of average molecular weight is from about 2,000 to about 40,000 daltons. Preferred average molecular weight ranges and terpolymer preparation processes are described in US Pat. No. 5,800,808, which is incorporated herein by reference.

In one aspect, the invention provides a terpolymer comprising tyrosine, alanine and lysine. The average mole fraction of amino acids in these terpolymers can vary, for example, tyrosine is present in a mole fraction of about 0.005 to about 0.250; Present in a molar fraction of 0.6;
It may be present in a mole fraction of about 0.5. The average molecular weight is about 2,000 to about 40,0
00 daltons, preferably between about 3,000 and about 35,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,
000 daltons.

In another aspect, the invention provides a terpolymer comprising tyrosine, glutamic acid and lysine. The average mole fraction of amino acids in these polypeptides can vary, for example, glutamic acid is present in a mole fraction of about 0.005 to about 0.300; tyrosine is present in about 0.005 to about 0.250. And lysine may be present in a mole fraction of about 0.3 to about 0.7. Average molecular weight is about 2
Between about 3,000 and about 40,000 daltons, preferably between about 3,000 and about 3
Between 5,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,000 daltons.

In another aspect, the invention provides a terpolymer comprising glutamic acid, alanine and lysine. The average molar fraction of amino acids in these polypeptides can vary, for example, glutamic acid is present in a molar fraction of about 0.005 to about 0.300; alanine is present in about 0.005 to about 0.600. And lysine may be present in a mole fraction of about 0.2 to about 0.7. Average molecular weight is about 2
Between about 3,000 and about 40,000 daltons, preferably between about 3,000 and about 3
Between 5,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,000 daltons.

In another aspect, the invention provides a terpolymer comprising tyrosine, glutamic acid and alanine. The average mole fraction of amino acids in these polypeptides can vary, for example, tyrosine is present in a mole fraction of about 0.005 to about 0.250; glutamic acid is present in about 0.005 to about 0.300. Present in a mole fraction of
And alanine may be present in a mole fraction of about 0.005 to about 0.800. The average molecular weight is between about 2,000 and about 40,000 daltons, preferably about 3,
Between 000 and about 35,000 daltons. In a more preferred embodiment, the average molecular weight is between about 5,000 and about 25,000 daltons.

In a further preferred embodiment, the molar fraction of amino acids in the terpolymer is also preferred for copolymer 1. The molar fractions of amino acids in copolymer 1 are glutamic acid (about 0.14), alanine (about 0.43), tyrosine (about 0.10) and lysine (about 0.34). The most preferred average molecular weight of Copolymer 1 is about 5,
Between 000 and about 9,000 daltons.

A more preferred terpolymer has a molar ratio of monomer, glutamic acid, alanine and tyrosine of about 0.21: about 0.65: about 0.14.

A more preferred terpolymer has a molar ratio of monomers, glutamic acid, alanine and lysine of about 0.15: about 0.48: about 0.36.

A more preferred terpolymer monomer, glutamic acid, tyrosine and lysine molar ratio is about 0.26: about 0.16: about 0.58.

A more preferred terpolymer has a molar ratio of monomers, tyrosine, alanine and lysine of about 0.10: about 0.54: about 0.35.

In one embodiment, the terpolymers of the invention are capable of binding MHC class II proteins, preferably associated with autoimmune diseases. Using any available method, one can determine whether a terpolymer binds to one or more MHC class II proteins. For example, after labeling a polypeptide with a reporter molecule (such as a radionuclide or biotin), mixing with a crude or pure MHC class II protein preparation and removing unbound polypeptide, the reporter molecule may be labeled with an MHC class II protein. If it is in contact with the protein, binding is detected.

[0038] In another embodiment, the terpolymers of the invention are capable of binding to MHC class II proteins associated with multiple sclerosis. The polypeptide of this embodiment has a similar or higher affinity to the antigen binding groove of the MHC class II protein associated with multiple sclerosis than Copolymer 1. Thus, contemplated polypeptides can inhibit the binding of myelin autoantigen or displace that binding from MHC class II proteins. One MHC class associated with multiple sclerosis
The II protein is HLA-DR4 (DRB1 ^* 1501).

In another embodiment, Copolymer 1 of the present invention, and terpolymers, are capable of binding MHC class II proteins associated with arthritic diseases, such as rheumatoid arthritis or osteoarthritis. The copolymer 1 or terpolymer of this embodiment has a higher affinity for the antigen-binding groove of the MHC class II protein associated with an autoimmune disease than the type II collagen 261-273 (SEQ ID NO: 3) peptide. are doing. Thus, the intended copolymer 1, or terpolymer, can inhibit the binding of type II collagen 261-273 peptide or replace it from the antigen-binding groove of MHC class II protein. Class II MHC proteins consist of approximately equal-sized α and β subunits, both of which are transmembrane proteins. The peptide bond cross-section is created by the amino terminal portion of both the α and β subunits. This peptide bond cross-section is the presentation of the antigen to T cells. There are at least three types of class II MHC molecules: HLA-DR, HLA-DQ and HLA-DP molecules. There are also numerous alleles that encode each type of these HLA molecules. Class II MHC molecules have excellent expression on the surface of antigen presenting cells such as B lymphocytes and macrophages.

The terpolymer of the present invention can be formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents,
Any or all of a sweetener and the like are included. Pharmaceutically acceptable carriers include, but are not limited to, a wide variety of materials such as flavorings, sweeteners and buffers and absorbents required to prepare certain therapeutic compositions. May be prepared from a wide range of materials.

The combination of such materials with pharmaceutically active substances is well known in the art.
Except insofar as any conventional material or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

The compositions of the present invention may be formulated as injectable solutions or suspensions, spray solutions or suspensions.

Therapeutic Methods Contemplated by the Invention The present invention is further directed to a method for treating and preventing an autoimmune disease in a mammal, comprising a copolymer 1, ie, an amino acid containing glutamic acid, tyrosine, lysine and alanine. Administering a therapeutically effective amount of a composition comprising at least three different amino acids, randomly polymerized in linear form, selected from the group consisting of: Providing a method comprising: In one embodiment, the polypeptide is Copolymer 1 or a terpolymer.

Autoimmune diseases contemplated by the present invention include either cell mediated diseases (eg, T cells) or antibody mediated (eg, B cell) disorders. Such disorders are, inter alia, arthritis, demyelination and inflammatory diseases. For example, autoimmune diseases that can be treated by the polypeptides include multiple sclerosis, autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmune uveo-retinitis, Crohn's disease, chronic Autoimmune thrombocytopenic purpura, colitis, contact hypersensitivity,
Diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic myxedema,
Examples include myasthenia gravis, psoriasis, pemphigus vulgaris, rheumatoid arthritis or systemic lupus erythematosus. The compositions can be used to treat one or more of these diseases.

As used herein, “arthritis disease” refers to a condition in which at least one symptom of rheumatoid arthritis affects at least one joint of a mammal, eg, the scapula, knee, hip, hip, spine or toes of the mammal. It is a disease seen. Examples of arthritis diseases are "polyarthritis", an arthritis disease affecting multiple joints, "juvenile arthritis", an arthritis disease in humans under the age of 21, and neutropenia, splenomegaly, weight loss, anemia It may include Felty's syndrome, which includes symptoms of sickness, lymph node damage as well as pigment spots on the skin.

In one embodiment, the intended polypeptide is an MH associated with an autoimmune disease
Any autoimmune disease can be treated with the polypeptide, as long as it binds to the C class II protein. One aspect of this embodiment is that antigenic peptides and MHC classes
A method comprising selecting a polypeptide that inhibits binding to a II protein, e.g., where step (a) is a class II for an MHC class II protein-peptide complex
Provided is a method further comprising selecting a heteropolymer that inhibits a specific T cell response, and wherein the antigenic peptide is associated with an autoimmune disease. In another aspect of the invention, the MHC class A method is provided wherein the II protein is associated with an autoimmune disease.

In another embodiment, the method for treating an autoimmune disease in a mammal further comprises inhibiting the proliferation or function of T cells responsive to the self antigen. RA is a T-cell mediated autoimmune disease that can be treated by the polypeptide. Autoimmune diseases and the pathological progression of the immune response are mediated by T cells. Upon binding and recognition of the antigen, the T cells proliferate, secrete cytokines, and additional inflammatory and cytotoxic cells increase at the site. The polypeptides inhibit T cell proliferation and T cell function, such as secretion of cytokines and proliferation to sites of inflammatory and cytotoxic cells. When the autoimmune disease is an arthritis disease, the self antigen is collagen, and the polypeptide can inhibit the proliferation and function of collagen-responsive T cells.

In another embodiment, the method for treating an autoimmune disease in a mammal comprises combining the polypeptide with an antigen presenting cell such as a macrophage, a dendritic cell of lymphoid tissue or an epidermal cell. included. When the appropriate antigen is presented, T cell proliferation and function is activated. It is believed that the polypeptide arrests or otherwise interferes with T cell activation by binding to antigen presenting cells.

[0049] In yet another embodiment, the method for treating an autoimmune disease in a mammal comprises binding the polypeptide to a major histocompatibility complex class II protein associated with the autoimmune disease. . Class II MHC proteins are well expressed on the surface of antigen presenting cells such as B lymphocytes and macrophages. These class II MHC proteins have a peptide binding cross-section where the antigenic peptide is presented to T cells. When the polypeptides bind to the major histocompatibility complex class II proteins, these polypeptides can stop or otherwise interfere with antigen presentation and / or T cell activation.

In another embodiment, the method for treating an autoimmune disease in a mammal comprises combining the polypeptide with a copolymer 1 reactive B cell antibody and / or a copolymer 1 reactive T cell. To be included. Copolymer 1 reactive _TH2 /
3T cells promote the therapeutic effect of Copolymer 1. By binding to Copolymer 1 reactive T cells, the polypeptides stimulate their T cell proliferation, secretion of anti-inflammatory cytokines, increasing the therapeutic benefit of treatment with the present methods. According to the present invention,
The polypeptides also bind to autoantigen-reactive antibodies and stop the antibodies from attacking the target tissue, thereby helping to prevent the development of an autoimmune disease. For example, if the polypeptide binds to an MBP-specific antibody, it will not bind to these antibodies MBP, thereby resulting in destruction of the myelin myelin MBP.

The polypeptide may be administered by any convenient route. In one embodiment, the polypeptide can be administered by injection to facilitate delivery to a tissue affected by the autoimmune disease. Thus, the polypeptides may be for example injected, ingested, inhaled or topically applied. The polypeptide of interest may be mixed with a cream, solution or suspension for topical administration. The polypeptide is preferably administered orally, topically or by injection without the addition of an adjuvant.

Useful Kits of the Invention Another aspect of the invention is a kit for assaying the binding of an analyte to an MHC protein, comprising a recombinantly produced water-soluble MHC produced in a non-mammalian cell.
A kit comprising a protein, means for detecting an analyte bound on the MHC protein, and instructions for use is provided. The MHC protein used in the kit was M
An MHC class II protein selected from the group consisting of HC class II HLA-DR1 protein, MHC class II HLA-DR2 protein and MHC class II HLA-DR4 protein. The kit may further comprise an autoantigenic peptide.

In a preferred embodiment, the MHC class II protein is produced in an invertebrate or bacterial cell, such as an insect or yeast cell, and thus has no peptide attached at the antigen cross section. Means for detecting the binding of an analyte to an MHC protein include radiation, fluorescence measurements, and methods commonly known in the art.
It may be a chemiluminescent, enzymatic or colorimetric means. In a preferred embodiment, the MHC protein is a class II HLA-DR1 or HLA-DR4
It is a protein. Further, the kit may comprise an autoantigenic peptide, such as a collagen II peptide or a peptide from myelin basic protein, a myelin poor dendritic protein, or a peptide from some other protein involved in an autoimmune disease. .

Synthesis of the Terpolymer of the Invention The terpolymer can be produced by any technique available to those skilled in the art. For example, the terpolymer can be prepared using dissolved amino acids at the desired mole fraction or under solid-state synthesis techniques under condensation conditions. The condensation conditions include a suitable temperature, pH and solvent conditions for condensing the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond. Condensing agents, such as dicyclohexylcarbodiimide, may be used to facilitate the formation of peptide bonds. Protecting groups may be used to protect the side-chain moieties and functional groups such as some amino or carboxyl groups to provide for unwanted side reactions.

For example, in the process disclosed in US Pat. No. 3,849,550, the N-carboxy anhydride of tyrosine, alanine, γ-benzylglutamate and N-trifluoroacetyl-lysine are dissolved in anhydrous dioxane. The addition of diethylamine as an initiator and polymerization at ambient temperature can be used. The γ-carboxyl group of glutamic acid can be deprotected with hydrogen bromide in glacial acetic acid. The trifluoroacetyl group can be removed from lysine with 1 molar piperidine. By selectively eliminating reactions related to any one of glutamic acid, alanine, tyrosine or lysine, the process can be carried out in Copolymer 1 with a desired amino acid, for example,
It can be readily appreciated by one of skill in the art that peptides and polypeptides containing only three of the four amino acids can be tailored to produce. For purposes of application, "ambient temperature" and "room temperature" are between about 20 to about 26 degrees Celsius (° C).
).

The average molecular weight of the terpolymer can be adjusted during polypeptide synthesis or after the terpolymer is prepared. To adjust the average molecular weight during polypeptide synthesis, the conditions or amounts of amino acid synthesis are adjusted so that synthesis stops when the polypeptide reaches the desired desired length. After synthesis, a polypeptide having the desired average molecular weight can be obtained by any available size selection procedure, for example, by chromatography of the polypeptide on a molecular weight sizing column or gel and collection of the desired average molecular weight range. The polypeptide may also be partially hydrolyzed, for example by hydrolysis with an acid or an enzyme, to eliminate high molecular weight species and later purified to eliminate the acid or enzyme.

In one embodiment, the present invention is a method of producing a terpolymer having a desired average molecular weight, comprising reacting a protected polypeptide with hydrobromic acid to obtain a desired average molecular weight profile. Methods comprising forming a polypeptide having the same. The reaction is performed at a time and temperature predetermined by one or more test reactions. During the test reaction, the time and temperature are varied to determine the average molecular weight range for a given batch of test polypeptide. The test conditions that give the optimal average molecular weight range for the batch of polypeptide are used for the batch. Thus, a polypeptide having the desired average molecular weight profile can be produced by a method comprising reacting the protected polypeptide with hydrobromic acid at a predetermined time and temperature in a test reaction.

In a preferred embodiment, a test sample of a given batch of protected polypeptide is reacted with hydrobromic acid at about 20-28 ° C. for about 10-50 hours. Perform the test reaction several times to determine the best conditions for the batch. For example, in one embodiment, the protected polypeptide is reacted with hydrobromic acid at about 26 ° C. for about 17 hours.

The following examples describe the invention in detail in terms of how certain particular embodiments thereof may be made, but the materials, equipment and processing steps are merely described. It is understood that the examples are intended to be illustrative. In particular, the invention does not limit the methods, materials, conditions, processing parameters, equipment, etc. described herein.

In this application, various publications, patents and patent applications are referenced. The teachings and disclosures of these publications, patents and patent applications are all incorporated herein by reference and fully describe the state of the art to which this invention pertains.

It should be understood and assumed by those skilled in the art that variations may be made on the principles of the invention disclosed herein, and such modifications are intended to be included within the scope of the invention.

[0062]

EXAMPLES Preparation protective polypeptide for manufacturing protective polypeptide of Example 1 polypeptides as described by Teitelbaum et el., Was dissolved in dioxane, amino acids tyrosine were blocked with N- carboxyanhydride (NCA), Prepared with alanine, glutamic acid and trifluoroacetyl lysine. 1
EUR. J. IMMUN. 242 (1971). The carboxylation group of glutamic acid is replaced with benzyl (BZ).
Block with a group.

The polymerization process is started by adding 0.01-0.02% diethylamine. The reaction mixture is stirred at room temperature for 20 hours and then poured into water. The protected polypeptide product is filtered, washed with water and dried. Removal of the γ-benzyl blocking group from glutamic acid residues can be achieved by protecting the protected polypeptide with 33% hydrobromic acid in glacial acetic acid at room temperature for 6-1
This is performed by treating with stirring for 2 hours. The product is poured into excess water, filtered, washed and dried to give the protected polypeptide.

Preparation of a Polypeptide Having a Molecular Weight of 7,000 ± 2,000 Da Treatment of a γ-benzyl protected polypeptide with 33% HBr in acetic acid allows the removal of a benzyl protecting group from the γ-carboxylate of a glutamic acid residue. Not only is removal guaranteed, but the polypeptide is cleaved into smaller polypeptides.

The time required to obtain a polypeptide having a molecular weight of 7,000 ± 2,000 daltons depends on the reaction temperature and the size of the protected polypeptide. The test reaction of each batch is carried out at a temperature between 20 and 28 ° C. for various times, for example 10 to 50 hours. Subtract the average molecular weight curve obtained over time. Molecular weight 7,000 ± 2,000
The time required to obtain ODa is calculated from the curve and the reaction is performed on a large scale.
On average, at 26 ° C., the time to obtain a mixture of polypeptides having a molecular weight of 7,000 ± 2,000 Da is 17 hours. The product is poured into excess water, filtered, washed and dried to yield a polypeptide having the desired average molecular weight range.

Preparation of Low Toxic Lysine Containing Polypeptide Protected trifluoroacetyl polypeptide (20 g) is dispersed in one liter of water and 100 g of piperidine is added. The mixture is stirred at room temperature for 24 hours and filtered. Dispense the unpurified polypeptide solution into dialysis bags and allow 1 until pH 8 is reached.
Dialyze against water at 0-20 ° C. The polypeptide solution is dialyzed against 0.3% acetic acid and then dialyzed again with water until the pH becomes 5.5 to 6.0. The solution is then concentrated and lyophilized.

Synthesis of poly (L-Tyr ^0.136 , L-Glu ^0.21 , L-Ala ^0.648 ) having a molecular weight of 7,600 TGA 1) Raw material: 18.74 g of L-Ala-NCA L-Tyr-NCA 6.5 g 5-BZ-L-Glu-NCA 13 g diethylamine 0.165 g dioxane 790 ml HBr / AcOH (33%) 480 ml phenol 4.8 g piperidine 13.2 ml deionized water acetic acid 2) Procedure L-Tyr-NCA (6. 5 g) in dioxane (211 ml) at 60 ° C.
And then filtered. 5-BZ-L-Glu-NCA (13 g) is put in dioxane (226 ml), stirred at 20 to 25 ° C. for 10 minutes, and then filtered. L-Ala-NCA (18.74 g) is placed in dioxane (350 ml), stirred at 20 to 25 ° C. for 10 minutes, and filtered.

An L-Tyr-NCA solution, 5-L was added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
The BZ-L-Glu-NCA solution and the L-Ala-NCA solution are charged. Diethylamine (0.165 g) in dioxane (2.5 ml) is charged to the reaction mixture and stirred at 20-25 ° C. for 20 hours. Add the reaction mixture to deionized water (1 L), filter and dry at 60 ° C. in vacuo. Yield -25.8 g.

A solution of phenol (4.8 g) in HBr / AcOH (33%; 480 ml) is stirred for 20 hours. HBr / A in 2L Erlenmeyer equipped with magnetic stirrer
A solution of cOH and poly [5-BZ-L-Glu, L-Ala, LL-Tyr] is charged and stirred at 26 ± 1 ° C. for 16 hours. The reaction mixture is deionized water (2 L)
And stir for 1 hour. The precipitate is filtered and washed with deionized water until the pH is 6. The product is dried in a circulating air oven at 40 ° C. for about 40 hours. Yield-24
g.

Piperidine (13.2 ml) in a 2 L Erlenmeyer equipped with a magnetic stirrer,
Deionized water (1200 ml) and poly [L-Glu, L-Ala, LLT
After stirring, the mixture was stirred at 20 to 25 ° C for 24 hours. The resulting solution is ultrafiltered through a 5000 dalton polyethersulfone membrane until two thirds of the original volume has been eliminated. Add fresh deionized water and return to original volume. HPL
This step is repeated 5 times until the impurity content is less than 1% by C. The resulting solution (total) is acidified with acetic acid to pH 4.4. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

Synthesis of molecular weight 8850GAL, poly [L-Glu ^0.153 , L-Ala ^0.479 , L-Lys ^0.365 ] 1) Raw material: L-Ala-NCA 14g N6-TFA-L-Lys-NCA 22.9 g 5-BZ-L-Glu-NCA 9.8 g diethylamine 0.12 g dioxane 850 ml HBr / AcOH (33%) 480 ml phenol 4.8 g piperidine 13.2 ml deionized water acetic acid 2) Procedure in dioxane (420 ml) N6-TFA-L-Lys-NCA (22.9
g) is stirred at 20-25 <0> 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) was added to 2
Stir at 0-25 ° C for 10 minutes and filter. N6-TFA-L-Lys-NCA solution, 5-BZ-L-Glu-NC in a 2 L Erlenmeyer equipped with a magnetic stirrer
A solution and L-Ala-NCA solution are charged. Diethylamine (0.12 g) in dioxane (2.5 ml) is added and the reaction mixture is stirred at 20-25 ° C. for 20 hours. Add the reaction mixture to deionized water (1 L), filter and dry at 60 ° C. in vacuo.

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

Piperidine (13.2 ml) was placed in a 2 L Erlenmeyer equipped with a magnetic stirrer,
Deionized water (1200 ml) and poly [L-Glu, L-Ala, N6-TF
[A-L-Lys], and then stirred at 20 to 25 ° C for 24 hours. The solution is ultrafiltered on a 5000 dalton exclusion limit polyethersulfone membrane until two thirds of the original volume has been eliminated. Add fresh deionized water and return to original volume.
This process is repeated 5 times (by HPLC) until the impurity content is less than 1%.
The resulting solution (total) is acidified with acetic acid to pH 4.4. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

[0074] Molecular weight 11,050TGL, poly ^{[L-Tyr 0.162, L-} Glu 0.2 59, L-Lys 0.579] Synthesis 1) Raw material: 5-BZ-L-Glu -NCA 10.34g N6-TFA-L-Lys-NCA 24.16 g L-Tyr-NCA 5.2 g Diethylamine 0.095 g Dioxane 810 ml HBr / AcOH (33%) 460 ml Phenol 4.6 g Piperidine 150 ml Deionized water Acetic acid 2) Procedure Dioxane (180 ml) L-Tyr-NCA (5.2 g) in
Heat for 0 minutes and filter. N6-TFA-LL in dioxane (450 ml)
Stir ys-NCA (24.16 g) at 20-25 ° C. for 10 minutes and filter. 5-BZ-L-Glu-NCA (10.35 g) in dioxane (180 ml) is stirred at 20-25 ° C. for 10 minutes and filtered.

N6-TFA-L-Lys-N was added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
A CA solution, an L-Tyr-NCA solution and a 5-BZ-L-Glu-NCA solution are charged. Charge diethylamine (0.095 g) in dioxane (2.5 ml) and stir the reaction mixture at 20-25 <0> C for 20 hours. Add the reaction mixture to deionized water (0.7 L), filter and dry at 60 ° C. in vacuo.

A solution of phenol (4.6 g) in HBr / AcOH (33%; 460 ml) is stirred for 20 hours. HBr / A in 2L Erlenmeyer equipped with magnetic stirrer
cOH and poly [5-BZ-L-Glu, L-Tyr, N6-TFA-LL
ys]. The mixture is stirred at 26 ± 1 ° C. for 16 hours, then the mixture is added to deionized water (1.5 L) and stirred for １ ／ hour. Filter the precipitate, pH
Wash with deionized water until pH is 5.5.

In a 2 L Erlenmeyer equipped with a magnetic stirrer, piperidine (150 ml), deionized water (1400 ml) and poly [L-Glu, L-Tyr, N6-TFA
-L-Lys] (50 g) and stirred at 20 to 25 ° C for 24 hours. Poly [
[L-Glu, L-Tyr, L-Lys] was obtained.
Is ultrafiltered through a 5000 dalton polyethersulfone membrane until exclusion is achieved. Return to original volume by adding fresh deionized water. This process is repeated 5 times (by HPLC) until the impurity content is less than 1%. The resulting solution (total) is acidified with acetic acid to pH 4.2. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

[0078] Molecular weight 20,000TAL, the synthesis of poly ^{[L-Tyr 0.102, L-} Ala 0.5 42, L-Lys 0.353] 1) material: L-Ala-NCA 14g N6 -TFA-L- Lys-NCA 22.9 g L-Tyr-NCA 4.9 g Diethylamine 0.1 g Dioxane 850 ml HBr / AcOH (33%) 500 ml Phenol 5 g Piperidine 162 ml deionized water acetic acid 2) Procedure L-Tyr-NCA in dioxane (170 ml) (4.9 g) at 60 ° C.
Heat for 0 minutes and filter. N6-TFA-LL in dioxane (417 ml)
Stir ys-NCA (22.9 g) at 20-25 ° C. for 10 minutes and filter. L-Ala-NCA (14 g) in dioxane (260 ml) was added at 20-25 ° C for 10
Stir for minutes and filter.

N6-TFA-L-Lys-N was added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
Charge CA solution, L-Tyr-NCA solution in dioxane and L-Ala-NCA solution. Charge diethylamine (0.1 g) in dioxane (2 ml),
The mixture is stirred at 20-25 ° C for 20 hours. The reaction mixture is deionized water (1 L)
), Filter and dry in vacuo at 60 ° C.

A solution of phenol (5 g) in HBr / AcOH (33%; 500 ml) was
Stir for 0 hours. HBr / AcO in 2L Erlenmeyer equipped with magnetic stirrer
A solution of H and poly [L-Ala, L-Tyr, N6-TFA-L-Lys] is charged and stirred at 26 ± 1 ° C. for 16 hours. The reaction mixture was then washed with deionized water (2 L).
) And stir for 1/2 hour. The precipitate is filtered and washed with deionized water until the pH is 5.5.

Piperidine (162 ml), water (1500 ml) and poly [L-Ala, L-Tyr, N6-TFA-LL were added to a 2 L Erlenmeyer equipped with a magnetic stirrer.
ys] (30 g) and stirred at 20 to 25 ° C for 24 hours. Poly [L-Al
a, L-Tyr, L-Lys] is ultrafiltered through a polyethersulfone membrane with an exclusion limit of 5000 daltons until two thirds of the original volume has been eliminated. Return to original volume by adding fresh deionized water. Impurity content is 1 (by HPLC)
This step is repeated 5 times until it is less than%. PH of the resulting solution (total) with acetic acid
Acidify to 4.4. Ultrafiltrate the solution until the pH is 5.5-6 and reduce the volume to one third. The resulting solution is lyophilized.

Example 2 In Vivo Testing of Low Molecular Weight Polypeptides Average molecular weights of 7.3 and 8.4 KDa (one copolymer greater than 40 KDa
. Three batches of Copolymer 1 having less than 5%) and 22 KDa (more than 5% of one copolymer greater than 40 KDa) were subjected to the toxicity tests described below. Five mice were used in each test group.

Method Copolymer 1 is dissolved in distilled water to obtain a solution of 2 mg / ml of the active ingredient. Each mouse is injected with 0.5 ml of the test solution into the lateral tail vein. The number of deaths and associated clinical signs of the mice for 48 hours were observed. 10 minutes, 24 hours and 48 after injection
Observations were recorded at time. At the end of 48 hours, if all animals are alive and show no bad signs, the batch is considered "non-toxic." However, if one or more mice die or show bad symptoms, the batch is considered "toxic."

Results When treated with Copolymer 1 polypeptide having an average molecular weight of 22 KDa, three of the five mice died after 48 hours. Therefore, this batch with a high average molecular weight is referred to as
Toxicity. One batch of copolymer having an average molecular weight of 7.3 and 8.4 KDa was both "non-toxic".

In Vitro Rat Basophilic Leukemia Cell Degranulation Assay Histamine (or serotonin) release from basophils is an in vitro model of immediate hypersensitivity. Rat basophilic leukemia cell line, RBL-2H ₀ is homogeneous, but it is easy to maintain in culture, there is a high sensitivity to test for degranulation, and a reproducible certain systems. EL Basumian, et al., 11 EUR. J.
IMMUNOL. 317 (1981). Physiological stimuli for histamine release include the association of antigens with membrane-bound IgE molecules that trigger a complex biochemical cascade. Degranulation is induced by non-IgE-mediated stimuli including various polypeptides and synthetic polymers such as polylysine. RP Siraganian, TRENDS IN PHARMACO
LOGICAL SCIENCES 432 (Oct, 1983). Thus, using the RBL degranulation test, a batch of that COP-1 that can cause substantial degranulation, thereby inducing unwanted local and / or systemic side effects. To select.

Methods Rat basophil leukemia cells (RBL-2H ₀ ) are fed [H ³ ] -serotonin and then incubated with 100 μg of COP-1 to be tested. The COP-1 batch to induce non-specific degranulation in medium [H ^3] - releasing serotonin. Radioactivity in the medium is measured with a scintillation counter, and total radiolabeled serotonin incorporated into the cells is determined in the pellet cells. % Degranulation is calculated as the percentage of serotonin released from all incorporated serotonin.

Results Four batches of Copolymer 1 with an average molecular weight between 8,250 and 14,500 were analyzed for both% of species above 40 kDa and RBL degranulation. The results are summarized in Table 1.

Table 1

[Table 1] As can be seen, when the percentage of the high average molecular weight species is low (less than 2.5%), the% serotonin release is low, and vice versa. These data indicate that a copolymer 1 polypeptide with a lower average molecular weight is preferred over a copolymer 1 polypeptide with a higher average molecular weight.

Example 3 Inhibition of EAE by Polypeptide Experimental Allergic Encephalomyelitis (EAE) can be inhibited by injecting Copolymer 1 in Freund's incomplete adjuvant before disease induction. This inhibition is believed to T _{H 2} type of copolymer 1-specific suppressor T cells that cross-react with myelin basic protein is mediated. Lando et al., 123 J. IMMUNOL.
2156 (1979); Aharoni et al., 17 EUR.J. IMMUNOL. 23 (1993); Aharoni et a
l., also 94 PROC. NATL. ACAD. SCI . USA 10821 (1997). that other researchers are associated with the therapeutic effect of Copolymer 1 also T _{H 2} cell induction in patients with multiple sclerosis Admit. Lahat et al., 244 J. Neuro. 129 (1997). In this example, EAE is inhibited by various polypeptides of the present invention.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

Ternary Copolymers Four terpolymers are available from Teva Pharmaceutical Industries (Petach Tikva, Israe).
Obtained from l). The properties of these terpolymers are as follows.

1) GAL terpolymer (SD-1689) is a polypeptide mixture containing the following molar fractions of the amino acids glutamic acid (0.153), alanine (0.479) and lysine (0.365) is there. The GAL average molecular weight ranges from about 4650 daltons to about 20,050 daltons. The average molecular weight of this GAL preparation is 8850 daltons.

2) TGA terpolymer (SD-1690) has the following molar fractions of the amino acids tyrosine (0.136), glutamic acid (0.210) and alanine (0.648)
And a polypeptide mixture comprising: The TGA average molecular weight ranges from about 1000 Daltons to about 40,000 Daltons. The average molecular weight of this TGA preparation is 7600
Dalton.

3) TAL terpolymer (SD-1691) is a polypeptide mixture containing the following molar fractions of the amino acids tyrosine (0.102), alanine (0.542) and lysine (0.353) is there. The TAL average molecular weight ranges from about 5700 daltons to about 34,400 daltons. The average molecular weight of this TAL preparation is 20,000 daltons.

4) GTL terpolymer (SD-1697) is a polypeptide mixture containing the following molar fractions of the amino acids glutamic acid (0.259), tyrosine (0.162) and lysine (0.579) is there. The GTL average molecular weight ranges from about 4,000 daltons to about 23,500 daltons. The average molecular weight of this GTL preparation is 1105
0 Daltons.

A control polypeptide consisting of a polypeptide mixture comprising a 1: 1: 1 mixture of the amino acids alanine, glutamic acid and tyrosine and having an average molecular weight of 26,700 Da is used. This polypeptide was obtained from Sigma Chemical Company (St. Louis, MO).
Obtain from

Induction of EAE 4 mg / ml H in female (SJL / J × BALB / c) FI mice 2 to 3 months old
Mouse spinal cord homogenate (3.5 mg / mouse) emulsified at a 1: 1 ratio with Freund's complete adjuvant (CFA) supplemented with 37Ra is injected into all four footpads. Immediately after and 48 hours later, pertussis toxin (0.25 ml, 250 ng, Sigma)
Is injected intravenously. Mice are examined daily for clinical signs of EAE from day 10 post-challenge and evaluated on a scale of 0-5 as described by Lando et al., 123 J. IMMUNOL. 2156 (1979).

Inhibition of EAE by Injection of Incomplete Adjuvant The polypeptide to be tested (10 mg / mouse) is injected subcutaneously in Freund's incomplete adjuvant (ICFA) into a few of the nodal regions. EAE is induced after 3 weeks as described above.

EAE Inhibition by Oral Administration In a second study, female Lewis rats were given 5 mg / kg guinea pig BP or co-administered in phosphate buffered saline (PBS) at intervals of 2 to 3 days before EAE induction. Polymer 1 is provided. EAE is 4 mg / ml Mycobacterium tuberculosis (H37Ra) (Difco, Lab, Det.
(Roit, Mich.) and induced 2 days after final feeding by injection of 25 μg guinea pig MBP emulsified 1: 1 with CFA. 0 total capacity in each of the two hind footpads
. Inject 1 ml. Control rats are mock receiving phosphate buffered saline.

The effect of oral administration of Copolymer 1 on the prevention of EAE in rats was determined by Higgins et al.
al., 140 J. IMMUNOL. 440 (1988) and compared to rats given guinea pig MBP.

Mice were examined daily for symptoms from day 10 post-challenge. EAE is evaluated as follows: 0 = no symptoms; 1 = fragile tail; 2 = hind limb paralysis; 3 = all limbs paralyzed;
= Moribund condition; and 5 = dead.

Results Inhibition of EAE by Injection of Incomplete Adjuvant Table 2 shows the effect of injecting the polypeptide of the present invention by ICFA before EAE induction. Of the four polypeptides tested, Copolymer 1 and TAL resulted in maximum EAE suppression. GTL also showed excellent inhibitory activity. GAL
And TGA were somewhat less potent.

Table 2 EAE suppression in mice by injection of the polypeptide of the invention

[Table 2] * MMS = average highest point D-copolymer 1 is d-lysine, d-tyrosine, d- in the molar ratio of copolymer 1.
It is a copolymer of glutamic acid and d-alanine.

Table 3 compares the efficacy of orally administered Copolymer 1 in preventing clinical signs of EAE in Lewis rats compared to rats receiving phosphate buffered saline (PBS) alone or guinea pig basic protein (GPBP). Is shown.

Table 3 EAE suppression in rats by oral administration of the polypeptide of the present invention

[Table 3] Each number is the cumulative result of three to five independent tests. The p value is the control (P
BS) shows the statistical significance of the difference from the group. The average highest score is calculated for all groups.

These data indicate that the polypeptides of the present invention are therapeutically effective when administered either orally or by infusion to reduce the signs and severity of EAE.

Example 4 Binding to Antigen Presenting Cells Some of the polypeptides efficiently bind to live antigen presenting cells.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

Biotinylation of Antigen Biotinylation of Copolymer 1 and the terpolymer are described in Frydkis-Hareli et al. 91 PRO.
Perform at 0 ° C. with biotin-N-hydroxysuccinimide (Sigma) according to C. NATL. ACAD. SCI. USA 4872 (1994).

Binding of Biotinylated Antigen to Antigen Presenting Cells Binding of biotinylated polypeptide to live antigen presenting cells of mouse and human origin was examined using fluorescently labeled streptavidin and FACS analysis. (SJ
L / J × BALB / c) adherent spleen cells from FI mice, or DR7. wII
EBV-transformed human B cells of haplotype ^{(1 × 10 8 / 100μl)} 0.1
The mixture was incubated at 37 ° C. for 20 hours with 50 μg of biotinylated copolymer 1 or ternary copolymer dissolved in 100 μl of PBS containing% BSA. Cells were then incubated with phycoerythrin-conjugated streptavidin (Jackson Immuno Research, West Grove, PA) at a cell suspension concentration of 0.5 μg / 100 μl at 4 ° C. for 3 hours.
Incubated for 0 minutes. After each incubation, the cells are
The plate was washed three times with PBS containing% BSA. The cells were then analyzed by flow cytometry using a FACS scan (Becton-Dickinson, Mountain View, CA). For each analysis, 5000 cells were tested. Dead cells show forward and side light scatter (f
orward and side-angle light scatter).

Epstein-Barr Virus (EBV) Transformed B Cell Line The EBV transformed B cell line was isolated from Teitelbaum et al. 89 PROC. NATL. ACAD. SCI.
Induced according to USA 137 (1992). About 20 × 10 ⁶ peripheral blood mononuclear cells were B95.8
The cells were cultured with the cell line supernatant at 37 ° C. for 1 hour. The cells are then washed and
The cells were cultured in RPMI medium containing% FCS and cyclosporin A (10 μg / ml) to deplete T cells.

Results Table 4 shows the binding of terpolymer and Copolymer 1 polypeptide to live antigen-presenting cells, including mouse spleen macrophages and human EBV-transformed B cell lines. Data showing both% binding and cell staining intensity (Table 4 I + II)
. TAL was even better than Copolymer 1 and bound with maximum efficiency. GAL and GTL also bound very successfully, and were the same or somewhat better than Copolymer 1. TGA bound well, but was somewhat inferior to Copolymer 1.

As represented by the bond strength (Table 4), TAL is (SJL / J × BALB)
/ C) _{F 1} mice of splenic macrophages and normal DR7. It bound with maximum efficiency to EBV transformed B cells from w11 donor.

Table 4 Binding of terpolymers to antigen presenting cells Antigen-presenting cells derived from mouse spleen macrophages

[0116]

[Table 4-1] ^* MFI = average fluorescence intensity II. Antigen presenting cells from human EBV transformed B cell line

[0117]

[Table 4-2] ^* MFI = average fluorescence intensity Example 5 This polypeptide binds to purified human leukocyte antigen (HLA) In this example, the polypeptide of the present invention binds to human B cells and HLA-DR1, HL
It shows binding to purified human lymphocyte antigen (HLA) with high affinity, such as A-DR2 and HLA-DR4 molecules.

Methods Cell Lines and Antibodies The homozygous EBV-transformed human B lymphocyte line used for immunoaffinity purification of HLA-DR1, -DR2 and -DR4 molecules was LG-2 (D
RB1), MGAR (DRB1-1501) and Preiss (DRB-04
01 / DRB4-0101).

The cells were 10% FCS, 2 mM glutamine, 50 U / ml penicillin G and 5
The cells were grown in roller bottles containing RPMI 1640 supplemented with 0 μg / ml streptomycin and stored at −80 ° C. as pellets. Anti-DR hybridoma LB3.1 (IgG2b) is grown in serum-free medium (Macrophage-SFM, Gibco BRL). See Gorga et al., 103 CELL. IMMUNOL. 160 (1986).

Protein Purification Immunoaffinity purification of HLA-DR1, -DR2 and -DR4 molecules was
Performed with slight modifications as previously reported by Gorga et al., 262 J. BIOL. CHEM. 16087 (1987). In short, LG-2, MGAR and Pries
S-cell-derived detergent-soluble membrane preparation was applied to a series of columns at a flow rate of about 11 ml / hr in the following order: Sepharose CL-6B (30 ml), normal mouse serum-Af
f-gel 10 (10 ml), protein A-Sepharose CL-4B (5 ml)
And LB3.1-protein A-Sepharose CL-4B (5 ml). Before passage through the LB3.1 immunoaffinity column, DR2a (DRB5 ^* 0
101) and Drw53 (DRB4 ^* 0101), the product of the DR gene associated with the DRB1 allele, were not removed from the MGAR and Priess lysates, and the DR2 and DR4 preparations were contaminated in amounts of 5-10%. Was. All subsequent steps were as previously described by Gorga et al., 262 J. BIOL. CHEM. 16087 (1987). The eluate was washed with 0.1% deoxycholate, 10 mM Tris.
-HCl, dialyzed against pH 8.0, Centriprep 30 membrane (Aamicon
). Protein concentration was determined by the bicinchonitric acid assay (P
ierce Chemical Co.).

Polypeptide and Control Antigen Methods Copolymer 1 One batch of copolymer having an average molecular weight of 5,800 daltons and 8,150 daltons, # 55495 and 52596, respectively, is Teva Pharmaceutical Industri
es (Petach Tikva, Israel). One batch 52596 of the copolymer had a molar ratio of 1 tyrosine: 1.5 glutamic acid: 4.3 alanine: 3.1 lysine.

Myelin Basic Protein and Hemagglutinin Control Peptide The MBP protein was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (1979). The peptides were purified by reverse phase HPLC. The peptide used was the sequence PKYVKQNTLKL
HA306-318 with AT (MW1718) (SEQ ID NO: 2) and sequence D
MBP84-102 with ENPVVHFFKNIVTPRTPP (MW2529) (SEQ ID NO: 1).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

Polypeptide Labeling Biotinylation of various polypeptides is performed as in Example 4. Unreacted biotin is dialyzed (Spectra / Por® membrane MWCO500, Spectrum Medic
al Industries).

Assay for Polypeptides that Bind to Class II MHC Protein Solution: The solution used for this assay is as follows. The binding buffer was 20 mM 2- [N-morpholino] ethanesulfonic acid (ME unless otherwise noted).
S) 1% n-octyl β-D-glucopyranoside, 140 mM NaCl,
0.05% NaN ₃ , pH 5.0. PBS is 150 mM sodium chloride, 7.5 mM sodium phosphate, dibasic, 2.5 mM sodium phosphate, monobasic, pH 7.2. TBS was 137 mM sodium chloride, 25 mM Tr.
is pH 8.0, 2.7 mM potassium chloride. TTBS is 0.0 for TBS
5% Tween-20 was added.

Microtiter assay plate preparation: 96-well microtiter immunoassay plates (PRO-BIND ™, Falcon) were added to PBS (100 total).
1 μg / well of affinity purified LB3.1 monoclonal antibody in μl) at 4 ° C. for 18 hours. The wells were then sealed with TBS containing 3% BSA at 37 ° C. for 1 hour and washed three times with TTBS. 50 μl of TBS / 1% BSA was added to each well before adding samples.

Binding reaction: detergent soluble HLA-DR1, -DR2 and -DR4 molecules (0.
5 μg / sample) in 50 μl of binding buffer at various concentrations with biotinylated peptide at 37 ° C. for 40 h, transfer to prepared microtiter assay plates and 37 ° C. to capture polypeptide-class II complex Incubated for 1 hour.

Inhibition reaction: 1.5 μM final concentration of biotinylated polypeptide in 50 μl binding buffer, unlabeled polypeptide and peptide HA30 used as inhibitor
6-318 (SEQ ID NO: 2) or MBP84-102 (SEQ ID NO: 1), and H
Co-incubated with LA-DR molecule at 37 ° C for 40 hours.

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 of streptavidin-conjugated alkaline phosphatase (1: 3000, BioRad) for 1 hour at 37 ° C., then p-nitrophenyl phosphate in triethanolamine buffer (BioRad) was added. The absorbance at 410 nm is monitored by a microplate reader (Dynatech MR4000).

Results Terpolymer Coupling with Class II MHC Proteins The detergent soluble HLA-DR1, HLA-DR2 and HLA-DR4 proteins were each homozygous EBV transformed B cell line LG-2 (DRB1 ^* 010
1), MGAR (DRB1 ^* 1501) and Priess (DRB1 ^* 040)
Purified from 1) as previously described by Fridkis-Hareli et al. 160 J. IMMUNOL. 4386 (1998). Three different preparations of Copolymer 1 bound to these molecules with high affinity. Id. To determine the affinity of the terpolymer for the HLA-DR protein, a binding assay of the biotinylated terpolymer was performed and compared to Copolymer 1. HLA-DR1, wherein the polypeptide is purified at a range of concentrations,
Incubate with HLA-DR2 and HLA-DR4 molecules at pH 5.0, then capture by class II specific mAbs and alkaline phosphatase-
Detection with streptavidin was performed.

Detergent Soluble HLA-DR1 and HLA-DR4 of TAL and Copolymer 1
The binding to the molecule is better than that of GAL, TGA or GTL. However, GTL and Copolymer 1 were based on the saturation binding curve (FIG. 2A) and the _Kα value calculated from the double reciprocal plot of the binding data (FIG. 2B, Table 5), indicating that other polypeptides were Better binding.

Biotinylated copolymer 1 and the following unlabeled inhibitors: copolymer 1, TAL, T
Competitive binding assays were performed on GA, GAL, TGL, MBP84-102 and HA306-318 polypeptides (FIG. 3). The MBP84-102 (SEQ ID NO: 1) peptide is a poor inhibitor of the binding between Copolymer 1 and HLA-DR2. Binding of biotinylated copolymer 1 to detergent soluble HLA-DR1 and -DR4 molecules is efficiently inhibited by unlabeled TGA, TAL, HA306-318 (SEQ ID NO: 2) and copolymer 1. However, the inhibition by TGL of the binding between biotinylated copolymer 1 and detergent soluble HLA-DR1 and -DR4 molecules is 10-fold lower as indicated by the amount of inhibition (IC ₅₀ ) (FIG. 3, Table 5). Similarly, G
AL is also a poor inhibitor of the binding of Copolymer 1 to HLA-DR1 and -DR4 molecules. Generally, the binding pattern with HLA-DR2 is the same as that seen with HLA-DR1 (Table 5). These results indicate that the three amino acid polypeptides, particularly TAL and TGA, bind to class II MHC molecules having the same affinity range as that of antigenic peptide and copolymer 1. Therefore, T
AL and TGA are effective competitors for Class II MHC molecules that bind Copolymer 1. Based on these binding capacities, the polypeptides are ordered 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> T
GA; (C) Binding to HLA-DR4: TAL> Copolymer 1 >>TGA>GTL>
GAL;

Table 5 Purified human HLA-DR1, -DR2 and -DR of the polypeptide
Affinity for 4 molecules

[Table 5] Copolymer 1 polypeptide having ^one average molecular weight 5,800; molecular weight 20,0
GAL having a molecular weight of 8,850; TGA having a molecular weight of 7,600; and HLA-DR1, -DR obtained by purifying TGL having a molecular weight of 11,050 in a certain concentration range.
2 and -DR4 molecules at pH 5.0, then class II
Capture with a specific mAb and detection with alkaline phosphatase-streptavidin were performed.

^Two detergent soluble HLA-DR1, -DR2 and -DR4 molecules were purified as described in Materials and Methods.

³ K _α , the dissociation constant at equilibrium, was calculated from the slope of the double reciprocal plot (FIG. 2B) and expressed as × 10 ⁻⁸ M.

^{4 The} IC ₅₀ , the inhibitory concentration giving 50% inhibition, was calculated based on the competitive binding assay (FIG. 3) and expressed as × 10 ⁻⁶ M.

[0137] _{The IC 50} values of ⁵ GAL is less than 1000μM could not be accurately determined (see FIG. 3).

Effect of Superantigen on Binding of Polypeptides to HLA-DR Molecules Bacterial superantigens SEA, SEB and TSST-1 inhibit Copolymer 1, which binds purified HLA-DR molecules only at very high concentrations Have been shown to do so. Frid
kis-Hareli et al. 160 J. IMMUNOL. 4386 (1998). Terpolymer and purified HL
To test the effect of these superantigens on binding to A-DR1, HLA-DR2 and HLA-DR4 molecules, competitive binding assays were performed on unlabeled SEA, SEB and TSST-1.

The binding of SEA, SEB and TSST-1 TAL to HLA-DR1, HLA-DR2 and HLA-DR4 is only inhibited by a high molar ratio of superantigen, eg 50-fold to inhibit TAL binding Amounts of superantigen are required (FIG. 4A). However, TGA and G
Binding to AL was more differentially inhibited by the superantigen (FIGS. 4B and C), indicating that these polypeptides bind to the lower affinity HLA antigen.

Example 6 Inhibition of T Cell Proliferation Induced by MBP In this example, it is demonstrated that proliferation of T cells activated by myelin basic protein (MBP) can usually be inhibited by co-exposure to the polypeptide. Is shown.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

[0142] Myelin basic protein peptide was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (1
979). The peptide was purified by reverse phase HPLC. The peptide used was the sequence PKY
HA306-3 having VKQNTLKLAT (MW1718) (SEQ ID NO: 2)
18 and the sequence DENPVVHFFKNIVTPRTPP (MW2529) (SEQ ID NO: 1).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

T Cell Lines and Clones Myelin basic protein (MBP) specific T cells were 4 mg / ml 10 days before
Emulsified with Freund's complete adjuvant to which Mycobacterium tuberculosis H37Ra was added
Originated from the spleen of a mouse immunized with 20 μg of the 84-102 peptide. The cells are cultured and a medium (RPMI, 2 mM glutamine, 1 mM sodium pyruvate, non-essential amino acids, 5 × 10 ⁻⁵ M 2) supplemented with 1% autoserum using the immunizing antigen (0.2 to 1 mg / plate) -In vitro selection in mercaptoethanol, 100 μg / ml penicillin, 100 μg / ml streptomycin). Four days later, cells were transferred to a medium containing 10% FCS and 10% of ConA-activated normal spleen cells.
Supernatant as well as T cell growth factor (TCGF) were added. Every 14-21 days cells were stimulated by 3 days of exposure to immunizing antigen appearing in syngeneic irradiation (3000 rad) spleen cells (50 × 10 ⁶ / plate) and then grown in TCGF medium. Cloning of T cells is performed by limiting dilution at 0.3 cells / well in microtiter plates in the presence of antigen (2-10 μg / well) and irradiated syngeneic spleen cells (50 × 10 ⁶ / well).

The human T cell line is Teitelbaum et al. 89 PROC. NATL. ACAD. SCI. USA 137 (1
992) derived from peripheral blood mononuclear cells. Approximately 5 × 10 ⁶ cells were incubated in each well of a 24-well culture plate with medium supplemented with 10% heat-inactivated autoserum with Copolymer 1 or MBP (50 μg / ml). After 7 days in culture, cells were harvested with 10% fetal calf serum and recombinant human interleukin-2 (20 units / m2).
l). Cells were grown continuously in this medium with regular exposure every 14 to 18 days to the antigen appearing on irradiated (3000 rad) autologous mononuclear cells.

Proliferation Assay Cells or clones of the T lineage were tested for their specific proliferative response 10-21 days after challenge. T cells (1.5 × 10 ⁴ ) were triplicated in 10% FCS medium with 5 × 10 ⁵ irradiated spleen cells and a final volume of 0.2 ml of the indicated antigen. At the end of the 48 hour incubation, 1 μCi [ ³ H] -thymidine (standard deviation <20% of mean cpm) was applied to the medium and harvested after 6-12 hours.

Inhibition Studies Inhibition of the T cell proliferative response was added to varying concentrations of Copolymer 1 and terpolymer,
The proliferation assay system was examined by adding a stimulating MBP antigen. Inhibition is calculated as% inhibition using equation 2:% inhibition = [1− (cpm with inhibitor / cpm without inhibitor)] × 100.

Results FIG. 1 shows how Copolymer 1 and the terpolymer result in T cell proliferation specific to certain myelin basic protein (MBP) peptides. Generally, T cells proliferate when exposed to the antigen they sensitize. Therefore M
BP and, in particular, certain antigenic peptides from MBP stimulate the proliferation of MBP-specific T cells.

Copolymer 1 and the terpolymer did not stimulate T cell proliferation that is specific for the 84-102 peptide of MBP. Instead, they significantly inhibited the proliferation of T cells exposed to this MBP antigen.

TAL was most effective at inhibiting the proliferation of T cells specific for the MBP84-102 antigen. The inhibition by TAL was even greater than that by Copolymer 1. TGA-induced T cell proliferation was lower. GAL and GTL inhibited T cell proliferation with a similar dose response as Copolymer 1.

Example 7 Terpolymer Recognized by Some Copolymer 1 Specific T Cells Ternary Copolymer Stimulates Some Copolymer 1 Specific T Cell Lines Let
It can secrete a cytokine, IL-4.

Method Copolymer 1 One batch of copolymer having an average molecular weight of 6000 Da and 5800 Da, # 02095 and 55495, respectively, was obtained from Teva Pharmaceutical Industries (Petach
Tikva, Israel).

The myelin basic protein peptide was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (197
9). The peptide was purified by reverse phase HPLC. The peptide used was the sequence PKYV
HA306-31 having KQNTLKLAT (MW1718) (SEQ ID NO: 2)
8 and MBP84-102 having the sequence DENPVVHFFKNIVTPRTPP (MW2529) (SEQ ID NO: 1).

Ternary Copolymer GAL, TGA, TAL, GTL and control polypeptides are as described in Example 3 above.

T Cell Lines and Clones Mouse T cell lines and clones were obtained from Aharoni et al., 23 Eur. J. Immunol. 17
(1993); Aharoni et al., 94 PROC. NATL. ACAD. SCI. USA 10821 (1997). The strain specific for copolymer 1 originated from the mouse spleen,
Copolymer 1 emulsified with Freund's incomplete adjuvant (Difco) 35 days before
5-10 mg was injected subcutaneously into each mouse to reduce sensitivity to EAE. The strain specific to copolymer 1 was 4 mg / ml of M. tuberculosis H37 10 days ago.
Obtained from lymph nodes of mice immunized with 1200 μg of copolymer emulsified with Freund's complete adjuvant (Difco) supplemented with Ra (Difco).

Using a copolymer 1 specific T cell line, the T cell clones were LN-1, LN-2,
S-3, 5-22-5, C-14 and C-52 were used. LN-2 antibody is CF
Derived from injected copolymer 1 in A (SJL / BALB / c) F 1 mice lymph node. The 5-22-5 antibody was obtained from Aharoni et al., 23 EUR. J. IMMUNOL. 17 (19
93); Aharoni et al., PROC. NATL. ACAD. SCI. USA 10821 (1997).
It was obtained from the injected copolymer 1 in A (SJL / BALB / c) F 1 mice spleen.

Human T cell clones were obtained from Teitelbaum et al. 89 PROC. NATL. ACAD. SCI. USA 13
7 (1992) derived from peripheral blood mononuclear cells. Approximately 5 × 10 ⁶ cells were used for copolymer 1
Alternatively, the cells were incubated in each well of a 24-well culture plate containing a medium supplemented with 10% heat-inactivated autologous serum together with MBP (50 μg / ml). After 7 days in culture, cells were transferred to a medium containing 10% fetal calf serum and recombinant human interleukin-2 (20 units / ml). Cells were grown continuously in this medium with regular exposure every 14 to 18 days to the antigen appearing on irradiated (3000 rad) autologous mononuclear cells. The C-52 T cell clone is also available from Teitelbaum et al. 89 PROC. NATL. ACA.
D. according to D. SCI. USA 137 (1992). Derived from w11 donor.

Proliferation Assay Cells or clones of the T lineage were tested for their specific proliferative response 10-21 days after challenge. T cells (1.5 × 10 ⁴ ) in triplicate in 10% FCS medium with 5 × 10 ⁵ irradiated spleen cells or human EBV transformed B cells (5 × 10 ⁴ ) and a final volume of 0.2 ml of the indicated antigen did. At the end of the 48 hour incubation, 1 μCi [ ³ H] -thymidine was applied to the medium and harvested after 6-12 hours. The average deviation of the triplicate culture was less than 20%.

<< P51 >> The cell line cross-reacted only with TAL. None of the cell lines cross-reacted with GTL or cross-reacted with AGT (1: 1: 1) that had the same amino acids as TGA but in a different mole fraction than TGA or Copolymer 1.

Table 6 Cross-reactivity of terpolymer with Copolymer 1 against Copolymer 1 specific T cell lines and clones Murine T cell lines and clones

[0161]

[Table 6-1] ^* Prol = measurement by proliferation. ^‡ IL-4 = measurement by interleukin-4 II. Human T cell clone

[0162]

[Table 6-2] EXAMPLE 8 Terpolymer Cross-Reacts with Anti-Copolymer 1 Antibody Antibody directed against Copolymer 1 crosses terpolymer lacking either tyrosine, glutamic acid or alanine React. However, terpolymers lacking lysine have not been effectively recognized by anti-copolymer 1 antibody.

Antibodies Mouse anti-MBP and anti-copolymer 1 monoclonal antibodies were obtained by fusion of SJL / J mouse MBP- or copolymer 1 immunized spleen cells with the NSO / 1 murine plasmacytoma cell line. Teitelbaum et al. Proc. Natl. Acad. Sci. USA 9528
(1991).

Radioimmunoassay Soft plastic microtiter plates were coated with Copolymer 1 or terpolymer (2 μg / ml). After incubation at room temperature for 16 hours, the plates were washed three times, 2% bovine serum albumin, 0.05% Tween 20, 0.1%
Saturated with PBS containing sodium azide, 10 mM EDTA and 5 units / ml heparin ("PBS buffer") for 2 hours. Monoclonal antibody supernatant (50 μl) was added to the wells during the 2 hour incubation and the wells were washed again with PBS buffer. ¹²⁵ I-labeled goat anti-mouse Fab antibody (1 ×
10 ⁵ cpm / well) was added during the overnight incubation at 4 ° C. After extensive washing, the radioactivity is measured with a gamma counter.

Methods ELISA Assays for Antibody Cross-Reactivity A standard ELISA assay is performed using microtiter plates coated with anti-copolymer 1 polyclonal antibody and a 2 μg / ml terpolymer preparation.

Copolymer 1 Copolymer 1 having the following amino acid composition was prepared by using Teva Pharmaceutical Industries (P
etach Tikva, Israel).

Amino acid molar fraction L-glutamic acid 0.141 L-alanine 0.427 L-tyrosine 0.095 L-lysine 0.338 terpolymer The four terpolymers of Example 1 were used. did.

Results Table 7 shows that anti-copolymer 1 polyclonal antibody cross-reacts with terpolymers lacking either tyrosine, glutamic acid or alanine. The binding to terpolymers lacking glutamic acid (TAL) is relatively high, as will be evident from its high average molecular weight. The tertiary copolymer lacking lysine has not been effectively recognized by the anti-copolymer 1 antibody. These data suggest that charged amino acids such as lysine play a role in the recognition and binding of Copolymer 1 and terpolymer.

Table 7

[Table 7] ^* Dalton ^‡ nd-not measurable Cross-reactivity between terpolymer and copolymer 1-reactive monoclonal antibody Cross-reactivity between terpolymer and copolymer 1 at B-cell response level Check using antibodies (mAbs). The monoclonal antibody is reactive with both Copolymer 1 and MBP (mAbs 2-2-18 and 3-1-45), or Copolymer 1 (mAbs 3-3-9 and 5-7-2). ) Is only reactive with Teitelbaum et al. 88 Proc. Natl. Acad. Sci.
USA 9528 (1991).

Table 8 shows that the terpolymers differed in their ability to bind to mAbs. TGA and GTL were not recognized by any of the copolymer 1 specific mAbs. On the other hand, TAL and GAL bound to copolymer 1 and MBP-specific mAbs having the same affinity as copolymer 1. GAL and T
AL differed only in binding to one mAbs, 5-7-2, which bound TAL but not GAL.

Table 8 Cross-reactivity of terpolymers with MBP- and copolymer 1-reactive B cell antibodies

[Table 8] 2-2-18 is an anti-mouse MBP monoclonal antibody cross-reactive with Copolymer 1 3-1-45 is an anti-copolymer 1 monoclonal antibody cross-reactive with MBP 3 -3-9 is an anti-copolymer 1 monoclonal antibody having no cross-reactivity with MBP 5-7-2 is an anti-copolymer 1 monoclonal antibody having no cross-reactivity with MBP Example 9 Copolymer 1 and terpolymer are combined with collagen in binding to human leukocyte antigen.
In this example, which competes and inhibits collagen-specific T cell responses, Copolymer 1, TAL, GAL, GTL and TGA bind rheumatoid arthritis-associated immune dominant collagen antigen in binding to MHC proteins;
CII 261-273 (SEQ ID NO: 3).

Methods Protein Expression and Purification Recombinant HLA-DR1 and HLA-DR4 molecules (DRA / DRB1 respectively) ^* 0101 and^*0401) is Stern, L. et al. 68 CELL
 465 (1992) and Dessen, A. et al. 7 IMMUNITY 473 (1997).
Was expressed in Drosophila S2 cells. Cells are 0-5%
Excell 401 medium (Sigma, St. Louis, MO) supplemented with fetal calf serum (Sigma)
) Were grown at 26 ° C. in roller bottles. CuSO₄To a final concentration of 1 mM
To induce cells, then incubate for another 4-5 days.
I did it. Immunoaffinity analysis of recombinant HLA-DR1 and HLA-DR4
Manufactured by Stern, L. et al. 68 CELL 465 (1992) and Dessen, A. et al. 7 IMMUNIT
Perform as previously reported by Y 473 (1997). Continuously collect the collected cell supernatant.
Protein A, Protein G and Protein A-LB3
. One column, followed by binding of HLA-DR to 50 mM 3-cyclohexyl
Elution with amino-1-propanesulfonic acid (CAPS), pH 11.5, 200
Neutralize with mm phosphate (pH 6.0). This eluate is centripre
Concentrate on a p10 membrane (Amicon). Protein concentration is bicinchoninic acid (bicin
choninic acid) assay (Pierce Chemical Co.).

Polypeptide Labeling The polypeptide and HA306-318 peptide were biotinylated as in Examples 4 and 7.

Class II MHC Protein Binding Assay In this assay, a recombinantly produced water soluble protein biotinylated polypeptide of the invention and varying amounts of unlabeled competitor polypeptide, collagen CII peptide or influenza virus HA Incubated with peptide. Assays were performed in 96-well microtiter immunoassay plates (PRO-BIN) coated with affinity-purified LB3.1 monoclonal antibody.
D ™, Falcon). Antibody coating was performed by adding 100 μl of 10.0 μg / ml LB3.1 monoclonal antibody to each well and incubating at 4 ° C. for 18 hours. The microtiter wells were then blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) at 37 ° C. for 1 hour,
Washed three times with TTBS. Add 1% BSA to each well before adding sample
Add 50 μl of TBS containing. 150 mM phosphate buffered saline (PBS)
Sodium chloride, 7.5 mM sodium phosphate, dibasic, 2.5 mM sodium phosphate, monobasic, 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 obtained by adding 0.05% Tween-2G to TBS. Other solutions used in this assay are described in Fridkis-Hareli et al. 160 J.
IMMUNOL. 4386 (1998).

Binding analysis revealed that the polypeptide of the present invention in which a water-soluble DR molecule was biotinylated, and various concentrations of unlabeled inhibitors (Copolymer 1, TAL, GAL, GTL, TGA, collagen CII 261-273 peptide or HA306 -318 peptide). Collagen type CII peptide 261-2
73 has SEQ ID NO: 3 (AGFKGEQGPKGEP) and has a molecular weight of 1516. The DR concentration used is 0.15 μM. The final concentration of biotinylated copolymer 1 or terpolymer is 1.5 μM. Incubation is for 40 hours at 37 ° C. in 50 μl of binding buffer pH 5.0.

The bound label is detected using streptavidin-conjugated alkaline phosphatase as follows. The plate was washed three times with TTBS and 100 μl of streptavidin-conjugated alkaline phosphatase (1: 3000, BioRad, Richmond, VA).
for 1 hour at 37 ° C., then p-nitrophenyl phosphate in triethanolamine buffer (BioRad) was added. The absorbance at 410 nm was measured using a microplate reader (MR4000, Dynatech, Chantil).
ly, VA).

T Cell Hybridoma and Antigen Presenting Cell (APC) Binding Assay To determine whether Copolymer 1 and ternary copolymer can inhibit the activation of T cells in response to collagen CII peptide, Examined. Mouse DR
One restriction 3.19 and 19.3 T cell hybridomas and mouse DR4 restriction 3838 and D3 T cell hybridomas were used. Rosloniec. EF, et
al., 185 J. EXP. MED. 1113-1122 (1997); Andersson, EC, et al., PROC.
NATL. ACAD. SCI. USA (1998). Antigen presenting cells (APCs) were L cells transfected with DR1 (Rosloniec. EF, et al., 185 J. EXP. MED. 1113-1122 (
1997), L4 cells transfected with DR4, and Price (DRB1 ^* 0401 / DRB4 ^* 0101). T cell stimulation tests were performed in 96-well microtiter plates in a total volume of 0.2 ml. Irradiated (3000-rad) APCs (2.5 × 10 ⁴ / well) were co-incubated with CII261-273 (40 μg / ml) and various concentrations of the polypeptide at 37 ° C. for 2 hours and then T cells ( 5 × 10 ⁴ / well) and incubation is continued at 37 ° C. for 24 hours. The supernatant (30
μl) and remove 1 with IL-2-dependent CTL-L (5 × 10 ⁴ / well).
After incubation for 2 hours, the cells were labeled with ³ H-thymidine (1 μCi / well) for 12 hours. Plates are collected and radioactivity is monitored using a 1450 microbeta plus liquid scintillation counter (Wallac, Gaithersburg, MD).

Results Class II MHC Protein Binding Assay Recombinant, water-soluble HLA-DR1 and -DR4 proteins produced in insect cells were largely free of bound autoantigens or other peptides. Thus, the data obtained from the insect cells that produced the protein more accurately indicates the actual binding affinity for the polypeptide. Fridkis-Hareli et al. 160 J. IMMUNOL. 43
86 (1998), the entire contents of which are incorporated herein by reference.

HLA-DR1, HLA-DR2 and copolymer 1 and terpolymer
And competitive binding to HLA-DR4 molecules is shown in FIG. Each copolymer 1
(YEAK, FIG. 5A, top panel) and binding to the terpolymer was determined by the amount of CII 261-273 (SEQ ID NO: 3) peptide required for 50% inhibition, as indicated by C
Stronger than that of the II261-273 (SEQ ID NO: 3) peptide. Also, as can be seen from the above, TAL has a higher affinity than HLA-
It bound to DR1 and HLA-DR4 molecules. Unlabeled TAL (YAK, FIG. 5A,
(Bottom panel) The kinetics of inhibition by the polypeptide was also somewhat better than that of the influenza virus peptide HA306-318 (SEQ ID NO: 2). However, the influenza virus peptide HA306-318 has CII261-2.
The binding to copolymer 1 and the binding to the terpolymer were inhibited more efficiently than the 73 peptide.

T Cell Hybridoma and Antigen Presenting Cell Binding Assay Results Copolymer 1 and terpolymer are DR1 restricted T by CII collagen peptide
It also inhibited cell activation (FIG. 6). T cell activation was detected by observing IL-2 production by DR1-restricted CII-specific T cell hybridomas. Various concentrations of the collagen peptide CII 261-273 (SEQ ID NO: 3) were co-incubated with one of the polypeptides and then T cells (indicated clone 3.1)
9 or 19.3) was added and the mixture was further incubated. The supernatant of the incubated cells is removed and the supernatant is treated with IL-2-dependent cytotoxic T.
By observing whether proliferation of lymphocytes (CTL-L) was induced,
Assayed for L-2. Circle filled with the degree of inhibition by TAL (●)
, A triangle (▲) filled with TGA, an open triangle (△) with GTL, and a square (■) filled with copolymer 1.
). The percent inhibition of CTL-L proliferation indicated on the vertical axis was calculated using Equation 1.

[0181] TAL is also the leading inhibitor. However, GTL and Copolymer 1 were also potent inhibitors of T cell activation by CII collagen peptide.
Inhibition of TGA activation was not very effective. Similar results were obtained with other batches of Copolymer 1 and terpolymer.

As shown in FIG. 7, similar inhibition of activation of DR4 restricted T cells was seen. IL
-2 production was used to assess the activation of DR4 restricted CII-specific T cell hybridomas (3838 and D3). It was found that the presence of various polypeptides inhibited IL-2 production, which inhibited DR4 restricted T cell activation. FIG. 7A
Shows the effect of co-incubating irradiated 3838 or D3 Price cells with a constant concentration of 40 μg / ml of collagen peptide CII261-273 (SEQ ID NO: 3) and various concentrations of the polypeptide at 37 ° C. for 2 hours. FIG. 7B
L-cells transfected with the gene encoding HLA-DR4 were treated with a constant concentration of 40 μg / ml of collagen peptide CII261-273 (SEQ ID NO: 3) and various concentrations of GAL, TAL, GTL, TGA and copolymer 1, 37
It shows the effect of incubating at 2 ° C for 2 hours. Then the T cells (indicated clone 3
838 or D3) was added and the samples were further incubated at 37 ° C. for 24 hours. The supernatant (30 μl) was then removed and the IL- induced by IL-2 production was removed.
Assayed for activation by proliferation of 2-dependent cytotoxic T lymphocytes (CTL-L). Each polypeptide mixture was tested in duplicate. The concentration of the polypeptide is shown on the horizontal axis. The degree of inhibition by TAL is indicated by filled circles (●), triangles filled by TGA (▲), triangles opened by GTL (△), and squares filled by copolymer 1 Shown by (■). The percent inhibition of CTL-L proliferation indicated on the vertical axis was calculated using Equation 1.

Example 10 Copolymer 1 Inhibits Activation of T Cells in Response to Myasthenia Gravis Antigenic Peptide
That method copolymer 1 copolymer 1 was obtained from Teva Pharmaceutical Industries (Petach Tikva, Israel ).

The myasthenia gravis-related peptide was synthesized using a solid phase technique on an Applied Biosystems peptide synthesizer. Barany et al., THE PEPTIDES 1 (1979).
Peptides were purified by reverse phase HPLC. The peptide used was the p259 peptide, Zisman et al., Hum Immunol 1995 Nov; 44 (3): 121-30; Brocke et
al., Immunology 1990 Apr, 69 (4): 495-500.

IL-2 Secretion Cell Line WCB Responsive to Myasthenia Gravis Peptide and / or Copolymer 1
The IL-2 secretion by AB was evaluated. Cells (1.5 × 10 ⁴ ) were incubated with the indicated antigen. IL-2 secretion by the cell line WCB AB was used as a measure of activation of that T cell line.

Results Table 9 shows that the p259 peptide stimulates T cell secretion. However, when Copolymer 1 is incubated with the p259 peptide, T cell secretion of IL-2 is inhibited in a dose-related manner. Copolymer 1 IL-2 at 100 μM
About 91% of the secretion was inhibited (Table 10 ), indicating that Copolymer 1 is a potent inhibitor of T cell activation.

Table 9 IL-2 secretion from WCB AB strain in response to p259

[Table 9] Table 13 WCB in response to Cop1 alone or 2 μM p259 + Cop1
IL-2 secretion from AB strain

[Table 10] Absorbance is the average of three samples. Confidence values were calculated for the absorbance of the blank (% CV = SD / (mean-blank) ^* 100).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. “GAL” is a random terpolymer of glutamic acid, alanine and lysine; “TGA” or “YEA” is a random terpolymer of tyrosine, glutamic acid and alanine; “GTL” or “YEK” And "YEAK" is Copolymer 1; a random terpolymer of glutamic acid, tyrosine and lysine;

[Detailed description of drawings]

FIG. 1 shows the effect of Copolymer 1 or terpolymer on the proliferation of T cells specific for certain myelin basic protein (MBP) peptides. Some of the terpolymers of the present invention comprise a myelin basic protein antigen, MBP 84-10.
2 (SEQ ID NO: 1) inhibits the growth of T cell lines. The following symbols: Copolymer 1 (●); GAL (□); TGA (△); TAL (○); GTL (×) were used. FIG. 1A shows growth inhibition of mouse T cell clone MBP-sp-1 specific for MBP 84-102 peptide (0.5 g / well). As a control, the growth of mouse T cell clone MBP-sp-1 stimulated with MBP 84-102 (SEQ ID NO: 1) without inhibitor was 21,145 cpm. FIG. 1B shows MBP 8
Human T cell clone GP against 4-102 peptide (0.125 g / well)
2 shows inhibition of the -25 response. MBP 84-102 without inhibitor (SEQ ID NO: 1)
The proliferation of the human T cell clone GP-25 stimulated by was 4442 cpm.

FIG. 2A compares the binding of the polypeptide to the Class II major histocompatibility molecules DR1 (top), DR2 (middle) and DR4 (bottom) and of Copolymer 1. Coupling with copolymer 1 (■) and the following biotinylated terpolymer: GAL
(□); TGA (▲); GTL (△); and binding by TAL (結合) were compared. The amount of Class II major histocompatibility molecule was kept constant at 0.5 μ / sample and the peptide concentration was varied between 0 and 8 μM as shown on the y-axis. Binding is 37 ° C,
pH 5.0 for 40 hours. Binding was detected by capturing the polypeptide-class II complex with the LB3.1 antibody and reacting with streptavidin-conjugated alkaline phosphatase followed by monitoring the absorbance at 410 nm to detect the amount of biotinylated polypeptide bound. .

FIG. 2B is a Lineweaver-Burk plot of the binding data obtained in FIG. 2A.

FIG. 3 shows the antagonistic inhibition of binding between Copolymer 1 and class II major histocompatibility molecules by the present polypeptide. Purified HLA-DR1 (top), HLA-DR2 (middle) and HLA-DR4 (bottom) molecules alone or with the following unlabeled polypeptide: copolymer 1 (■); GAL (□); TGA (▲); in the presence of one of GTL (△); and TAL (◆), a fixed amount (1.5 μM
) Was incubated with the biotinylated copolymer 1). The inhibition by these polypeptides was compared to the inhibition by the myelin basic protein antigen HA 306-318 (O) (SEQ ID NO: 2). Binding was at 37 ° C., pH 5.0 for 40 hours. The amount of unlabeled polypeptide was varied between 0.1 and 1000 μM as shown on the y-axis. The binding rate was calculated by the formula 1:% inhibition = 100− (signal in the presence of competitor−background) × 100 / (
(% Signal without competitor-background).

FIG. 4A: TAL with bacterial superantigens SEA, SEB and TSST-1
Figure 5 shows competitive inhibition of binding of to class II major histocompatibility molecules. Purified HLA-DR
1 (top), HLA-DR2 (middle) and HLA-DR4 (bottom) molecules at increasing concentrations of unlabeled bacterial superantigen SEA (□); SEB (■); or TSST-1 (●)
Was incubated with a fixed amount of biotinylated TAL in the presence of Binding is 37
C., pH 5.0 for 40 hours. As shown on the y-axis, the amount of superantigen was 0.1 to 10
It was varied between 00 μM. The binding rate is shown as% inhibition according to equation 1.

FIG. 4B. TGA with bacterial superantigens SEA, SEB and TSST-1.
Figure 5 shows competitive inhibition of binding of to class II major histocompatibility molecules. Purified HLA-DR
1 (top), HLA-DR2 (middle) and HLA-DR4 (bottom) molecules at increasing concentrations of unlabeled bacterial superantigen SEA (□); SEB (■); or TSST-1 (●)
Was incubated with a fixed amount of the biotinylated polypeptide TGA in the presence of Binding was at 37 ° C., pH 5.0 for 40 hours. The amount of superantigen was varied between 0.1 and 1000 μM as shown on the y-axis. The binding rate is the inhibition% calculated by the above equation 1.
As shown.

FIG. 4C. GAL with bacterial superantigens SEA, SEB and TSST-1.
Figure 5 shows competitive inhibition of binding of to class II major histocompatibility molecules. Purified HLA-DR
1 (top) and HLA-DR2 (bottom) molecules at increasing concentrations of unlabeled bacterial superantigen SE
A (□); SEB (■); or TSST-1 (●) were incubated with a fixed amount of biotinylated polypeptide GAL. Binding is 37 ° C, pH5.
0 was 40 hours. The amount of superantigen was varied between 0.1 and 1000 μM as shown on the y-axis. The binding rate is shown as% inhibition calculated by the above equation 1.

FIG. 5A shows inhibition of binding between a labeled molecule and a purified MHC class II protein by the terpolymer of the present invention. FIG. 5A shows inhibition of binding to MHC HLA-DR1 protein.

FIG. 5B shows inhibition of binding to MHC HLA-DR4 protein. As unlabeled competitors, terpolymers of the invention, influenza virus hemagglutinin (HA) peptides 306-318 (SEQ ID NO: 2), and type II collagen (CII) peptides 261-273 (SEQ ID NO: 3) Is mentioned. The concentration of unlabeled competitor is shown on the horizontal axis. In each panel, CII 261-273 (SEQ ID NO: 3)
) Is indicated by an open circle (○), inhibition by HA 306-318 (SEQ ID NO: 2) is indicated by a filled circle (●), and inhibition by each of the terpolymers of the present invention is indicated by an open triangle or Shown as a square, or a filled triangle or square. The degree of inhibition by GTL is indicated using an open triangle (△), the one with TAL is filled with a triangle (▲), the one with TGA is opened with a square (□), and the one with copolymer 1 is filled. Are indicated by squares (■). Observed,
The binding rate shown on the vertical axis was calculated as% inhibition using the above formula 1.

FIG. 6 shows inhibition of IL-2 production by DR1-restricted CII-specific T cell hybridomas in the presence of various polypeptides of the invention. Irradiated L57.
23 cells (fibroblasts transfected with the gene encoding HLA-DR1) were incubated with collagen peptide CII261-273 (40 μg / ml) and various concentrations of the polypeptide shown on the horizontal axis in duplicate, at 37 ° C. Co-incubated for 2 hours, then T cells (indicated clones 3.19 or 19.3) were added and the mixture was further incubated at 37 ° C. for 24 hours. Supernatant (30 μl
) Was then removed and assayed for IL-2-induced activation by proliferation of IL-2-dependent cytotoxic T lymphocytes (CTL-L). The degree of inhibition by TAL is indicated by filled circles (●), triangles filled by TGA (▲), triangles opened by GTL (△), and squares filled by copolymer 1 Shown by (■). CT shown on vertical axis
The% inhibition of LL proliferation was calculated according to equation 1.

FIG. 7A shows DR4 restricted CII in the presence of various polypeptides of the invention.
FIG. 9 shows inhibition of IL-2 production by specific T cell hybridomas (3838 and D3). FIG. 7A shows that irradiated 3838 or D3 Price cells were treated at a constant concentration of 4;
The effect of co-incubating with 0 μg / ml of collagen peptide CII 261-273 (SEQ ID NO: 3) and various concentrations of each of the present polypeptides at 37 ° C. for 2 hours is shown.

FIG. 7B shows that L cells transfected with the gene encoding HLA-DR4 were cultured at a constant concentration of 40 μg / ml collagen peptide CII261-27.
3 (SEQ ID NO: 3) and various concentrations of each of the polypeptides at 37 ° C.
Shows the effect of incubating for hours. Then T cells (indicated clone 3838)
Or D3) was added, the samples were further incubated at 37 ° C. for 24 hours, and the supernatant was assayed as described in FIG. Each polypeptide was tested in duplicate. The concentration of the polypeptide is shown on the horizontal axis. In this drawing, the same reference numerals as those in FIG. 6 are used.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/06 A61P 29/00 19/02 101 29/00 37/00 101 C07K 5/11 ZNA 37/00 7/06 C07K 5/11 ZNA A61K 37/02 7/06 37/66 G (31) Priority claim number 60 / 102,960 (32) Priority date October 2, 1998 (1998.10.2) (33) Priority claim country United States (US) (31) Priority claim number 60/108, 184 (32) Priority date November 12, 1998 (November 12, 1998) (33) Priority claim country United States (US) (31) Priority claim number 60 / 123,675 (32) Priority date March 9, 1999 (1999.3.9) (33) Priority claim country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, R, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG) , AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant President and Fellows of Harvard College United States, Mass. 02138 Cambridge, Mass., 1350, Massachusetts Avenue 1350, Hol York Center, Sweet 727 (72) Inventor Aharoni, Lina Israel, 76346 Rehoboth, Hess Street 24 Be (72) Inventor Teitelbaum, Dvora Israel, 76209 Rehoboth, Skorunik Street 5 (72) Inventor Arnnon, Ruth Israel , 76100 Rehoboth, Meonot Shine 9 (72) Inventor Serra, Michael Israel, 76100 Rehoboth, Meonot Wicks 2 (72) Inventor Fridkis-Haleri, Masha United States, Mass. 02138 Cambridge, Divini Avenue 7 (72) Inventor Strominger, Jacques El. United States 02173 Massachusetts, USA 02173 Lexington, Mass. 2030 F term (reference) 4C084 AA02 AA07 AA19 BA01 BA17 BA18 BA20 DA12 DA16 DA23 MA52 MA56 MA65 MA66 NA14 ZA02 ZA33 ZA55 ZA66 ZA81 ZA89 ZB02 ZB15 ZC02 ZC06 ZC35 4H045 AA10 BA13 BA16 BA17 EA22 FA42 GA10 HA03 HA04 HA05

Claims (156)

[Claims] 1. A terpolymer consisting essentially of tyrosine, alanine and lysine randomly polymerized into a polypeptide. 2. The ternary copolymer according to claim 1, which is substantially free of glutamic acid. 3. The terpolymer of claim 1, wherein tyrosine is about 0.1%.
Alanine is present in a mole fraction of about 0.3 to about 0.6; and lysine is present in a mole fraction of about 0.1 to about 0.5. Terpolymer present. 4. The terpolymer of claim 1, wherein tyrosine is about 0.1%.
A terpolymer in which alanine is present in a mole fraction of about 0.54; and lysine is present in a mole fraction of about 0.35. 5. A terpolymer consisting essentially of glutamic acid, tyrosine and lysine, which is randomly polymerized into a polypeptide. 6. The terpolymer according to claim 5, which is substantially free of alanine. 7. The terpolymer of claim 5, wherein glutamic acid is present in a mole fraction of about 0.005 to about 0.300; and tyrosine is present in about 0.005 to about 0.
. A terpolymer wherein the lysine is present in a mole fraction of about 0.3 to about 0.7; 8. The terpolymer of claim 5, wherein glutamic acid is present in a mole fraction of about 0.26; tyrosine is present in a mole fraction of about 0.16; and lysine Is present in a molar fraction of about 0.58. 9. A terpolymer consisting essentially of glutamic acid, alanine and lysine randomly polymerized into a polypeptide. 10. The terpolymer of claim 9, wherein the terpolymer is substantially free of tyrosine. 11. The terpolymer of claim 9, wherein glutamic acid is present in a mole fraction of about 0.005 to about 0.300; alanine is present in about 0.005 to about 0.600. A terpolymer in which lysine is present in a mole fraction of about 0.2 to about 0.7. 12. The terpolymer of claim 9, wherein glutamic acid is present in a mole fraction of about 0.15; alanine is present in a mole fraction of about 0.48; and lysine Is present in a molar fraction of about 0.36. 13. A terpolymer consisting essentially of tyrosine, glutamic acid and alanine randomly polymerized into a polypeptide, wherein the tyrosine is present in a mole fraction of about 0.005 to about 0.250. The glutamic acid is about 0.00
5 to about 0.300 mole fraction; and said alanine is present in about 0.005 to about 0
. Terpolymer present in a molar fraction of 800. 14. The terpolymer of claim 13, wherein the tyrosine is present in a mole fraction of about 0.14; and the glutamic acid is present in a mole fraction of about 0.21; A terpolymer wherein said alanine is present in a molar fraction of about 0.65. 15. The terpolymer of claim 13, wherein the terpolymer is substantially free of tyrosine. 16. A pharmaceutical composition for the treatment of an autoimmune disease, comprising three different amino acids selected from the group of tyrosine, glutamic acid, alanine and lysine, randomly polymerized into a polypeptide. A pharmaceutical composition comprising a therapeutically effective amount of a terpolymer and a pharmaceutically acceptable carrier. 17. The pharmaceutical composition according to claim 16, wherein said terpolymer consists essentially of tyrosine, alanine and lysine. 18. The pharmaceutical composition according to claim 17, wherein the terpolymer is substantially free of glutamic acid. 19. The method according to claim 19, wherein the tyrosine is present in a mole fraction of about 0.005 to about 0.250; the alanine is present in a mole fraction of about 0.3 to about 0.6; 18. The pharmaceutical composition according to claim 17, wherein the composition is present in a mole fraction of 0.1 to about 0.5. 20. The pharmaceutical composition of claim 17, wherein the tyrosine is present in a mole fraction of about 0.10; the alanine is present in a mole fraction of about 0.54; A pharmaceutical composition wherein lysine is present in a mole fraction of about 0.35. 21. The pharmaceutical composition according to claim 16, wherein said terpolymer consists essentially of glutamic acid, tyrosine and lysine. 22. The pharmaceutical composition according to claim 21, wherein said polypeptide is substantially free of alanine. 23. The pharmaceutical composition according to claim 21, wherein said glutamic acid is present in a mole fraction of about 0.005 to about 0.300;
And a lysine from about 0.3 to about 0.7.
A pharmaceutical composition which is present in a molar fraction of 24. The pharmaceutical composition of claim 21, wherein said glutamic acid is present in a mole fraction of about 0.26; said tyrosine is present in a mole fraction of about 0.16; A pharmaceutical composition wherein lysine is present in a mole fraction of about 0.58. 25. The pharmaceutical composition according to claim 16, wherein the terpolymer consists essentially of glutamic acid, alanine and lysine. 26. The pharmaceutical composition according to claim 25, wherein said polypeptide is substantially free of tyrosine. 27. The pharmaceutical composition of claim 25, wherein said glutamic acid is present in a mole fraction from about 0.005 to about 0.300;
From about 0.2 to about 0.700; and from about 0.2 to about 0.7.
A pharmaceutical composition which is present in a molar fraction of 28. The pharmaceutical composition of claim 25, wherein said glutamic acid is present in a mole fraction of about 0.15; said alanine is present in a mole fraction of about 0.48; A pharmaceutical composition wherein lysine is present in a mole fraction of about 0.36. 29. The pharmaceutical composition according to claim 16, wherein said tyrosine,
Essentially a terpolymer from glutamic acid and alanine, said tyrosine being about 0
. Glutamic acid is present in a molar fraction of from about 0.005 to about 0.250;
And alanine is present in a molar fraction of from about 0.005 to about 0.300;
A pharmaceutical composition which is present in a molar fraction of 800. 30. The pharmaceutical composition of claim 29, wherein the tyrosine is present in a mole fraction of about 0.14; the glutamic acid is present in a mole fraction of about 0.21; A pharmaceutical composition wherein alanine is present in a molar fraction of about 0.65. 31. The pharmaceutical composition according to claim 29, which is substantially free of lysine. 32. The pharmaceutical composition according to claim 16, wherein the terpolymer has a molecular weight of about 2,000 to about 40,000 daltons. 33. The pharmaceutical composition according to claim 16, wherein the terpolymer has a molecular weight of about 4,000 to about 9,000 daltons. 34. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is a B cell mediated autoimmune disease. 35. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is a T cell mediated autoimmune disease. 36. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is arthritis. 37. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is a demyelinating disease. 38. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is an inflammatory disease. 39. The pharmaceutical composition according to claim 16, wherein the autoimmune disease is multiple sclerosis, autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmunity. Uveal retinitis, chronic autoimmune thrombocytopenic purpura, colitis, contact hypersensitivity, diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis A pharmaceutical composition which is pemphigus vulgaris, rheumatoid arthritis or systemic lupus erythematosus. 40. A method of treating an autoimmune disease in a mammal, comprising administering a therapeutically effective amount of a ternary copolymer polypeptide containing three different amino acids, randomly polymerized into the polypeptide. A method selected from the group of tyrosine, glutamic acid, alanine and lysine. 41. The method of claim 40, wherein said terpolymer copolymer consists essentially of tyrosine, alanine and lysine. 42. The method of claim 40, wherein the terpolymer comprises essentially of glutamic acid, tyrosine and lysine. 43. The method of claim 40, wherein said terpolymer polypeptide consists essentially of glutamic acid, alanine and lysine. 44. The method of claim 40, wherein said terpolymer polypeptide consists essentially of tyrosine, glutamic acid and alanine, and wherein said tyrosine is from about 0.005 to about 0.250. The glutamic acid is present in a mole fraction of about 0.005 to about 0.300; and the alanine is present in a mole fraction of about
. The process wherein the molar fraction is from 005 to about 0.800. 45. A method for treating an autoimmune disease, comprising administering a therapeutically effective amount of a polypeptide consisting essentially of the amino acids tyrosine, glutamic acid, alanine and lysine, wherein the multiple sclerosis is treated. Not including method. 46. The polypeptide of claim 4, wherein the polypeptide inhibits T cell activation.
46. The method of claim 45 or claim 45. 47. The method of claim 40 or claim 45, wherein said polypeptide activates a T cell suppressor mechanism. 48. The method of claim 40, wherein said polypeptide inhibits activation of T cells responsive to myelin basic protein. 49. The method of claim 40 or claim 45, wherein said polypeptide inhibits activation of T cells responsive to type II collagen peptide. 50. The method of claim 40 or claim 45, wherein the polypeptide binds to a class II MHC protein. 51. The method of claim 40 or claim 45, wherein said polypeptide binds to HLA-DR1. 52. The method of claim 40 or claim 45, wherein said polypeptide binds to HLA-DR2. 53. The method of claim 40 or claim 45, wherein said polypeptide binds to HLA-DR4. 54. The method of claim 40 or claim 45, wherein said polypeptide binds to an antigen presenting cell. 55. The method according to claim 40, wherein the autoimmune disease is multiple sclerosis. 56. The method according to claim 40 or claim 45, wherein the autoimmune disease is autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmune uveal retina. Disease, chronic autoimmune thrombocytopenic purpura, colitis, contact irritability, diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis, heaven A method which is pox, rheumatoid arthritis, or systemic lupus erythematosus. 57. The method of claim 40 or claim 45, wherein the polypeptide has a molecular weight of about 2,000 to about 40,000 daltons. 58. The method of claim 40 or claim 45, wherein the polypeptide has a molecular weight of about 4,000 to about 12,000 daltons. 59. A synthetic peptide having an amino acid sequence comprising at least three amino acids selected from the group consisting of aromatic amino acids, negatively charged amino acids, positively charged amino acids, and aliphatic amino acids, wherein the peptide has a length of at least 7 amino acids. A synthetic peptide which is an amino acid residue and can bind to an MHC class II protein associated with an autoimmune disease. 60. The synthetic peptide according to claim 59, wherein the aromatic amino acid is selected from the group consisting of tyrosine, valine and phenylalanine. 61. The synthetic peptide of claim 59, wherein the positively charged amino acid is lysine and the sequence comprises lysine-tyrosine; lysine-valine; or lysine-phenylalanine. 62. The synthetic peptide according to claim 59, wherein the negatively charged amino acid is glutamic acid and the sequence comprises glutamic acid-lysine-tyrosine; glutamic acid-lysine-valine; or glutamic acid-lysine-phenylalanine. . 63. The synthetic peptide of claim 59, wherein the aliphatic amino acid is alanine and the sequence is glutamic acid-lysine-tyrosine-alanine; glutamic acid-lysine-valine-alanine; or glutamic acid-lysine-phenylalanine. -A synthetic peptide comprising alanine. 64. The synthetic peptide of claim 59, wherein the sequence comprises an amino-terminal alanine, and alanine-glutamic-lysine-tyrosine-alanine; alanine-glutamic-lysine-valine-alanine; or alanine. -A synthetic peptide comprising glutamic acid-lysine-phenylalanine-alanine. 65. The synthetic peptide of claim 59, wherein the aliphatic amino acid is alanine and the sequence is lysine-glutamic-tyrosine-alanine; lysine-tyrosine-alanine-glutamic acid; lysine-glutamic-valine- A synthetic peptide comprising: alanine; lysine-valine-alanine-glutamic acid; lysine-glutamic acid-phenylalanine-alanine; or lysine-phenylalanine-alanine-glutamic acid. 66. The synthetic peptide according to claim 59, wherein the aliphatic amino acid is alanine and the sequence is lysine-tyrosine-alanine-alanine; lysine-lysine-tyrosine-alanine; lysine-valine-alanine- A synthetic peptide comprising: alanine; lysine-lysine-valine-alanine; lysine-phenylalanine-alanine-alanine; or lysine-lysine-phenylalanine-alanine. 67. The synthetic peptide according to claim 59, wherein said peptide further comprises two alanine residues and the sequence is alanine-lysine-tyrosine-alanine-glutamic acid; glutamic acid-alanine-lysine- Tyrosine-alanine; alanine-lysine-valine-alanine-glutamic acid; glutamic acid-alanine-lysine-valine-alanine; alanine-lysine-phenylalanine-alanine-glutamic acid; or glutamic acid-alanine-lysine-phenylalanine-alanine. 68. The synthetic peptide according to claim 64, wherein the autoimmune disease is arthritic. 69. The synthetic peptide of claim 68, wherein the arthritic condition is rheumatoid arthritis. 70. The composition of claim 59, wherein said peptide is between 7 and 100 amino acid residues in length. 71. A composition which is a synthetic peptide having therapeutic activity in a patient suffering from an autoimmune disease, wherein said peptide has an amino acid sequence of glutamic acid,
At least one amino acid of each of lysine and alanine amino acids;
A composition having one amino acid selected from the group consisting of tyrosine, valine and phenylalanine. 72. The composition of claim 71, wherein said peptide is 7-100 amino acids in length. 73. The composition of claim 71, wherein said peptide is 7 to 50 amino acids in length. 74. The composition of claim 69, wherein said peptide is 7 to 25 amino acids in length. 75. The composition of claim 69, wherein said peptide is 7 to 15 amino acids in length. 76. The composition of claim 59, wherein the composition is formulated as a unit dose in a pharmaceutically acceptable carrier. 77. The composition of claim 59, wherein the composition is substantially pure. 78. The composition according to claim 59, wherein the type II collagen 261-type forms an antigen-binding groove of an MHC class II protein associated with an autoimmune disease.
A composition having a higher affinity than the 273 peptide. 79. The composition of claim 71, wherein the composition comprises an amino acid analog at a residue position and in an amount sufficient to inhibit protease degradation of the patient's peptide. 80. AKEYAAAAAAKAAAAA (SEQ ID NO: 25), AA
EYAAAAAAKAAAAA (SEQ ID NO: 26), AAKYAEAAAAKAAAAA
A (SEQ ID NO: 27), and EAKYAAAAAAKAAAAA (SEQ ID NO: 28)
An isolated peptide composition having a sequence selected from the group consisting of: 81. The isolated peptide of any of claims 80, wherein tyrosine (Y) is replaced with valine (V) or phenylalanine (F). 82. AEKYAAAAAAKAAAAA (SEQ ID NO: 29), AK
EYAAAAAAKAAAAA (SEQ ID NO: 25), KEYAAAAAAAKAAAAA
A (SEQ ID NO: 30), AEEYAAAAAAAKAAAAA (SEQ ID NO: 31), AA
EYAAAAAAKAAAAA (SEQ ID NO: 26), EKYAAAAAAAKAAAAA
A (SEQ ID NO: 32), AAKYEAAAAAKAAAAA (SEQ ID NO: 33), AA
KYAEAAAAKAAAA (SEQ ID NO: 27), EAAYAAAAAAAKAAA
A (SEQ ID NO: 34), EKKYAAAAAAKAAAAA (SEQ ID NO: 35), EA
KYAAAAAAAKAAAA (SEQ ID NO: 28), AKYEAAAAAAAAAAA
A (SEQ ID NO: 55), AAEYKAAAAAAAAAAA (SEQ ID NO: 37), AA
KYEAAAAAAAAAAA (SEQ ID NO: 38), AAKYAEAAAAAAAAA
A (SEQ ID NO: 39), AEYAKAAAAAAAAAAAA (SEQ ID NO: 40), AE
KAYAAAAAAAAAAA (SEQ ID NO: 41), AYKAEAAAAAAAAAAA
A (SEQ ID NO: 42) and AKYAEAAAAAAAAAAAA (SEQ ID NO: 43)
An isolated peptide composition having a sequence selected from the group consisting of: wherein the peptide has high affinity for MHC class II proteins. 83. An isolated peptide according to any of the sequences of claim 82, wherein tyrosine (Y) is replaced by valine (V) or phenylalanine (F). 84. An isolated peptide composition having an amino acid sequence capable of inhibiting a patient's immune response to a self-antigen, comprising MHC class II D
A peptide composition wherein the position of the amino acid sequence of the peptide corresponding to the antigen binding pocket in the peptide binding groove of the R protein is identified as a specific amino acid. 85. The isolated peptide composition of claim 84, wherein:
A composition wherein the autoantigen is associated with a condition selected from the group consisting of multiple sclerosis and arthritis. 86. The isolated peptide composition of claim 84, wherein:
A composition wherein the MHC class II DR protein is selected from the group consisting of an MHC class II HLA-DR1 protein and an MHC class II HLA-DR4 protein. 87. The isolated peptide composition of claim 84, wherein:
A composition wherein the MHC class II DR protein is an MHC class II HLA-DR2 protein. 88. The isolated peptide composition of claim 84, wherein:
A composition wherein the amino acid residue having a sequence portion corresponding to the P1 pocket in the MHC class II peptide binding groove is selected from the group consisting of tyrosine, valine, and phenylalanine. 89. The amino acid residue at the first amino acid position in the sequence corresponding to the P1 pocket in the MHC class II peptide binding groove is alanine.
5. The isolated peptide composition of claim 4. 90. The isolated peptide composition of claim 84, wherein:
A composition wherein the amino acid residues located beyond the first 8 amino acid residues of the sequence corresponding to the P1 pocket in the MHC class II peptide binding groove are selected from the group consisting of lysine and alanine. 91. A pharmaceutical preparation comprising a first peptide sequence and a second peptide sequence, wherein the first peptide has an amino acid sequence different from that of any one of claims 84 in a pharmaceutically acceptable carrier. A mixture of a second peptide, wherein the first
A pharmaceutical preparation wherein the sequence of the above further has a lysine residue and the second sequence has an alanine residue at an amino acid position 8 residues ahead of the amino acid corresponding to the P1 pocket of the MHC class II peptide binding groove. 92. A method for treating a patient with an autoimmune disease, comprising: (a) a synthetic heteropolymer having at least two different amino acids, wherein a first charged amino acid and a second hydrophobic amino acid are polymerized in a linear form. Selecting a therapeutic agent comprising a polymer and a pharmaceutically acceptable carrier; and (b) administering the therapeutic agent to a patient with an autoimmune disease, provided that the autoimmune disease is polymorphic. In the case of sclerosis, the synthetic heteropolymer is other than copolymer 1). 93. The method of claim 92, wherein in step (a) the charged amino acid is selected from the group consisting of lysine, glutamic acid and aspartic acid. 94. The method of claim 92, wherein in step (a),
A method comprising a third amino acid selected from the group consisting of charged amino acids and different from the second amino acid. 95. The method of claim 92, wherein said hydrophobic amino acid is selected from the group consisting of valine, leucine, isoleucine, alanine, phenylalanine and tyrosine. 96. The method of claim 95, wherein said heteropolymer comprises the amino acids lysine, alanine and tyrosine. 97. The method of claim 95, wherein said heteropolymer comprises the amino acids lysine, glutamic acid, alanine and phenylalanine. 98. The method of claim 95, wherein said heteropolymer comprises the amino acids lysine, glutamic acid, alanine and valine. 99. The method of claim 98, wherein the heteropolymer of step (a) is polymerized in a mole ratio of at least 3 moles of lysine per mole of tyrosine and at least 4 moles of alanine per mole of tyrosine. A method containing lysine, alanine and tyrosine. 100. The method of claim 99, wherein the lysine: alanine:
A method wherein the molar ratio of tyrosine is 4.0: 5.0: 1.0. 101. The method of claim 95, wherein the heteropolymer of step (a) is an amino acid polymerized in a glutamic acid: lysine: alanine 1.5: 4.0: 5.0 molar ratio. One that contains. 102. The method of claim 93, wherein the heteropolymer comprises an amino acid polymerized in a glutamic acid: alanine: tyrosine 1.5: 5.0: 1.0 molar ratio. . 103. The method of claim 93, wherein said heteropolymer comprises an amino acid polymerized in a glutamic acid: lysine: tyrosine 1.5: 4.0: 1.0 molar ratio. 104. The method of claim 92, wherein step (a) further comprises selecting a heteropolymer that inhibits binding of the antigenic peptide to the MHC class II protein. 105. The method according to claim 92, wherein step (a) comprises MHC
A method further comprising selecting a heteropolymer that inhibits a class II-specific T cell response to a class II protein-peptide complex. 106. The method of claim 104, wherein said antigenic peptide is associated with an autoimmune disease. 107. The method of claim 105, wherein the MHC class II protein is associated with an autoimmune disease. 108. The method of claim 92, wherein step (b) further comprises adding at least one additional therapeutic agent to the formulated heteropolymer and carrier. 109. The method of claim 108, wherein step (b) is performed wherein the antibody, enzyme inhibitor, antibacterial, antiviral, steroid, non-steroidal anti-inflammatory, antimetabolite, cytokine and The method further comprising selecting an additional therapeutic agent from the group consisting of soluble cytokine receptors. 110. The method of claim 108, wherein step (b) further comprises selecting an additional therapeutic agent that is an inducer of cytokine synthesis in the patient. 111. The method of claim 109, wherein step (b) further comprises selecting a cytokine from the group consisting of interferon-β, interleukin-4 and interleukin-10. 112. The method of claim 109, wherein step (b) further comprises selecting an enzyme inhibitor from the group consisting of a protease inhibitor and a cyclooxygenase inhibitor. 113. The method of claim 108, wherein step (a) comprises administering to the patient by a route selected from the group consisting of intravenous, intramuscular, intraperitoneal, subcutaneous, oral, and transdermal administration. A method further comprising selecting a pharmaceutically acceptable carrier suitable for administration. 114. A method of producing a therapeutic heteropolymer composition, comprising: (a) a member selected from the group consisting of charged amino acids, aliphatic amino acids and aromatic amino acids.
(B) polymerizing the amino acids selected in step (a) to form a random linear heteropolymer; (c) combining the antigenic peptide and the MHC class II protein in step (b). A heteropolymer which inhibits the binding of the antigenic peptide to the MHC class II protein in a pharmaceutically acceptable carrier. A method that includes obtaining an object. 115. The method of claim 114, wherein step (c) further comprises obtaining a heteropolymer that inhibits binding between an antigen that is a self antigen and an MHC class II protein. 116. The method of claim 115, wherein the self antigen in step (c) is a collagen type II peptide 261-273 and a class II M
The method wherein the HC protein is MHC HLA-DR1 or MHC HLA-DR4. 117. The method according to claim 115, wherein the self antigen in step (c) is myelin basic protein peptide 84-102 and the class II MHC protein is MHC HLA-DR2. 118. The method of claim 116, wherein step (c) comprises the step of combining collagen type II peptide 261-273 with a class II MHC HLA-DR1 or MHC HLA-DR4 protein at a concentration of at least 20 micromolar. A method further comprising obtaining a 1.5 micromolar concentration of the heteropolymer that inhibits binding by 50%. 119. The method of claim 117, wherein step (c) is at a concentration of at least 20 micromolar myelin basic protein peptide 84-10.
Inhibits the binding of Class 2 MHC HLA-DR2 protein by 50%,
A method further comprising obtaining a 1.5 micromolar concentration of the heteropolymer. 120. The method of claim 1, further comprising, prior to step (a), recombinantly producing a class II MHC protein in a non-mammalian cell.
15. The method according to 14. 121. A method for obtaining an MHC class II binding motif amino acid sequence in a synthetic peptide heteropolymer mixture having therapeutic activity in a patient, comprising: (a) binding the synthetic heteropolymer mixture to an MHC class II protein molecule. Forming a heteropolymer-MHC protein complex; (b) removing the amino terminal amino acid residue of the heteropolymer protruding from the heteropolymer-MHC protein complex by peptidase enzymatic digestion, Terminating the terminus to the terminus of the MHC protein complex; and further (c) eluting the arranged heteropolymer from the MHC protein by dissociating the complex, and removing the heteropolymer having a binding motif arranged at the amino terminus. Releasing. 122. The method according to claim 121, wherein the further step (d)
) Comprises determining the amino terminal sequence of the sequenced heteropolymer to identify the binding motif. 123. The method according to claim 122, wherein the further step (e)
C) comparing the amino terminal sequence of the sequenced heteropolymer with the amino acid sequence of the synthetic heteropolymer composition. 124. The method of claim 121, wherein said MHC class
A method wherein the II protein is associated with an autoimmune disease. 125. The method of claim 124, wherein said autoimmune disease is an arthritic condition or a demyelinating condition. 126. The method of claim 122, wherein said further step (e) comprises synthesizing a plurality of peptide preparations each having a binding motif amino acid sequence. 127. The method according to claim 126, wherein the further step (f)
C) determining the affinity of each of the synthesized peptides for MHC class II. 128. A method of treating a patient with arthritis, comprising: obtaining a therapeutic composition that is a pure heteropolymer comprising amino acids; and an effective amount of the composition to improve the arthritis condition in the patient. Administering to the subject. 129. The method of claim 128, wherein said effective amount is at least 5 mg per day. 130. The method of claim 128, wherein said effective amount is at least 10 mg per day. 131. The method of claim 128, wherein said effective amount is at least 15 mg per day. 132. The method of claim 128, wherein said effective amount is at least 20 mg per day. 133. The method of claim 128, wherein the effective amount is substantially in the range of 25-400 μg / kg patient per day. 134. A kit for assaying the binding of an analyte to an MHC protein, comprising a water-soluble MHC protein recombinantly produced in a non-mammalian cell, a reaction chamber containing the analyte and the MHC protein, an analyte and the MHC protein.
A kit comprising a means for detecting binding to a protein, a container, and instructions for use. 135. The kit of claim 134, wherein said MHC protein is selected from the group consisting of MHC class II HLA-DR1, MHC class II HLA-DR2 and MHC class II HLA-DR4 proteins.
A kit that is a C class II protein. 136. The invention of claim 134, further comprising an autoantigenic peptide.
The kit according to 1. 137. A pharmaceutical composition for treating an autoimmune disease, comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 A pharmaceutical composition comprising the isolated copeptide of Claim 1 and a pharmaceutically acceptable carrier. 138. A pharmaceutical composition for treating an autoimmune disease, which is a therapeutically effective drug selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 28 and SEQ ID NO: 41. A pharmaceutical composition comprising an amount of an isolated copeptide and a pharmaceutically acceptable carrier. 139. A pharmaceutical composition for treating an autoimmune disease, comprising a therapeutically effective amount of an isolated copeptide selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 41. . 140. A pharmaceutical composition for treating an autoimmune disease, comprising isolating a therapeutically effective amount selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. And a pharmaceutically acceptable carrier. 141. A method for treating an autoimmune disease, comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 4 SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 A method comprising administering a copeptide. 142. A method for treating an autoimmune disease, comprising a therapeutically effective amount of a member selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 28 and SEQ ID NO: 41. A method comprising administering a copeptide. 143. A method for treating an autoimmune disease, comprising administering a therapeutically effective amount of a copeptide selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 41. 144. A method for treating an autoimmune disease, comprising administering a therapeutically effective amount of a copeptide selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. A method that includes: 145. The method of any one of claims 141-144, wherein said copeptide inhibits T cell activation. 146. The method of any one of claims 141-144, wherein said copeptide activates a T cell suppressor mechanism. 147. The method of any one of claims 141-144, wherein said copeptide inhibits activation of T cells responsive to myelin basic protein. 148. The method of any one of claims 141-144, wherein said copeptide inhibits activation of T cells responsive to collagen type II peptide. 149. The method of any one of claims 141-144, wherein said copeptide binds to a class II MHC protein. 150. The method of any one of claims 141-144, wherein said copeptide binds to HLA-DR1. 151. The method of any one of claims 141-144, wherein said copeptide binds to HLA-DR2. 152. The co-peptide of claim 1, wherein said co-peptide binds to HLA-DR4.
151. The method according to any one of 41 to 144. 153. The method of any one of claims 141-144, wherein said copeptide binds to antigen presenting cells. 154. The method according to any one of claims 141 to 144, wherein the autoimmune disease is autoimmune hemolytic anemia, autoimmune ovitis, autoimmune thyroiditis, autoimmune grape. Membrane retinopathy, chronic autoimmune thrombocytopenic purpura, colitis, contact hypersensitivity, diabetes mellitus, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic myxedema, multiple sclerosis, myasthenia gravis The method is psoriasis, psoriasis, pemphigus vulgaris, rheumatoid arthritis or systemic lupus erythematosus. 155. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, Sequence No. 15, Sequence No. 16, Sequence No. 17, Sequence No. 18, Sequence No. 19, Sequence No. 20, Sequence No. 21, Sequence No. 22, Sequence No. 23, Sequence No. 24, Sequence No. 25, Sequence No. 26, Sequence No. 27 SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, Sequence No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 5 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or encoding Kopepuchido consisting essentially of SEQ ID NO: 57 An isolated nucleic acid. 156. The method of any one of claims 40 or 45, wherein said autoimmune disease is rheumatoid arthritis.