IE912701A1 - Novel immunosuppressant peptides - Google Patents

Abstract

Peptide compositions are provided which bind MHC molecules of interest and inhibit T cell activation. The peptides are of 4-25 amino acid residues in length, and have a core binding region comprising, in the direction from the N- to the C- terminus, a hydrophobic L-amino acid or amino acid mimetic, a spacer sequence of 2-6 residues and a Thr or Thr mimetic. At least one residue is a D-amino acid or an amino acid mimetic. The compositions may be used to treat diseases associated with particular Dr alleles, including autoimmune diseases such as rheumatoid arthritis.

Description

The present invention relates to the use of novel peptides which bind to major histocompatibility complex (MHC) molecules and modulate an immune response.

An integral part of the mammalian immune response is the ability of T cells to recognize protein antigens. The T cells are presented with processed antigen complexed with the MHC molecules expressed on the surface of certain cells. Antigen presentation appears to be a major, if not the primary, function of MHC molecules.

Both helper and cytotoxic T cells are believed to recognize antigens by interacting with MHC-antigen complexes. MHC Class I molecules (e.g., HLA-A, -B and -C molecules in the human system) are involved in generating a T-killer cell response, in which cells bearing the eliciting antigen are attacked. Class II molecules (HLA-DP, -DQ and -DR in humans) present antigen to T-helper cells, and thus control the expansion and maturation of a selected population of B cells, resulting in the production of antigen-specific antibodies. Thus, Class I and Class II MHC molecules play a critical role in regulating an individual's immune response to a protein antigen.

Autoimmune diseases are characterized by an immune response against self antigens. In essence, autoimmune disease results when an individual's immune response is no longer able to distinguish self antigens from non-self (foreign) antigens. It is thought that self-reactive B cells exist in the body for many potential autoantigens, but that the reactivity of the self-reactive B cells and other immune cells is controlled by MHC molecules. - 2 - Case 126-1001 Several autoimmune diseases have been shown to be associated with particular MHC alleles. One of the most notable associations has been a rheumatoid syndrome involving the spinal column, ankylosing spondylitis, and the allele HLA-B27. Another, the haplotype HLA-DR2, is common in the autoimmune disease multiple sclerosis. Hashimoto's disease, which affects the thyroid, tends to be associated with HLA-DR5. Other studies have shown that individuals having HLA-DR1, and/or DR4w4 and/or DR4wl4 alleles are genetically predisposed towards rheumatoid arthritis. Research suggests that the corresponding MHC proteins play a key role in such diseases, perhaps by binding with a self-antigen and presenting it to T cells.

Current treatment for autoimmune disease such as rheumatoid arthritis consists primarily of treating the symptoms, but not intervening in the etiology of the disease. Broad spectrum chemotherapeutic agents are typically employed, which agents are often associated with numerous undesirable side effects.

The inadequate treatments presently available for rheumatoid arthritis illustrate the urgent need for agents to treat autoimmune diseases that avoid nonspecific suppression of an individual's overall immune response yet do not cause serious side effects. Such agents should also be economical to produce and possess favourable pharmacologic properties for example a relatively long half-life, thereby facilitating lover dosages and/or less frequent administration. Peptides which inhibit HLA-DR restricted T cell activation by binding to HLA-DR molecules, such as DR1, DR4w4, or DR4wl4 offer these advantages.

For example, as HLA-DR alleles have been associated with rheumatoid arthritis, antigens which bind to the DR molecule may serve as a source of binding peptides. Influenza hemagglutinin (HA) is a protein identified as specifially reacting with HLA-DR, and portions thereof may be screened for reactivity to provide a sequence which binds the appropriate DR molecule, such as DR1, DR4w4 or DR4wl4, and then screened for the ability to inhibit or otherwise modulate an antigen-specific immune response such as might be associated with the disease. The MHC molecule with which the peptide is being tested may be - 3 - Case 126-1001 isolated from cells which express it, or the cells may be used intact.

A large number of cells with defined MHC molecules, particularly MHC Class II molecules, are known and readily available. A variety of assay procedures may be employed in the screening protocol and are also well known in the art. Typically, for instance, the peptide may be labelled, e.g., with a radionuclide, enzyme, fluorescer, etc. and specific binding activity determined.

Using the procedure outlined herein, a partial sequence of HA, amino acid residues 307 to 319 (designated here as peptide A or HA307-319), has been identified which binds to both DR1 and DR4w4 MHC molecules. The sequence of this peptide is as shown in Sequence Identifier No.l.

The biological activity of the peptide, i.e. the ability to inhibit antigen-specific T cell activation, may also be assayed in a variety of systems. In one cellular assay, an excess of peptide is incubated with an antigen-presenting cell of known MHC expression, (e.g., DR1) and a T cell clone of known antigen specificity (e.g., tetanus toxin 830-843) and MHC restriction (again, DR1), and the antigenic peptide itself (i.e., tetanus toxin 830-843). The assay culture is incubated for a sufficient time for T cell proliferation, such as four days, and proliferation is then measured using standard procedures, such as pulsing with tritiated thymidine during the last 18 hours of incubation. The percent inhibition, compared to the controls which received no inhibitor, is then calculated.

The capacity of peptides to inhibit antigen presentation in an in vitro assay may be correlated to the capacity of the peptide to inhibit an immune response in vivo. In vivo activity may be determined in animal models, for example, by administering an antigen known to be restricted to the particular MHC molecule recognized by the peptide, and the immunomodulatory peptide. T lymphocytes are subsequently removed from the animal and cultured with a dose range of antigen. Inhibition of stimulation is measured by conventional means, e.g., pulsing with [3H]-thymidine, and comparing to appropriate controls.

Once a peptide which binds to the selected MHC molecule is - 4 - Case 126-1001 identified, a core binding region of the antigen may be determined by sythesizing overlapping peptides or by employing N-terminal or C-terminal deletions (truncations) or additions. By core binding region as used herein is meant a sequence of amino acids or amino acid mimetics important in maintaining a binding affinity of the peptide for an MHC molecule. The core binding region may contain contact residues having characteristics essential for binding. Thus, the core binding region comprises those residues of the peptide that define the specificity of the peptide-MHC interaction. In the determination of a core binding region, a series of peptides with single amino acid substitutions may be employed to determine the effect of electrostatic charge, hydrophobicity, etc. on binding. For instance, with peptide A, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions may be made along the length of the peptide revealing different patterns of sensitivity towards various DR molecules. For example, the DR4w4 molecule preferentially binds to peptides that bear little or no electrostatic charge in a core region consisting of residues 309-314 inclusive. Notably, substitution of Tyr 309 of peptide A with either Glu or Arg results in substantially decreased binding affinity.

Within the core region, critical contact sites, i.e., those residues (or their functional equivalents) which must be present in the peptide so as to retain the ability to bind an MHC molecule and inhibit the presentation to the T cell, may be identified by single amino acid substitutions, deletions, or insertions. In addition, one may also carry out a systematic scan with a specific amino acid (e.g., Ala) to probe the contributions made by the side chains of critical contact residues. For example, Ala substitutions along the length of HA peptide A reveals that binding to the DR1 molecule is relatively unaffected by such substitutions, except for an Ala substitution of position Tyr-309. Binding to DR4w4 is relatively unaffected by Ala substitutions, except at positions Tyr-309 and Thr-314.

Peptides of the invention which are relatively insensitive to single amino acid substitutions with neutral amino acids, except at essential contact sites, have been found to tolerate multiple substitutions. Particularly preferred multiple substitutions are small, - 5 - Case 126-1001 relatively neutral moieties such as Ala, Gly, Pro, or similar residues. The substitutions may be homo-oligomers or hetero-oligomers. The number and types of residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule may also be achieved by such substitutions, compared to the affinity of the parent peptide. In any event, such spacer substitutions should employ amino acid residues or other molecular fragments chosed to avoid, for example, steric and charge interference which might disrupt binding.

The effect of single amino acid substitutions may also be probed using D-amino acids. Such substitutions may be made using well known peptide synthesis procedures. Single D-amino acid substitutions in peptide A reveal that those made to the core region produce a peptide having substantially decreased affinity for MHC DR1, whereas those D-form substitutions made outside the core region are relatively well tolerated.

Using the strategies outlined above, certain minimal requirements for peptide binding to HLA-DR have been identified. In particular, a peptide of the present invention will comprise a core binding region comprising a hydrophobic L-amino acid or amino acid mimetic, a spacer sequence of from 2 to 6 residues, and a Thr or Thr mimetic residue, wherein at least one residue is a D-amino acid or an amino acid mimetic.

Accordingly, the present invention provides peptides of formula I Rx-Rj-R3-R«-Rs I in which, reading from left to right in the direction from the N to the C terminus; R^ is a spacer sequence of from 0 to 7 amino acid or amino acid mimetic residues, R2 is a hydrophobic L-amino or L-amino acid mimetic residue, R3 is a spacer sequence of from 2 to 6 L-amino acid or L-amino acid mimetic residues, - 6 - Case 126-1001 R4 is Thr or a Thr mimetic residue, and R5 is a spacer sequence of between 0 and 10 amino acid or amino acid mimetic residues, provided that at least one residue is a D-amino acid or an amino acid mimetic.

In certain embodiments, the peptides of the invention display at least one of the following features 1) and 2): 1) if Ri is present, the last residue of Rx, together with R2, constitutes an isostere of Χχ-Phe; 2) R3 comprises 2-5 residues, two of which constitute an isostere of X2-Gln; wherein Χχ is a L- or D-amino acid mimetic and X2 can be any L-amino acid or L-amino acid mimetic.

In one preferred embodiment of the peptides according to the invention, the last residue of Rx (Χχ) is positively charged. Preferred residues are Ala and ala; the isostere of Χχ-Phe is typically ala-Phe, Ala-Phe, ala-Cha, Ala-Cha, ala-Ada or Ala-Ada, where Cha is cyclohexylalanine and Ada is adamantylamine.

If an isostere of X2-Gln is present, it is preferably Arg-Gln.

The term spacer sequence refers to sequences of amino acids or amino acid mimetics, which are substantially uncharged or positively charged under physiological conditions and which may have linear or branched side chains.

By amino acid mimetic as used herein is meant a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a peptide of the present invention without adversely interfering to a significant extent with, or in some circumstances actually enhancing, the interaction of the peptide with its MHC receptor. Peptides incorporating amino acid mimetics may be synthesized and screened for - 7 - Case 126-1001 effect on binding in a variety of assays, e.g., in a competition assay with an unsubstituted parent peptide. However, the term Thr mimetic, or in general X mimetic where X is any specific natural amino acid, includes other natural amino acids which may substitute for the specific amino acid without adversely affecting binding, as described above. For a general discussion of peptide mimetics, see, Morgan and Gainor, Ann. Repts. Med. Chem. 24:243-252 (1989).

The term residue refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.

The term isostere as used herein refers to a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications (i.e., amide bond mimetics) well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. Several peptide backbone modifications are known, including ip[CH2S], ψ[0Η2ΝΗ], ♦[CSNH2], *[NHC0], *[C0CH2] and ψ[(Ε) or (Z) CH=CH]. In the nomenclature used above, ψ indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets.

Other possible modifications include an N-alkyl (or aryl) substitution (ip[C0NR]), backbone crosslinking to construct lactams and other cyclic structures, or retro-inverso amino acid incorporation (ψ[ΝΗΟΟ]). By inverso is meant replacing L-amino acids of a sequence with D-amino acids, and by retro-inverso or enantio-retro is meant reversing the sequence of the amino acids (retro) and replacing the L-amino acids with D-amino acids. For example, if the parent peptide is Thr-Ala-Tyr, the retro modified form is Tyr-Ala-Thr, the inverso form is thr-ala-tyr, and the retro-inverso form is tyr-ala-thr. Compared to the parent peptide, a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in a retro-inverso isomer with a topology that closely resembles the parent peptide and is able to bind the selected - 8 - Case 126-1001 MHC molecule. See Goodman et al. Perspectives in Peptide Chemistry pp. 283-294 (1981).

Various amino acid mimetics (as defined above) may be incorporated in the isostere. The side chain of the individual residues may also be modified in a number of ways. For instance, the side chain of a lysine residue isostere may be lengthened or shortened. Other modifications include the alkylation of NH2 of lysine with a dihydroxypropyl radical. The isosteres described above may comprise either a peptide bond or a peptide bond mimetic, but preferably comprise a peptide bond.

It should be understood that the nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxy group to the right (the C-terminus) of each amino acid residue. In relation to a sequence or part of a sequence, the term first refers to the residue nearest to the N-terminus, and the term last to that nearest the C-terminus. In the formulae representing specific embodiments of the present invention, the amino- and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified.

In the formulae, each amino acid residue is generally represented by the standard designation, in which the L-form of an amino acid residue is represented by a three-letter symbol having a capital first letter and the D-form for those amino acids having D-forms is represented by a three-letter symbol having a lower case initial letter. Glycine has no asymmetric carbon atom and is simply referred to as Gly. It is to be understood that where a sequence is defined as consisting only of L-amino acids, that sequence may contain Gly, or other amino acids lacking an asymmetric carbon atom.

The peptides of the invention contain at least one residue selected from D-amino acids and amino acid mimetics, preferably at least one D-amino acid. Such residues are preferably located at one or both of the termini, as this is found to improve the stability of the peptide in vivo. The peptides of the present invention preferably have a human serum half life at least about two times that of peptide A, - 9 - Case 126-1001 preferably about four to ten times greater. Peptides with half lives greater than ten times that of peptide A are particularly preferred. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. A preferred method for determining stability is to measure half life in the 25% human serum assay described in detail below.

Certain preferred embodiments of the peptides of the present invention comprise modifications to the N- and C-terminal residues to alter the physical or chemical properties of the peptide, such as, for example, to affect binding, stability, bioavailability, ease of linking, etc.

The N-terminal residue can be modified to include alkyl, cycloalkyl, aryl, arylalkyl, and acyl groups. The N-terminal residue may be linked to a variety of moieties other than amino acids such as polyethylene glycols (such as tetraethylene glycol carboxylic acid monomethyl ether), pyroglutamic acid, succinoyl, methoxy succinoyl, benzoyl, phenylacetyl, 2-, 3-, or 4-pyridylalkanoyl, aroyl, alkanoyl (including acetyl and cycloalkanoyl e.g. cyclohexylpropanoyl), arylakanoyl, arylaminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, alkyloxycarbonyl (carbamate caps), and cycloalkoxycarbonyl, among others.

Preferred modifications of the C-terminus include modification of the carbonyl carbon of the C-terminal residue to form a carboxyterminal amide or alcohol (i.e., as reduced form). In general, the amide nitrogen, covalently bound to the carbonyl carbon on the C-terminal residue, will have two substitution groups, each of which can be hydrogen, alkyl or an alkylaryl group (substituted or unsubstituted). Preferably the C-terminal is an amido group, such as -C0NH2, -CONHCH3, -C0NHCH2C6H5 or -C0N(CH3)2, but may also be 2-, 3-, or 4-pyridylmethyl, 2-, 3-, or 4-pyridylethyl, carboxylic acid, ethers, carbonyl esters, alkyl, arylalkyl, aryl, cyclohexylamide, piperidineamide and other mono or distubstituted amides. Other moieties that can be linked to the C-terminal residue include piperidine-4-carboxylic acid or amide and cis- or trans- 4-amino- cyclohexanecarboxylic acid or Case 126-1001 amide.

In forming C-terminal amidated compounds of the present invention, the compound can be synthesized directly, for example, using an amine and an oxime resin, as described by Kaiser et al., Science 243:187 (1989), or using a methyl benzhydryl amine (MBHA) polystyrene resin. Alternatively, the amidated compounds may be chemically amidated subsequent to peptide synthesis using means well known in the art, or enzymatically amidated. C-terminal alcohols and corresponding ethers can be prepared by methods well known to those skilled in the art.

Fully protected peptides suitable for C-terminal amidation are conveniently prepared by solid phase synthesis using the SASRIN resin in conjunction with an FMOC synthesis protocol.

A typical synthesis procedure is as follows. The carboxy-terminal acid is coupled to a Sasrin resin (Bachem, Switzerland) using dicyclohexylcarbodiimide and 4-dimethylaminopyridine. Subsequent chain elongation is carried out using an Applied Biosystems 430A peptide synthesizer. After removal of the fluorenylmethyloxycarbonyl (Fmoc) protecting group from the alpha-amino group of the resin-bound amino acid by treatment with a 50 Z solution of piperidine in dimethylformamide, the resin is coupled with a four fold excess of the hydroxybenzotriazole ester of the appropriate amino acid. Chain elongation is continued according to the same protocol. The final amino acid is incorporated with an alpha-amino-tert-butoxycarbonyl (t-BOC) protecting group. The peptide is then cleaved from the Sasrin resin by treatment with 1 X trifluoroacetic acid in dichloromethane. Upon neutralization with pyridine and evaporation of the solvent, a fully protected peptide with a free carboxy terminus is obtained. This compound is then coupled to an amine of choice using hydroxybenzotriazole and dicyclohexylcarbodiimide as the condensing agents. Finally, amino terminal and side chain protecting groups are removed by treatment with trifluoroacetic acid.

In a preferred embodiment, the group Ri contains 1 or 2 residues, with the first residue being a D-amino acid, preferably Ac-ala, phe or ala, wherein the prefix Ac- before the three-letter symbol for an amino acid signifies an acetylated N-terminus on that amino acid. The second - 11 - Case 126-1001 residue, if present, is preferably Ala or ala. The group may also comprise at least one N-alkyl or α-alkyl amino acid residue. Particularly preferred groups Rx are ala, ala-Ala, Ac-ala and Ac-ala-Ala.

R2 is a hydrophobic residue and is thought to be a binding site contact residue. Although its selection is generally dependent on the particular MHC molecule of interest, the R2 residue is preferably selected from the group of Tyr, Phe, Trp, Met, and their amino acid mimetics, such as 6-2-thienyl-alanine, a-amino-4-phenylbutyric acid, phenylglycine, 3-(2-naphthyl)alanine, 3-(l-naphthyl)alanine, β-1-adamantylalanine, β-9-anthracenylalanine, 3-cyelohexylalanine, 2- amino-3(l,l'-biphenyl-4-yl)-propanoic acid, 2-aminoindane-2carboxylic acid, tetrahydroisoquinoline carboxylic acid, l-(2-propanoic acid)-3-amino-benzazepine-2-one, and 3-(4-pyridyl)-alanine. More preferably, particularly when the MHC molecule is DR1, DR4w4, and DR4wl4, R2 is Phe, cyclohexylalanine, or β-1-adamantylalanine. If R2 is absent, R2 is preferably cyclohexylpropanoyl.

R3 usually comprises 4 residues, preferably composed of Gln-Ser-Gln-Thr, Gln-Arg-Gln-Thr, Ala-Arg-Ala-Thr, Ala-Ala-Ala-Thr, Ala-Ala-Ala-Ala, Ala-Ala-Ala-Arg, Ala-Ala-Ala-Lys or Ala-Asp-Ala-Ala, of which the last four are particularly preferred.

R4 is also thought to be a binding site contact residue. It is preferably Thr, but may also be a Thr mimetic, such as His, Gly, Val, Pro, Ser, alkyl substituted His, hydroxyproline, hydroxyvaline, 3- acetyl-2,3-diaminopropanoic acid, 4-acetyl-2,4-diaminobutanoic acid, allothreonine, and the like.

R5 is preferably 3-6 residues long, more preferably 4-5 residues long, the last residue being a D-amino acid or an unnatural amino acid not found in proteins. Preferred sequences are Lys-Ala-Ala-Pip-NH2, Ala-Ala-Ala-Ala-ala, Lys-Ala-Ala-Ala-ala, Leu-Lys-Leu-Ala-thr, Leu-Lys-Ala-Ala-ala, and Ala-Lys-Ala-Ala-ala, wherein Pip-NH2 is piperidine-4-carboxamide.

The peptide has a core binding region composed only of L-amino - 12 - Case 126-1001 acids. The core binding region will typically include, in the direction from the N- to the C-terminus, a hydrophobic L-amino acid (preferably Phe) or L-amino acid mimetic, a spacer sequence of from 2 to 6 residues, and a Thr or Thr mimetic residue, corresponding to R2, R3 and R4 respectively. Preferred sequences of the core binding region are Cha-Ala-Ala-Ala-Ala-Thr, Cha-Ala-Ala-Ala-Lys-Thr, Phe-Ala-Ala-Ala-Ala-Thr, Phe-Ala-Ala-Ala-Lys-Thr, or Phe-Gln-Arg-Gln-Thr-Thr, where Cha is cyclohexylalanine.

Ri, R3, and R5, as previously described, are spacer sequences.

They comprise neutral and positively charged residues. The neutral residues of the spacer sequences typically include, among others, Ala, ala, Gly, Pro, β-Ala, substituted β-ala (e.g. a-methyl, β-methyl, a-benzyl, or β-benzyl substituted), PABA, or other neutral spacers of non-polar amino acids (those with hydrophobic side groups at physiologic pH values) such as aminoisobutyric acid (Aib), Val, Leu, lie, Het, Phe, or Trp; or other neutral polar amino acids (those residues with hydrophilic, uncharged side groups at physiologic pH), such as Ser, Thr, Asn, Gin. A spacer sequence may also include amino acid mimetics such as ethylene glycol or propylene glycol.

Positively charged residues may be incorporated in the spacer sequences to increase aqueous solubility. The presence of these residues generally increases solubility to at least about lpg/ml and in some cases, higher than lmg/ml at pH 7.4. Positively charged spacer residues include, among others, Lys, homo-Lys, Arg, N-alkyl or Ν,Ν-dialkyl-Lys, homo-Arg, N-,N-dialkyl-homo-Arg, N-alkyl or N,N-dialkyl-Arg, Ν,Ν,Ν-trialkylammonium salts, ornithine (Orn), 2,3-diaminopropanoic acid (dap), 2,4-diaminobutanoic acid (dab), and may have, for example, molecules such as dimers of ethylene glycol (polyethylene glycol or propylene glycol (polypropylene glycol) attached thereto, e.g., to Lys, to further enhance solubility.

R3 is a spacer sequence within the core binding region. As discussed above, the core binding region (R2 to R4) of a peptide of the present invention does not contain D-amino acids. Thus, the above description of the residues in Rx and R5 applies to R3 except that all the R3 amino acid residues are in the L form. - 13 - Case 126-1001 In addition to the significances given above for R2, R2 may, if RL is absent, be any of a number of non-amino acid hydrophobic moieties which form a hydrophobic cap at the N-terminus. These moieties are preamino acid mimetics which do not interfere with recognition of the peptide by the MHC molecule and increase serum half life by increasing peptidase resistance of the peptide.

The N-terminal hydrophobic moiety can be linked to the amino terminus (e.g., an Rx residue; R2 , if Rx is absent; or R3, if the hydrophobic moiety is R2) through any of a number of linkages such as peptide backbone modifications known to those skilled in the art (see, Spatο1a, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983). Linkages suitable in the present invention include: R- , R-CO- , R-CS- , R-S02- , R-0-C0- , R-NH-CO- , R-O-CS- , R-S-CO- , R-NH-CS- , and R-S-CS- , where R is the hydrophobic moiety.

A wide variety of N-terminal caps (e.g. amino acid mimetics) can be used so long as MHC binding is not affected and serum half life is improved. Examples of suitable N-terminal moieties include aryl (including phenyl, 1-naphthyl, and 2-naphthyl); arylCi_7alkyl; styryl; C4_8cycloalkyl; and C4_ecycloalkylCi_7alkyl groups, which may be mono-, di-, or tri-substituted by substituents selected from hydroxy, methoxy, ethoxy, phenoxy, amino, dimethylamino, chloro, bromo, methyl and methylthio; adamantyl and adamantylCi_7alkyl moieties.

Further N-terminal caps include groups of formula II R'-(CH2)n- II in which R' is a heterocyclic group selected from pyridyl, pyrazinyl, triazinyl, thiazolyl, oxazolyl, imidazolyl and isoxazolyl, which may be mono- or di-substituted by substituents selected from hydroxy, methoxy, - 14 - Case 126-1001 ethoxy, phenoxy, amino, dimethylamino, chloro, bromo, methyl and methylthio; and n = 0 to 7.

Peptides of the present invention possess various combinations of the above features. For instance, virtually any residue of peptide A may be substituted with one of the D-amino acids or amino acid mimetics discussed above, although, as discussed above, the core binding region should consist exclusively of L-amino acids. Alternatively, one or both terminal residues may be modified as described above. In certain embodiments both terminal residues are modified D-amino acids, for instance, the C-terminal residue is amidated and the N-terminal residue is acetylated. In addition, the C-terminal residue can be an isostere of the dipeptide Ala-Ala (such as piperidine-4-carboxylic acid or amide and cis-4-aminocyclohexanecarboxylic acid or amide) and the N-terminal residue can be a D-amino acid. In other embodiments Rx may be absent and R2 is a hydrophobic amino acid mimetic (e.g. cyclohexylpropanoyl) vhich forms a hydrophobic cap. Any combination of the modifications and substitutions discussed can be used so long as MHC binding is not adversely affected.

Peptides or analogs thereof having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to inhibit the preselected immune response. For instance, the peptides can be modified by extending, decreasing or substituting in the compound's amino acid sequence, e.g., by the addition or deletion of amino acids on either the amino terminal or carboxy terminal end, or both, of the sequences disclosed above.

The peptides or analogs of the invention can be modified by altering the order or composition of certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites, may generally not be altered without an adverse effect on biological activity. The non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-ot-amino acids, or their D-isomers, but may include non-protein amino acids as well, such as β-, γ-, or δ-amino It 912701 - 15 - Case 126-1001 acids, β-, γ-, or δ-imino acids (such as piperidine-4-carboxylic acid) as well as many derivatives of L-a-amino acids. As discussed, a peptide of the present invention may generally comprise either L-amino acids or D-amino acids, but not D-amino acids within the core binding region.

The peptides and analogs thereof of the present invention which bind to MHC molecules and inhibit or block MHC restricted antigenspecific T cell activation comprise at least 4 amino acid residues or the conformational equivalent thereof, more usually at least 6, preferably at least 8 and more preferably at least 11 residues long. They usually will not exceed 25 residues or the equivalent thereof in length, preferably no more than 18, more preferably 14 amino acid residues or the equivalent thereof in length. As mentioned above, it is understood that non-critical amino acids may be substituted with other amino acids or with amino acid mimetics; in instances where spacer molecules are substituted for non-essential residues, the approximate length of the resulting peptide should generally not exceed that which may be accommodated by the binding domain of the selected MHC molecule.

The peptides of the present invention and pharmaceutical compositions thereof are particularly useful for administration to mammals, particularly humans, to treat a variety of conditions involving autoimmune diseases. For instance, although rheumatoid arthritis can be treated with conventional chemotherapeutic agents as mentioned above, the peptides of the invention as described herein may be used to treat the symptoms of rheumatoid arthritis or prevent their occurrence in identified patients or those patients at risk of developing the disease. Since the immunomodulatory peptides which bind to selected MHC molecules are more selective than conventional agents, and generally will be less toxic, they will be more effective and less likely to cause complications than the conventional broad spectrum agents. Of course, if necessary or desirable, the peptides may be administered in conjunction with the traditional chemotherapeutic agetns, and may be used to lover the dose of such agents to levels substantially below those typically associated with toxicity.

Other autoimmune associated disorders for which the peptides of - 16 - Case 126-1001 the invention may be employed to relieve the symptoms of, treat or prevent the occurrence or reoccurrence of include, for example, Sjogren syndrome, scleroderma, polymyositis, dermatomyositis, systemic lupus erythematosus, juvenile rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis (antibodies to acetylcholine receptors), bullous pemphigoid (antibodies to basement membrane at dermalepidermal junction), pemphigus (antibodies to mucopolysaccharide protein complex or intracellular cement substance), glomerulonephritis (antibodies to glomerular basement membrane), Goodpasture's syndrome, autoimmune hemolytic anemia (antibodies to erythrocytes), Hashimoto's disease (antibodies to thyroid), pernicious anemia (antibodies to intrinsic factor), idiopathic thrombocytopenic purpura (antibodies to platelets), Grave's disease and Addison's disease (antibodies to thyroglobulin), and the like.

The dose of the immunomodulatory peptides of the invention for treatment of autoimmune disease will vary according to, e.g., the peptide composition, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician. For example, for the treatment of rheumatoid arthritis with a peptide of the present invention the dose is in the range of about 50 yg to 2,000 mg/day, preferably 5 to 700 mg/day for a 70 kg patient. Administration should begin at the first sign of symptoms or shortly after diagnosis, and continue at least until symptoms are substantially abated and for a period thereafter. In established cases loading doses followed by maintenance doses may he required.

The pharmaceutical compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The compositions are suitable for use in a variety of drug delivery systems. Preferably, the pharmaceutical compositions are administered parenterally, e.g. intravenously. Thus, the invention provides compositions for parenteral administration which comprise an immunomodulatory peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier e.g. water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by - 17 - Case 126-1001 conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of immunomodulatory peptides in the pharmaceutical formulations can vary widely, i.e., from less than about 0.01%, usually at or at least about 5% to as much as 50 to 75% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of peptide. Two or more peptides of the invention may be combined to form a peptide cocktail under certain circumstances for increased efficacy. The peptides of the invention may also be used in conjunction with other pharmaceutically active agents.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, preferably 25-75%.

For aerosol administration, the immunomodulatory peptides are preferably supplied in finely divided form along with a conventional non-toxic surfactant and a suitable propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably l%-10%; and of surfactant from 0.1%-20% by weight, preferably 0.25%-5%. - 18 - Case 126-1001 The compositions containing the immunomodulatory peptides can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as therapeutically effective dose. Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.1 mg to about 2,000 mg of peptide per day for a 70 kg patient, with dosages of from about 0.5 mg to about 700 mg of peptide per day being more commonly used. It must be kept in mind that the materials of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations.

In prophylactic applications, compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of a particular autoimmune disease to enhance the patient's own immunoregulatory capabilities. Such an amount is defined to be a prophylactically effective dose. In this use, the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.1 mg to about 500 mg per 70 kg patient, more commonly from about 0.5 mg to about 200 mg per 70 kg of body weight.

The peptides may also find use as diagnostic reagents. For example, a peptide may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In such instances cells bearing the selected MHC antigen, e.g., white blood cells, will be obtained from the individual, incubated in vivo with the peptide(s), an immunostimulatory peptide, and a collection of T cells from the same individual. The proliferation of the T cells can then be assayed in the presence or absence of said immunomodulatory peptide or peptide mixture. - 19 Case 126-1001 EXAMPLES Selection and Screening of Peptide Library This Example illustrates the method used to identify peptides which bind human MHC HLA-DRl and -DR4w4. A library of about one hundred eighty synthetic peptides is tested. The library screened is nonredundant in the sense that care is taken to eliminate amino acid sequences too closely related; overlapping peptides are allowed but only if they overlap less than 502 of total residues.

Cells. EBV-transformed homozygous cell lines are used as a source of DR molecules. Cell lines are routinely monitored for DR expression by FACS analysis. Their DR types are confirmed by serological typing (Terasaki et al., Amer. J. Clin. Path. 69:103-120 (1978)) and RFLP analysis (Schreuder, J. Expt. Med. 164:938 (1986)). Cell lines used are maintained in vitro by culture in RPMI 1640 medium (Flow Labs, McLeon, VA), supplemented with 2 raM L-glutamine (Gibco, Grand Island, NY), 50 mM 2-mercaptoethanol (Sigma Chemical Co., St. Louis, M0), 100 ug/ml streptomycin, 100 U/ml penicillin (Gibco, Grand Island, NY), and 102 heat-inactivated fetal calf serum (Hazelton Biologies Inc., Lenexa, KS) or horse serum (Hazelton Biologies Inc.). Large quantities of cells are grown in stationary cultures (250 cc flasks). Cells are lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl pH 8.5, containing 22 Rennex, 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates are cleared of nuclei and debris by centrifugation at 10,000 x g for 20 minutes.

Affinity purification of DR molecules. DR molecules are purified essentially as described by Gorga et al. (Gorga et al., J. Biol. Chem. 262:16087-16094 (1987)) using the monoclonal antibody LB3.1 (Gorga et al., Cell. Immunol. 103:160-173 (1986)), covalently coupled to protein A-Sepharose CL-4B. Epstein-Barr virus-transformed human B cell lines, homozygous at the DR locus, are used as a source of DR molecules. The LG-2 (DR1) and Preiss (DR4, Dw4) lines were obtained from Dr. J.C.

Gorga (Harvard University, Cambridge, MA), and MAT (DR3), Beh (DR4, Dv4) and SWEIG (DR5) from Dr. G. Nepom (Virginia Mason Research Center, Seattle, VA). Aliquots of cell lysates equivalent to approximately 10 g - 20 - Case 126-1001 of cells are passed sequentially through the following columns: Sepharose CL-4B (10 ml), protein-A-Sepharose (5 ml), W6/32-protein-ASepharose (10 ml), LB3.1-protein-A-Sepharose (15 ml), using a flow rate of 30 ml/h. The columns are washed with 10 column volumes of 10 mM Tris-HCl pH 8.0, 0.1X Rennex (5 ml/hr; 2 column volumes of PBS and 1% octylglucoside. The DR is eluted with 0.05 M diethylamine in 0.15 M NaCl containing 1% octylglucoside (pH 11.5), immediately neutralized with 2 M glycine pH 2.0 and concentrated by ultrafiltration through an Amicon YM-30 membrane. Protein content is evaluated by a BCA protein assay (Pierce) and confirmed by SDS PAGE electrophoresis.

Peptide synthesis. Peptides are synthesized on an Applied Biosystems (Foster City, CA) 430A peptide synthesizer according to well known protocols. The synthesis of the peptide ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNHi (Example 10 below) is typical. The hydroxybenzotriazole ester of tert-butyloxycarbonyl-dalanine (Boc-d-alanine) is prepared by treatment of Boc-d-alanine with equimolar amounts of hydroxybenzotriazole and dicyclohexylcarbodiimide in dimethylformamide for thirty minutes at room temperature. The mixture is then filtered to remove precipitated dicyclohexylurea. The resulting solution is added to a methylbenzhydrylamine resin and allowed to react for 60 minutes with gentle shaking or rocking. The resin is thoroughly washed with dimethylformamide followed by dichloromethane. The ^-butyloxycarbonyl protecting group of the resin-bound alanine is then removed by mixing the resin together with a 50 X solution of trifluoroacetic acid in dichloromethane for 30 min., followed by thorough washing with dichloromethane. The resulting resinbound amine trifluoroacetate salt is neutralized by treatment with a 20 % solution of diisopropylethylamine in dichloromethane.

Chain elongation is accomplished by repeating the above sequence of 1) amino acid activation, 2) coupling and 3) deprotection for each amino acid residue. Upon completion of the chain elongation, the resin is dried and the peptide is cleaved from the resin and completely deprotected by exposure to hydrogen fluoride in the presence of anisole for 90 minutes at 0°C. The hydrogen fluoride is evaporated with a nitrogen stream and the peptide is washed with cold diethylether. Finally the peptide is redissolved in 25 X aqueous acetic acid and the - 21 - Case 126-1001 solvent is removed by lyophilization. The synthesis of peptides containing amino acid mimetics is carried out in the same way. Suitably protected amino acid mimetics are incorporated according to the above procedures. The peptides are then purified by reverse-phase highperformance liquid chromatography (HPLC). The purity of the peptides is substantiated by amino acid sequence and/or composition analysis. They are routinely >95X pure after HPLC. Peptides are radiolabeled as described below.

DR-peptide binding assay. Purified DR molecules (10-1,000 nM) are incubated with 10 nM 125I-radiolabeled peptide for 48 hours in the presence of added protease inhibitors. HPLC-purified peptides are radioiodinated (125I) using the Chloramine T method, (Buus et al., Science 235:1353-1358 (1987)). The final concentrations of protease inhibitors are: 1 mM PMSF, 1.3 mM 1,10-phenanthroline, 73 μΜ pepstatin A, 8 mM EDTA, 6mM N-ethyl maleimide, and 200 yM Na-p-tosyl-L-lysine chloromethyl ketone (TLCK). The final detergent concentration in the incubation mixture is 0.05X NP-40. Several of the peptides screened are hydrophobic and require DMSO to maintain solubility and reduce peptide adsorption to surfaces. In these instances peptide stock solutions are prepared in neat DMSO, and the final DMSO concentration in the incubation mixture is adjusted to 5%. Control experiments demonstrate that the ID 50X values obtained with or without DMSO are similar. The DR-peptide complexes are separated from free peptide by gel filtration on a Sephadex G50 (Pharmacia Fine Chemicals, Piscataway, NJ) column 23 x 1.3 cm), as previously described. Id. The columns are eluted using PBS (0.5X NP-40, 0.1X NaN3) collected in 1 ml fractions, which are then assayed for radioactivity. The fraction of peptide bound to DR (a) relative to the total amount of offered peptide is determined as the ratio of peptide in the void volume to the total peptide recovered. The a is corrected by subtracting the negative control (125I-peptide incubated for two days at room temperature in the presence of protease inhibitors and 120 yg/ml of unlabeled peptide, but in the absence of any DR). Competitive inhibition by unlabeled peptide is used to determine ID 50X values.

Peptide HA 307-319, (Peptide A) having good binding activity for both DR1 and DR4w4 molecules, is chosen from the screening assays for lb 912701 - 22 - Case 126-1001 further study. The DR1, DR2, Dr4w4 and DR4wl4 binding capacities of a series of peptide A analogs with single or multiple amino acid substitutions are determined as described above. In addition, the peptides are tested for their ability to block, an antigen-specific, HLA-DR restricted T cell proliferative response. The protocol is as follows: Cellular Assay for Inhibition of Antigen Presentation An EBV positive, DR1+ homozygous typing cell line, LK-2, is washed 3X with Hank's Balanced Salt Solution (BBSS), suspended to 5 x 10®/ml in HBSS, and fixed with 0.5% p-formaldehyde (w/v) at room temperature for 20 minutes. The fixed LG-2 cells are washed IX with HBSS, washed 2X with RPMI media supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, antibiotics, and 10% human sera type AB (Complete Media, CM), resuspended at 10®/ml in CM, and then plated as 100 yl into individual wells of a 96 well microtiter dish. The fixed LG-2 cells are then pulsed simultaneously for 2 hours at 37°C with 50 pi of test peptide and 50 ul of stimulatory peptide which have been dissolved in CM. The amount of stimulatory peptide added to each test well is constant and is calculated so as to yield 60-80% of the maximal T cell proliferative response. The potential inhibitor peptide is evaluated in duplicate at several concentrations corresponding to multiples of the stimulatory peptide concentration, usually 3X, 10X, 50X, and 250X. At the end of the two hour incubation the plates are centrifuged, and the media carefully aspirated and replaced with fresh medium. The plates are washed in this manner three times in order to ensure complete removal of unbound peptide. At the end of the washes 50 yl of CM remains in the well. In separate wells, increasing concentrations of stimulatory peptide are assayed in the absence of inhibitory peptide in order to obtain dose-response curves for the stimulatory peptide and to ensure that the amount of stimulatory peptide used in test wells is truly limiting.

Antigen-specific T cells are washed 2X in CM, suspended to 2 x 10s/ml in CM, and plated as 150 ul into the wells containing the peptide-pulsed LG-2 cells. The cultures are incubated for three days at 37°C, and pulsed with 0.1 pCi/well of 3H-thymidine during the last 16 - 23 - Case 126-1001 hours of culture. At the termination of the cultures the cells are harvested onto glass fiber and the amount of 3H-thymidine, which has been incorporated by the responder T cell, determined using liquid scintillation counting.

The inhibitory activity of each peptide is determined relative to the peptide of Example 29 below. For each test peptide the amount of inhibition is plotted as a function of the inhibitor concentration, and this graph is used to determine a 50% inhibitory dose. The inhibitory activity is then expressed as the ratio of the 50% inhibitory dose of the standard peptide (determined in a parallel experiment) to that of the test peptide. These data are presented in the last three columns of Table 1. This inhibitory value constitutes an additional means by which the MHC binding peptides can be compared.

Stability Assays To analyze stability of the peptides, the half life of the peptides is determined using a 25% human serum (v/v) assay. Using the procedure described below, the 25% human serum half life of peptide A is determined to be about 100 minutes. The protocol is as follows. Pooled human serum (Type AB, non-heat inactivated) is obtained from Irvine Scientific and is delipidated by centrifugation before use.

Human serum is diluted to 25% with RPMI tissue culture media. All chemicals (reagent or HPLC grade) are purchased commercially from Sigma or Aldrich and used without further purification.

Typically, 1 ml of reaction solution in a 1.5 ml Eppendorf tube is temperature-equilibrated at 37 ± 1°C for 15 minutes before adding 5 yl of peptide stock solution (10 mg/ml in DMSO) to make the final peptide concentration 40yg/ml. The initial time is recorded. At known time intervals 100 yl of reaction solution is removed and added to 200 yl of either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then spun at 14,000 rpm (Eppendorf centrifuge) for 2 minutes to pellet the precipitated serum proteins.

The following controls are carried out for each run: i) each set - 24 - Case 126-1001 of stability experiments includes a degradation study on a reference study on a reference peptide (Peptide A), carried out in separate solution, ii) peptide stability is also determined in precipitation supernatant containing 4% (or greater) trichloroacetic acid to ensure that the peptides do not undergo acid-catalyzed degradation while awaiting HPLC sample analysis, iii) sample recovery upon TCA or ethanol precipitation of serum proteins is determined by comparison of the peptide peak area at time = 0 with a peptide stock solution of known concentration in DMSO/HjO (1:1). Generally, 6% TCA shows the highest peptide recovery, but where less than 90% recovery in TCA is observed, EtOH is used; all peptides show 85% or greater recovery in their optimal precipitation media and iv) peptide degradation is carried out under conditions where the degradation rate constant is independent of peptide concentration (demonstrated, for example, by lowering the peptide concentration or using radiolabeled peptide only), and proportionately dependent on biological media concentration.

Peptide analysis is carried out by reversed-phase HPLC using stability-specific chromatography conditions. Most samples are analyzed using a 5 pm 25 x 0.4 cm Vydac C-18 column and a 0 —> 50 % gradient (0-0.08 % trifluoroacetic acid in water —> 0.08 % trifluoroacetic acid in CH3CN) over 30 minutes (flow = 1 mL/minute, detection = 214 nm, AUFS = 0.2) at room temperature. Where peptide coelutes with a media peak or one of the peptide degradation product peaks, a different brand of C-18 column (for example, Waters Bondapak) is used to effect separation. HPLC linearity is determined for a representative group of peptides up to 50 ug/mL (three-fold higher than the concentration actualy assayed for in the biological media Examples) in DMS0/H20 (1:1). This mixed solvent system is chosen to minimize peptide loss due to adhesion to the reaction vessel. HPLC stability-specificity is carried out by traditional methods including: separate analysis of non-precipitated media peaks, analysis of peptide peak shape and peak absorbance ratioing at 50 % peptide remaining, and kinetic analysis where first order loss of parent peptide should be observed (degradation of the parent peptide cannot display a first order decay curve [when run under pseudo first order conditions] if B or C [of same retention time and similar molar absorptivity] build up to any appreciable extent). - 25 - Case 126-1001 Kinetic analysis is carried out by least squares analysis of log (integration peak area) versus time. When necessary, correction is made for small, interfering media peaks that coelute with the parent peptide (subtraction of background). Most reactions are followed for at least two halflives (except for the most stable peptides) and all pseudo first order plots are linear showing correlation coefficients > .98 (8 points).

EXAMPLES 1-48 The following peptides were synthesized and tested: Case 126-1001 Ex.No. ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNHj tyr-Ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Ala-Lys-Ala-Ala-alaNH2 ala-Ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 tyr-Ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH2 tyr-Ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH2 tyr-Ala-Ala-Cha-Ala-Ala-Ala-Lys-Tyr-Ala-Ala-Ala-Ala-alaNH2 Ac-tyr-Ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Leu-Ala-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH2 Ac-tyr-Ala-Ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 tyr-Ala-Ala-Phe-Ala-Ala-Ala-Lys-Tyr-Ala-Ala-Ala-Ala-ala ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Ala-Lys-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Leu-Ala-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Lys-Ala-Ala-Thr-Ala-Lys-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-ala Ac-ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-alaNH2 Ac-ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH2 Ac-ala-Ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Lys-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Ala-Lys-Ala-Ala-ala tyr-Ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Lys-Ala-Ala-Ala-alaNH2 Ac-tyr-Ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH2 tyr-Ala-Ala-Cha-Ala-Ala-Ala-Ala-Hpr-Lys-Ala-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-alaNH2 tyr-Ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-alaNH2 ala-Ala-Cha-Ala-Ala-Ala-Ala-Hpr-Lys-Ala-Ala-Ala-alaNH2 tyr-Ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Leu-Ala-Ala-Ala-alaNH2 ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 Ac-tyr-Ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-ala Ac-tyr-Ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Ala-Lys-Ala-Ala-alaNH2 Ac-tyr-Ala-Ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-alaNH2 Ac-tyr-Ala-Arg-Phe-Gln-Arg-Gln-Thr-Thr-Leu-Lys-Ala-Ala-ala tyr-Ala-Ala-Phe-Gln-Arg-Gln-Thr-Thr-Leu-Lys-Ala-Ala-ala Ac-tyr-Ala-Arg-Phe-Gln-Ser-Gln-Thr-Thr-Leu-Lys-Ala-Lys-thr peg-Tyr-Ala-Arg-Phe-Gln-Ser-Gln-Thr-Thr-Leu-Lys-Ala-Lys-ThrNH2 ala-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH2 tyr-Ala-Arg-Ada-Ala-Ser-Gln-Thr-Thr-Leu-Lys-Ala-Lys-thrNH2 ala-Ala-Phe-Ala-Ala-Ala-Ala-Dpl-Lys-Ala-Ala-Ala-alaNH2 Ada-Ala-Arg-Gln-Thr-Thr-Leu-Lys-Ala-Ala-alaNH2 ala-Dpl-Phe-Ala-Ala-Ala-Ala-Hpr-Leu-Dpl-Ala-Ala-Tol ala-Ala-Phe-Ala-Ala-Ala-Ala-Hpr-Ala-Opl-Ala-Ala-Tol tyr-Ala-arg-Ada-Ala-Arg-Gln-Thr-Thr-Leu-Lys-Ala-Ala-alaNH2 ala-ala-Phe-Ala-Ala-Ala-Ala-Hpr-Leu-Dpl-Ala-Ala-Tol ala-Dpl-Phe-Ala-Ala-Ala-Ala-Thr-Leu-Dpl-Ala-Ala-Tol ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Leu-Dpl-Ala-Ala-Tol ala-Dpl-Phe-Ala-Ala-Ala-Ala-Thr-Ala-Dpl-Ala-Ala-Tol ala-Ala-Cha-Ala-Ala-Ala-Ala-Pro-Lys-Ala-Ala-Ala-alaNH2 Notes Ac- at start signifies an acetylated N-terminus NH2 at end signifies a carboxyamide modified C-terminus Cha = L-cyclohexylalanine Hpr trans-4-hydroxyproline Ada = L-adamantylalanine Dpi = N-(dihydroxypropyl)-L-lysine Tol = L-threoninol peg = tetraethyleneglycol carboxylic acid monomethyl ether. - 27 - Case 126-1001 TABLE 1 Ex. No. Stability t% (min)1 MHC Binding Assay Cellular Assay DRl2 DR4w42 DR4wl43 DRl4 DR4w4 DR4wl4 1 356 ++ ++ ++ ++ ++ ++ 2 240 ++ ++ ++ ++ ++ + + 3 265 ++ ++ ++ ++ ++ ++ 4 260 ++ ++ ++ ++ ++ ++ 5 287 ++ ++ ++ ++ ++ + + 6 411 ++ ++ ++ ++ ++ ++ 7 300 + ++ ++ + ++ + 8 445 ++ ++ + + ++ ++ + + 9 n.d. ++ ++ ++ ++ ++ + 10 383 ++ ++ ++ ++ ++ + 11 750 ++ ++ ++ ++ + + ++ 12 531 ++ ++ + + ++ ++ + 13 335 ++ ++ ++ + + ++ 14 495 ++ ++ ++ + ++ + 15 575 ++ ++ ++ ++ ++ + 16 246 ++ ++ ++ + ++ + 17 627 ++ ++ ++ + ++ + 18 314 ++ ++ ++ ++ ++ n.d. 19 575 + ++ ++ ++ ++ ++ 20 531 ++ ++ ++ ++ ++ ++ 21 329 ++ ++ ++ ++ + + 22 265 ++ ++ ++ ++ ++ + 23 n.d. ++ ++ ++ ++ + n.d. 24 300 ++ ++ ++ ++ ++ ++ 25 665 ++ ++ ++ + ++ ++ 26 300 ++ ++ ++ ++ ++ n.d. 27 216 ++ ++ ++ ++ ++ + 28 226 ++ ++ ++ + ++ + 29 687 ++ ++ ++ + + + 30 406 ± ++ ++ + ++ + 31 n.d. ± ++ ± ± + + 32 n.d. + ++ ++ + + + 33 400 + ++ ++ + + ± 34 252 ++ ++ ++ + + + 35 265 ++ ++ ++ + + + 36 n.d. ± ++ ++ + + + 37 560 + ++ ++ + ++ n.d. 38 n.d. ++ ++ ++ ++ ++ n.d. 39 n.d. + ++ ± ++ ++ n.d. 40 n.d. ++ ++ ++ + ++ n.d. 41 n.d. + ++ ++ + ++ n.d. 42 n.d. ++ ++ ++ + ++ n.d. 43 n.d. ++ ++ ++ + ++ n.d. 44 n.d. ++ ++ ++ + ++ n.d. 45 n.d. ++ ++ ++ ++ ++ n.d. 46 n.d. ++ ++ ++ + ++ n.d. 47 n.d. ± ++ ++ + + n.d. 48 n.d. ++ ++ ± ++ ± n.d. - 28 - Case 126-1001 Notes to Table 1: 1) determined in 25% human serum 2) Binding affinity relative to peptide A =1, where affinity greater than 1.0 is 0.51 to 1.0 is 0.1 to 0.5 is ±, and less than 0.1 is KD of peptide A is approx. 5.6 nM vs DR1 and 42 nM vs DR4w4. 3) Binding affinity relative to peptide B = 1; notation as above. KD of peptide B is approx.67 nM. Peptide B is the peptide having the sequence set out in Seq. Id. No.2. 4) Inhibition of antigen presentation is relative to peptide of Example 29 = 1, where inhibition greater than 1.0 is 0.51 to 1.0 is 0.1 to 0.5 is ±, and less than 0.1 is The 50% inhibitory dose for the peptide of Example 29 is approx. 10 uM vs DR1, 30 yM vs DR4w4, and 30 yM vs DR4wl4. - 29 - Case 126-1001 EXAMPLES 49-50 These examples show that the carboxy-terminal residues can be replaced by dipeptide isosteres. In the peptides below, two C-terminal alanine residues have been replaced by either piperidine-4-carboxylamide (PipNH2) or cis-4-aminocyclohexanecarboxylamide (AccNH2) These substitutions do not dramatically alter the DR binding affinities of these peptides relative to the parent peptide. Trans-4-aminocyclohexanecarboxylic acid would be equally well tolerated.

Ex.49 ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Lys-Ala-Ala-PipNH2 Ex.50 ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Lys-Ala-Ala-AccNH2 The results are presented in Table 2, below. The binding assays are performed as described above. The binding affinity of the test peptides is presented relative to either peptide A or peptide B.

TABLE 2 Ex. No. MHC Binding Assay DR11 DR4W41 DR4wl42 ++ ++ ++ ++ ++ + 1) Relative to peptide A 2) relative to peptide B EXAMPLES 51-52 Ex.51 tyr-Ala-Lys-Phe-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-Tol Ex.52 tyr-Ala-Phe-Gln-Arg-Gln-Thr-Thr-Leu-Lys-Ala-Ala-Tol These examples (as veil as Examples 41,42 and 44-47) show that incorporation of reduced amino acid residues (amino alcohols) at the C-terminus of DR binding peptides does not adversely affect their binding affinities (Table 3). - 30 - Case 126-1001 it 91270Ί The binding assays are performed as described above. The binding affinity of the test peptides is presented relative to either peptide A or peptide B.

TABLE 3 Ex. No. MHC Binding Assay DR11 DR4W41 DR4wl42 + ++ ++ + ++ ++ 1) Relative to peptide A 2) relative to peptide B EXAMPLES 53-60 These examples show that R2 may be replaced by a hydrophobic end cap comprising an amino acid mimetic without affecting the binding (Table 4).

Ex.53 Chp-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.54 Ada-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.55 Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.56 Aac-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.57 Ppa-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.58 ada-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.59 2Na-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Ex.60 INa-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Phe-ala Chp = 3-cyclohexylpropionic acid Ada = L-adamantyl alanine Cha = L-cyclohexylalanine Aac = adamantylacetic acid Ppa = 3-phenylpropionic acid ada = D-adamantylalanine 2Na = 2-naphthoic acid INa = 1-naphthoic acid - 31 - Case 126-1001 TABLE 4 Ex. No. MHC Binding Assay DR11 DR4V41 DR4vl42 53 ++ ++ ++ 54 ++ ++ ++ 55 ++ ++ ++ 56 + ++ ++ 57 + ++ ++ 58 + ++ + 59 + ++ ++ 60 ++ ++ 1) Relative to peptide A 2) relative to peptide B - 32 - Case 126-1001

Claims (22)

1. ) Ri is other than 0 residues and the last residue of R x , together with R 2 , constitutes an isostere of Χι-Phe; or 1 A peptide of formula I Ri-R 2 -R 3 -R 4 -Rs I in which, reading from left to right in the direction from the N to the C terminus; Ri is a spacer sequence of from 0 to 7 amino acid or amino acid mimetic residues, R 2 is a hydrophobic L-amino or L-amino acid mimetic residue, R 3 is a spacer sequence of from 2 to 6 L-amino acid or L-amino acid mimetic residues, R 4 is Thr or a Thr mimetic residue, and Rs is a spacer sequence of between 0 and 10 amino acid or amino acid mimetic residues, provided that at least one residue is a D-amino acid or an amino acid mimetic. 2. ) R 3 comprises 2-5 residues, two of which constitute an isostere of X 2 -Gln; wherein X x is a L- or D-amino acid mimetic and X 2 can be any L-amino acid or L-amino acid mimetic.

2. A peptide according to Claim 1 in which:

3. A peptide according to Claim 1 or Claim 2 in which the last residue of Ri (Xi) is Ala or ala.

4. A peptide acording to Claim 2 in which the isostere of X 2 -Gln is Arg-Gln.

5. A peptide acording to Claim 1 in which the group R x contains 1 or 2 residues, composed of ala, ala-Ala, Ac-ala, or Ac-ala-Ala. - 33 - Case 126-1001

6. A peptide acording to Claim 1 in which the R 2 residue is selected from the group of Tyr, Phe, Trp, Met, and their amino acid mimetics.

7. A peptide according to Claim 6 in which R 2 is Phe, Tyr, cyclohexylalanine, or adamantylalanine.

8. A peptide acording to Claim 1 in which R3 comprises 4 residues, composed of Ala-Ala-Ala-Ala, Ala-Ala-Ala-Arg, or Ala-Ala-Ala-Lys.

9. A peptide acording to Claim 1 in which R 4 is Thr.

10. A peptide acording to Claim 1 in which R 5 is 3-5 residues long, the last residue being a D-amino acid.

11. A peptide acording to Claim 10 in which R 5 is Ala-Ala-Ala-Ala-ala, Lys-Ala-Ala-Ala-ala, Leu-Lys-Leu-Ala-thr, Leu-Lys-Ala-Ala-ala, or Ala-Lys-Ala-Ala-ala.

12. A peptide acording to Claim 1 which has a core binding region corresponding to R 2 , R3 and R4 having the sequence Cha-Ala-Ala-Ala-Ala-Thr, Phe-Ala-Ala-Ala-Ala-Thr or Phe-Ala-Ala-Ala-Lys-Thr, where Cha is cyclohexylalanine.

13. A peptide according to Claim 1 having the sequence ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Lys-Ala-Ala-PipNH 2 ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Lys-Ala-Ala-AccNH 2 ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH 2 ala-Ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-Ala-alaNH 2 ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH 2 tyr-Ala-Ala-Cha-Ala-Ala-Ala-Ala-Thr-Leu-Lys-Ala-Ala-alaNH 2 ala-Ala-Phe-Ala-Ala-Ala-Ala-Thr-Ala-Lys-Ala-Ala-alaNH 2 , or tyr-Ala-Ala-Phe-Gln-Arg-Gln-Thr-Thr-Leu-Lys-Ala-Ala-alaNH 2 , where Cha is cyclohexylamine, PipNH 2 is piperidine-4-carboxyl amide, and AccNH 2 is cis-4-amino-cyclohexanecarboxamide.

14. A pharmaceutical composition comprising a peptide according to Case 126-1001 any one of the preceding claims in association with a pharmacologically acceptable diluent or carrier.

15. A method of treating autoimmune disease in a patient comprising administering an effective dose of a compound according to any one of Claims 1-13.

16. A method according to Claim 15 in which the autoimmune disease is rheumatoid arthritis.

17. A peptide according to any one of Claims 1-13 for use as a pharmaceutical.

18. The use of a peptide according to any one of Claims 1-13 for the preparation of a medicament for the treatment of autoimmune disease.

19. The use of Claim 18 where the autoimmune disease is rheumatoid arthritis.

20. A peptide as defined in Claim 1, substantially as described herein by way of Example.

21. A pharmaceutical composition comprising a peptide according to Claim 20.

22. Use of a peptide according to Claim 20, substantially as described herein by way of Example.