WO1997025344A1 - Clip analogues and autoimmune disease - Google Patents

Abstract

The present invention relates generally to amino acid sequences and analogues thereof and more particularly to amino acid sequences and analogues thereof corresponding to CLIP peptides which facilitate or effect immunosuppression or immunomodulation in a human or animal subject. The present invention further provides pharmaceutical compositions comprising CLIP peptide sequences and derivatives and analogues thereof which interact with MHC class II molecules, either non-specifically or haplotype-specifically, thereby preventing, inhibiting, delaying or otherwise suppressing an immune response when administered to an animal. The present invention is particularly useful in the treatment of autoimmune disorders or any prophylactic or other medical treatment wherein the suppression of an immune response is desirable, for example following transplantation procedures. Furthermore, high-affinity haplotype-specific CLIP analogues of the present invention permits the development of diagnostics reagents for determining the contribution of a particular MHC class II haplotype to an autoimmune disease and more preferably, diagnostic reagents for determining an individual's haplotype and genetic predisposition to an autoimmune disease.

Description

CLIP ANALOGUES AND AUTOIMMUNE DISEASE

FIELD OF THE INVENTION

The present invention relates generally to genetic sequences and analogues thereof and more particularly to genetic sequences and analogues thereof which facilitate or effect immunosuppression, preferably by interacting with major histocompatability complex (MHC) class II molecules. The present invention further provides pharmaceutical compositions comprising genetic sequences and analogues thereof which interact with MHC class II molecules, thereby preventing, inhibiting, delaying or otherwise suppressing an immune response when administered to an animal. The present invention is particularly useful in the treatment of autoimmune disorders or any prophylactic or other medical treatment wherein the suppression of an immune response is desirable, for example following transplantation procedures.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence identity numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

BACKGROUND TO THE INVENTION

Invariant chain (Ii) associates with MHC class II molecules and performs a number of crucial functions in antigen presentation (Sant and Miller, 1994; Cresswell, 1994).

Acting as a molecular chaperone, this transmembrane glycoprotein escorts newly- assembled α/β class II complexes from the endoplasmic reticulum into the endosomal system, using a localisation signal mapped to the N-terminal cytoplasmic domain (Anderson et al , 1993). During this manoeuvre, the association between Ii and the α/β class II complex prevents premature binding of an antigenic peptide with the MHC class II molecule (Roche and Cresswell, 1991; Teyton et al , 1990). In the acidic environment of the endosomal system, Ii dissociates from the MHC class II molecule and is proteolytically degraded, leaving the class II binding site available for binding the antigenic peptide (Blum and Cresswell, 1988).

Class II-associated Ii peptides (CLIPs) comprise a core of approximately 14-24 amino acid residues, derived from the region comprising amino acid positions 81 to 104 of the invariant chain protein Ii. The amino acid sequence of the CLIP region (ie. amino acid residues 81 to 104 inclusive) is as follows:

L P K P P K P V S K M R M A T P L L M Q A L P M

Riberdy et al. (1992) found that derivatives of the CLIP region, in which the N-terminus is truncated by up to 3 amino acid residues, or the C-terminus is truncated by 1 amino acid residue are found associated with the human class II molecule haplotype HLA-DR3, isolated from T2.DR3 cells. Sette et al. (1992) also found that approximately 80% of the peptides bound to HLA-DR3 molecules in DR-expressing transfected 721.174 cells comprised residues 80-103 of Ii. Thus, it would appear from these data that residues 84- 103 of Ii at least, are required to bind to the human MHC class II haplotype HLA-DR3.

Using computerised modelling, Lee and McConnell (1995) predicted the structure of CLIP-MHC complexes and concluded that amino acid residues 89-100 contact the cleft of the human MHC class II molecule HLA-DR3 and the murine MHC class II molecules I-A^u, I-A^k and I-A^d.

The association of various autoimmune diseases with MHC class II restriction suggested to the inventors that specific MHC class II molecules may sei-ve as targets for immunomodulation in the treatment of autoimmune diseases. However, an effective immunomodulator of MHC class II activity must be stable to the activity to degradative enzymes including proteases, it must be preferably haplotype-specific as required and capable of binding to MHC class II molecules with high affinity.

Although CLIP molecules have been shown to inhibit the binding of antigenic peptides to MHC class II molecules, until the present invention a means of conferring haplotype- specificity on the CLIP molecule whilst retaining high-affinity binding thereto was hitherto unknown. In particular, a means of preventing proteolysis of CLIP molecules in vivo has not been a straightforward procedure. Furthermore, until the present invention, the efficacy of CLIP molecules as immunomodulators in the treatment of autoimmune diseases in humans such as multiple sclerosis and diabetes or the equivalent diseases in animals, were unknown.

SUMMARY OF THE INVENTION

In accordance with the present invention, CLIP analogues have been developed which are capable of binding with high-affinity either to several different MHC class II haplotype molecules, or in a haplotype-specific manner. The development of high- affinity haplotype-specific CLIP analogues are useful in the specific inhibition of specific antigen-MHC class II interactions and possess enormous potential in the treatment of autoimmune disorders such as diabetes and multiple sclerosis. Furthermore, the high- affinity haplotype-specific CLIP analogues of the present invention permits the development of diagnostic reagents for determining the contribution of a particular MHC class II haplotype to an autoimmune disease and more preferably, diagnostic reagents for determining an individual's haplotype and genetic predisposition to an autoimmune disease. The present invention also provides high-affinity haplotype-non-specific CLIP analogues which are at least useful in the suppression of immune responses generally, such as during and/or following transplantation procedures, in particular bone marrow transplantation. Accordingly, one aspect of the present invention provides an isolated CLIP peptide, CLIP analogue or a derivative thereof comprising a sequence of at least 9 amino acid residues derived from amino acid residues 86-104 of the invariant chain protein Ii.

A second aspect of the present invention provides an isolated CLIP peptide, CLIP analogue or a derivative thereof which is capable of binding to an MHC class II molecule of mammalian origin.

A third aspect of the present invention provides an isolate CLIP peptide, CLIP analogue or a derivative thereof which comprises a sequence of amino acids of at least 9 residues in length derived from amino acid residues 86-104 of the invariant protein Ii, which is capable of binding to an MHC class II molecule, preferably selected from the list comprising murine I-A^u, I-A\ I-A^8? and I-A^d or human haplotypes HLA-DR, HLA-DP or HLA-DQ, in particular HLA-DR3, amongst others.

A further aspect of the present invention is directed to an isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof.

The present invention also contemplates a method for identifying a specific MHC class II molecule haplotype in a biological sample, said method comprising isolating said biological sample and contacting same or purified or partially-purified a fraction thereof with a binding-effective amount of an isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof and then detecting said binding.

The present invention further extends to diagnostic kits for determinhig the presence of an MHC class II molecule haplotype in a biological sample, comprising several first compartments, each of which is adapted to contain an isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof, optionally labelled with a reporter molecule. A further aspect of the invention is directed to an isolated CLIP peptide, CLIP analogue or a derivative thereof which comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 1-54 or is at least 70% identical thereto.

A further aspect of the present invention extends to a pharmaceutical composition for use in a human or a domesticated animal to reduce, delay, inhibit or otherwise attenuate an immune response therein, said composition comprising an isolated CLIP peptide, CLIP analogue or a derivative thereof in combination with a pharmaceutically-acceptabie carrier, diluent or excipient.

The present invention extends to pharmaceutical compositions comprising combinations of CLIP peptides, CLIP analogues or derivatives thereof which are more effective than single CLIP molecules in attenuating the immune response of a human or domesticated animal.

A further aspect of the present invention extends to a method of delaying, inhibiting, reducing or otherwise attenuating an immune response in a human or domesticated animal comprising administration thereto of an immunomodulatingly-effective amount of a pharmaceutical composition comprising CLIP peptide, CLIP analogue or a derivative thereof in combination with a pharmaceutically-acceptabie carrier, diluent or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation showing competition for presentation by L-alanine- substituted CLIP analogues. Peptides comprising amino acids 86-104 of Ii, singly- substituted with alanine at various positions (eg. lys-ala at position 86 is designated K86A) were tested for their ability to compete with the antigenic peptides MBP-(Acl-II), HEL-(46-61) and Ova-(323-339), for presentation to a T-cell hybridoma. Multiple concentrations of CLIP analogue were used in competition assays. Antigenic T-cell stimulation was measured as supernatant IL-2 activity causing pH] thymidine O 97/25344 1^»CT/AU96/00812

- 6 - inco oration in the IL-2-dependent cell line HT-2. The data indicates 7-cell stimulation in the presence of 50μm CLIP analogue (panel a) or 25μm CLIP analogue (panels b and c).

Figure 2 is a graphical representation showing competition for presentation on cells expressing I-A^u (panels a and d), I-A^k (panels b and e) or I-A^d (panels c and f), by L- analine-substituted CLIP analogues substituted at position 93 (panels a-c) or position 99 (panels d-f). Peptide concentrations were as described in the legend to Figure 1.

Figure 3 is a photographic representation of a western blot showing I-A^u polypeptide levels following exposure of purified I-A^u molecules to pH 4.5-5.0 at 37°C for 1 hour, in the presence or absence of a CLIP peptide comprising amino acid residues 86-104 of Ii or the antigenic peptide Ova-(323-339). Aggregated (M_r= 110 kDa) and compact (M_r=65kDc) forms of I-A^u are shown with appropriate molecular weight markers.

Figure 4 is a graphical representation showing competition for presentation to cells expressing wild-type or mutated I-A^u molecules, by CLIP peptide comprising amino acid residues 86-104 of Ii. Panel a shows competition for presentation on the site-specific mutants D59K I-A^u and D59A I-A^u, which contain lysine or alanine respectively, instead of aspartate at position 59 in the α chain of the MHC class II molecule I-A^u. Panel b shows competition on the site-specific mutants T86S I-A^u and T86L I-A^u, which contain serine or leucine respectively, instead of threonine, at position 86 in the β chain of I-A^u. The antigenic peptide MBP-(Ac 1 = 11) (25μm) and CLIP peptide (400 m) were coincubated with T-cell hybridoma in the presence of antigen presenting cells expressing either wild-type or mutated I-A^u. Supernatants were harvested and tested for IL-2 activity. pH] thymidine incoφoration D59K I-A^u , 28, 089 ± 547; D59A, 22590 ± 673. In panel b; normal I-A^u, 59, 714 ± 9512; T86S I-A^u, 44629 ± 6946; T86L I-A^u, 35221 ± 2657.

Figure 5 is a graphical representation showing competition for presentation on cells expressing I-A^u (panel a), I-A^k (panel b) or I-A^d (panel c) by D-alanine -substituted CLIP analogues. Peptides comprising amino acids 86-104 of Ii, singly-substituted with D- alanine at various positions (eg. lys-D-ala at position 86 is designated K.86) were tested for their ability to compete with the antigenic peptides MBP-(Ac 1-11) (5 m), HEL- (46- 61) (1.5μm) and Ova-(323-339Y) (0.05μm) for presentation to a T-cell hybridoma. Multiple concentrations of CLIP analogue were used in competition assays. Antigenic T-cell stimulation were performed essentially as described in the legend to Figure 1. The data indicate T-cell stimulation in the presence of 50μm (panel a), lOOμm (panel b) or 12.5_/ m (panel c) of D-alanine-substituted CLIP analogue. As a control, L-alanine- substituted CLIP peptides were tested similarly against each MHC class II haplotype (panels d-f). Data in control experiments indicate T-cell stimulation in the presence of 50μm L-alanine-substituted CLIP for I-A^u (panel d), 25μm CLIP for I-A^k (panel c) or 6μm CLIP for I-A^d (panel f).

Figure 6 is a graphical representation showing competition for binding to cell surface I-A class II molecules by D-alanine-substituted CLIP analogues. Peptides comprising amino acids 86-104 of Ii, singly-substituted with D-alanine at various positions (eg. lys-D-ala at position 86 is designated K86) were tested as competitors of an unsubstituted, biotinylated CLIP peptide (20μm), for cell surface binding to I-A^u (panel a), I-A^k (panel b) or I-A^d (panel c). Multiple concentrations of CLIP analogue were used in competition assays. Data show median fluorescence intensity of stained cells less background, using a single concentration of unbiotinylated CLIP analogue at which the unsubstituted CLIP 86-104 peptide gave approximately 50% inhibition of binding (125μm for I-A^u, 200μm for I-A^k and 62.5μm for I-A^d).

Figure 7 is a graphical representation of circular dichroism (CD) spectra obtained for D- alanine-substituted CLIP analogues. Representative CD scans are shown for K86D-Ala and A94 D-Ala CLIP analogues in panel a. Unsubstituted CLIP comprising amino acid residues 86-104 of Ii and K86D-Ala exhibit essentially the same CD spectra. Panel b shows a difference CD for A94 D-Ala and K86 D-Ala, revealing abrogation of α-helix- forming propensity in the A94 D-ala CLIP analogue. Figure 8 is a graphical representation showing competition for binding to I-A class II molecules by truncated and frame-shift CLIP analogues. In panel a, CLIP analogues comprising N-terminal and C-terminal truncations from positions 86 and 104 respectively of Ii, were tested as competitors against a biotinylated CLIP comprising amino acids 86- 104 of Ii (50μm). In panel b, various 15-residue CLIP analogues were tested for competitors against the same biotinylated CLIP peptide. Multiple concentrations of CLIP analogue were used in competition assays. Data show median fluorescence intensity of stained cells less background, using a single concentration on unbiotinylated CLIP analogue at which the CLIP 86-104 peptide gave approximately 50% inhibition of binding (i.e. 250μm for I-A^u and I-A^k, 125μm for I-A^d).

Single-letter and three-letter abbreviations used for amino acid residues in the specification are defined in Table 1.

TABLE 1

Amino Acid Three-letter One-letter Abbreviation Symbol

Alanine Ala

Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C D-alanine Dal X Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp w Tryosine Tyr Y Valine Val V DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides an isolated CLIP peptide, CLIP analogue or a derivative thereof comprising a sequence of at least 9 amino acid residues derived from amino acid residues 86-104 of the invariant chain protein Ii.

The inventors have discovered that the affinity of binding of a CLIP peptide, CLIP analogue or a derivative thereof to an MHC class II molecule, is dependant upon the total length of the peptide region which contacts the peptide binding groove of said MHC class II molecule. For example, although peptide molecules as small as 9 amino acid residues in length bind to the murine MHC class II haplotypes I-A^u, I-A^k , I-A^g7 and I-A^d , optimum binding to said class II haplotypes is detectable using CLIP peptides, CLIP analogues or derivatives which are at least 14 amino acids in length.

Accordingly, a preferred embodiment of the present invention provides an isolated CLIP peptide, CLIP analogue or derivative thereof comprising a sequence of at least 10-13 amino acid residues in length derived from amino acid residues 86-104 of the invariant chain protein Ii. More preferably, said isolated CLIP peptide, CLIP analogue or derivative thereof is at least 14-19 amino acid residues in length.

Hereinafter the term "CLIP peptide" shall be used to define a peptide molecule comprising a sequence of amino acids which is derived from amino aicid residues 81 to 104 of the invariant chain protein Ii, which associates with an MHC class II molecule during its transport from the endoplasmic reticulum to the endosome. Preferably, and in the context of the present mvention, the term "CLIP peptide" shall be taken to define a peptide molecule comprising a sequence of amino acids which is derived from amino acid residues 86-104 of Ii, or an analogue or derivative thereof, which is at least capable of competing with an antigenic peptide for binding to a MHC class II molecule.

The term "CLIP analogue" or "CLIP peptide analogue" or similar term as used herein shall be taken to refer to an isolated peptide molecule comprising a sequence of amino acid residues which is at least 70% identical to a CLIP peptide, in particular a CLIP peptide derived from amino acid residues 86-104 of Ii or a homologue or derivative thereof, wherein said peptide molecule further comprises one or more amino acid substitutions, deletions or insertions.

Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which an amino acid residue is contained in a CLIP peptide replaced with another naturally-occurring amino acid of similar character, for example Glyr~Ala, Val-Ile-leu, Asp«Glu, Lys- Arg, Asn^«Gln or Phe~Tφ~Tyr. Preferably, however the substitutions encompassed by the present invention are "non-conservative", in which an amino acid residue which is present in CLIP peptide is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid present in a CLIP peptide is substituted wid a non-conventional amino acid, in particular norleucine, norvaline, α-aminobutyric acid or D-alanine, amongst others. Non-conventional amino acids encompassed by the invention include, but are not limited to those listed in Table 2. Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N- terminus and/or the C-terminus of a CLIP peptide or CLIP analogue, or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxy-terminal fusions and of the order of 1-4 amino acid residues.

A CLIP peptide or CLIP analogue as referred to herein may readily be made using peptide synthetic techniques well-known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substituent mutations at pre-determined sites using recombinant DNA technology, for example by Ml 3 mutagenesis, are also well-known. The manipulation of nucleic acid molecules to produce variant peptides, polypeptides or proteins which manifest as substitutions, insertions or deletions are well-known in the art.

Derivatives of a CLIP peptide or CLIP analogue include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, homopolymers or heteropolymers comprising two or more copies of the subject CLIP peptides or CLIP analogues, are within the scope of the invention. Procedures for derivatizing peptides are well-known in the art.

TABLE 2

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N- methy lalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspaπic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-meti ylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methy lornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtφ D-omithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle D-tryptophan Drrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D- α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtφ N-cyclohexylglycine Nchex D-α- methyl tyros ine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe D-N-memylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-( l-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-meti ylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtφ D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylorni thine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtφ N-( 1 -methy lethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(_/7-hydroxyphenyl)glyc ine Nhtyr L-r-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-r-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mrrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine N mhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethy 1) g ly cine l-carboxy-l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane

The CLIP peptides and CLIP analogues described herein may be derivatized further by the inclusion or attachment thereto of a protective group which prevents, inhibits or slows proteolytic or cellular degradative processes which normally degrade said CLIP peptide or CLIP analogue. Such derivatization of the CLIP molecules of the invention may be useful where said CLIP molecule is required for a pharmaceutical puφose. Examples of chemical groups suitable for this puφose include, but are not limited to, any of the non- conventional amino acid residues listed in Table 2, in particular a D-stereoisomer or a methylated form of a naturally-occurring amino acid listed in Table 1. Most particularly, a derivatized CLIP peptide will comprise at least one D-alanine residue. Additional chemical groups which are useful for this puφose are selected from the list comprising aryl or heterocyclic N-acyl substituents, polyalkylene oxide moieties, desulphatohirudin muteins, alpha-muteins, alpha-aminophosphonic acids, α-aminobutyric acid, L- norleucine, L-norvaline, water-soluble polymer groups such as polyethylene glycol attached to sugar residues using hydrazone or oxime groups, benzodiazepine dione derivatives, glycosyl groups such as beta-glycosylamine or a derivative thereof, isocyanate conjugated to a polyol functional group or polyoxyethylene polyol capped with diisocyanate, amongst others. Accordingly, a CLIP peptide or CLIP analogue which has been derivatized thus, has a longer half-life than a CLIP molecule which comprises the same amino acid sequence but is un-derivatized. Similarly, a CLIP peptide, CLIP analogue or a derivative thereof may be cross-linked or fused to itself or to a protease inhibitor peptide, to reduce susceptibility of said CLIP molecule to proteolysis.

In a most preferred embodiment of the present invention, there is provided an isolated CLIP peptide, CLIP analogue or derivative thereof comprising a sequence of amino acids of at least 9 amino acid residues in length derived from amino acid residues 86-104 of the invariant protein Ii, wherein said sequence is further at least 70% identical to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-54.

For the puφoses of nomenclature, the amino acid sequences set forth in SEQ ID NO: 1- 54 are CLIP peptide and CLIP analogue molecules which are derived from, or correspond to, the region comprising amino acid residues 86-104 of the invariant chain protein Ii. In particular, SEQ ID NO: 1 is a CLIP peptide corresponding to amino acid residues 86-104 of the invariant chain protein Ii, which competes efficiently with antigenic peptides for presentation by MHC class II molecules to T cells, in particular murine I-A^u, I-A^k , I-A^g7 and l-A . SEQ ID NOs: 2-54 are CLIP analogues and derivatives. SEQ ID NOs: 2-19 inclusive correspond to CLIP analogues in which each amino acid residue of the 19 amino acid peptide molecule, wim the exception of alanine at position 9 (amino acid residue 94 in Ii), has been singly-substituted with L-alanine. In the CLIP analogues set forth in SEQ ID NOs: 20-22, the amino acid at position 8 of the CLIP peptide set forth in SEQ ID NO: 1 has been substituted with the non- conventional amino acid residues α-aminobutyric acid (SEQ ID NO: 20), L-norvaline (SEQ ID NO: 21) or L-norleucine (SEQ ID NO: 22). In SEQ ID NOs: 23-25, the amino acid residue at position 14 of the CLIP peptide set forth in SEQ II) NO: 1 has been substituted with o-aminobutyric acid (SEQ ID NO: 23), L-norvaline (SEQ ID NO: 24) or L-norleucine (SEQ ID NO: 25). The amino acid sequences set forth in SEQ ID NOs: 26-44 correspond to derivatized CLIP peptide molecules comprising the region of Ii corresponding to amino acids 86-104 inclusive, wherein a single amino acid residue has been substituted with the non-conventional amino acid D-alanine. The amino acid sequences set forth in SEQ ID NOs: 45-54 correspond to derivative analogues of the CLIP peptide set forth in SEQ ID NO: 1 wherein amino acids have been removed from the amino- and/or carboxy- ter mini thereof.

The CLIP peptides and analogues set forth in SEQ ID NOs: 1-54 are provided for exemplification only. The present invention extends further to CLIP analogues comprising amino acid residues 86-104 of Ii or a part thereof wherein more than a single amino acid residue is altered relative to SEQ ID NO: 1, the only requirement being that said analogue successfully competes for binding of an antigenic peptide to its cognate MHC class II molecule and comprises a sequence of amino acids which is at least 70% identical to a sequence set forth in SEQ ID NOs: 1-54.

A CLIP analogue or a derivative mereof, or a derivative of a CLIP peptide, as generally contemplated by the present invention further encompasses peptides which are capable of competing for the binding of an antigenic peptide to an MHC class II molecule. Preferably, a CLIP analogue or derivative thereof, or a derivative of a CLIP peptide, binds with higher affmity to an MHC class II molecule than a corresponding CLIP peptide.

The ability of a CLIP peptide, analogue or derivative thereof to compete with an antigenic peptide for binding to an MHC class II molecule, is a measure of the affinity of said CLIP molecule for the class II molecule. Whilst not being bound by any theory or mode of action, it seems likely that the CLIP peptide, analogue or derivative occupies the class II peptide binding site, thereby physically precluding entry of the antigenic peptide. In so doing, antigen presentation is inhibited in the presence of the CLIP peptide, analogue or derivative and the immune response to the antigen is attenuated.

Accordingly, a second aspect of the present invention provides an isolated CLIP peptide, CLIP analogue or a derivative thereof which is capable of binding to an MHC class II molecule of mammalian origin.

According to this aspect of the invention, the MHC class II molecule may be any class II haplotype produced by a cell which is mammalian in origin. In a preferred embodiment of the present invention, the CLIP peptide, analogue or derivative thereof is able to compete for the binding of an antigenic peptide to an MHC class II molecule which is murine or human in origin.

In a most particularly preferred embodiment, the MHC class II molecule is selected from the list comprising murine haplotypes I-A^u, I-A\ I-A^g7 and I-A^d and human haplotypes HLA-DR, HLA-DP and HLA-DQ, especially haplotype HLA-DR3.

Also according to this aspect of the invention, the antigenic peptide may be any antigenic peptide which is capable of binding to an MHC class II molecule as contemplated herein, for example myelin basic protein [MBP-(Ac 1-11)], HEL-(46-61) or Ova-(323-339) which bind to murine class II haplotypes I-A^u, I-A^k, I-A⁸⁷ and I-A^d, respectively. It is known to mose skilled in the art that specific antigenic peptides which bind to particular class II molecules may be involved in, or associated with, the aetiology of different autoimmune diseases. The present invention extends to CLIP peptides, analogues and derivative which competes with any antigenic peptide, in addition to MBP-(Acl-l l),

HEL-(46-61) or Ova-(323-339), the only requirement being that said antigenic peptide is capable of eliciting an immune response in a mammalian species in the absence of a

CLIP peptide, analogue or derivative thereof, as determined by assaying specific T-cells.

In a preferred embodiment of the present invention said CLIP peptide CLIP analogue or derivative is further capable of inhibiting the binding of an antigenic peptide to an MHC class II molecule when said CLIP molecule is bound to said MHC class II molecule.

In a more preferred embodiment, the present invention provides a CLIP peptide, CLIP analogue or a derivative thereof which is capable of binding to an MHC class II molecule of mammalian origin and inhibiting the binding of an antigenic peptide to said MHC class II molecule, wherein said CLIP peptide, CLIP analogue or derivative comprises a sequence of amino acids substantially as set forth in any one of SEQ ID NOs: 1-54 or is at least 70 % identical thereto.

According to the embodiments described herein, a CLIP peptide, CLIP analogue or a derivative thereof inhibits the binding of an antigenic peptide to an MHC class II molecule in vitro if it is capable of reducing antigenic T-cell stimulation by said antigenic peptide by at least 10-50%, preferably by at least 50-80% , more preferably by at least 80-90% and even more preferably by at least 90-99% including 100%, when present at a molar concentration which is in the range of equimolar to 250-fold the concentration of antigenic peptide, or in accordance with the Examples provided herein. Those skilled in the an will appreciate that die peptide concentration required to effectively inhibit the binding of an antigenic peptide may vary depending upon the assay conditions used, in particular pH salt concentration, T-cell hybridoma line or antigenic peptide. Furthermore, as in vivo concentrations of antigenic peptide cannot be established easily using state-of-the-art technology, a CLIP peptide, CLIP analogue or derivative thereof is said to inhibit the binding of an antigenic peptide in vivo if it at least capable of such inhibition in vitro or, alternatively, if it is capable of reducing T-cell stimulation by said antigenic peptide in an animal, by at least 10%, preferably by at least 10-50%, more preferably by at least 50-80%, still more preferably by at least 80-90% and even more preferably by at least 90-99% including 100%, when administered to said animal.

In an even more preferred embodiment, the isolated CLIP peptide, CLIP analogue or a derivative thereof further possesses immunomodulatory activity.

The term "immunomodulatory activity" as used herein shall be taken to refer to the ability of a CLIP peptide, CLIP analogue or a derivative thereof to reduce, delay, inhibit or otherwise attenuate an immune response, either in vitro or in vivo, to a foreign or a self antigen. As used herein, a CLIP peptide, CLIP analogue or a derivative thereof possesses immunomodulatory activity if it is at least able to reduce, inhibit, delay or otherwise attenuate any process involved in the elicitation of a T-cell immune response, either in vitro or in vivo, in particular antigen presentation.

In a particularly preferred embodiment, the present invention provides an isolated CLIP peptide, CLIP analogue or a derivative thereof which is capable of binding to an MHC class II molecule of mammalian origin, wherein said CLIP peptide, CLIP analogue or derivative thereof further possesses immunomodulatory activity in T-cell responses which are involved in human autoimmune diseases, for example diabetes or multiple sclerosis, or the equivalent diseases in animals, such as EAE or IDDM.

According to the embodiments described herein, a CLIP peptide, CLIP analogue or a derivative thereof which binds to an MHC class II molecule or at least competes with antigenic peptide for binding to said MHC class II molecule, possesses an affinity thereto which is at least equivalent to the affinity of a CLIP peptide which comprises a sequence of amino acids set forth in SEQ ID NO: 1 , preferably 1- to 2- fold higher, more preferably 2- to 5-fold higher and still more preferably greater than 5-fold higher than the affmity of the CLIP peptide set forth in SEQ ID NO: l.

A further aspect of the present invention provides an isolate CLIP peptide, CLIP analogue or a derivative thereof which comprises a sequence of amino acids of at least 9 residues in length derived from amino acid residues 86-104 of the invariant protein Ii, which is capable of binding to an MHC class II molecule, preferably selected from the list comprising murine I-A^u, I-A^k, I-A*⁷ and 1-A^d or human haplotypes HLA-DR, HLA- DP and HLA-DQ, especially haplotype HLA-DR3, amongst others.

Preferably, said isolated CLIP peptide, CLIP analogue or derivative mereof is at least 10-13 amino acid residues in length, more preferably at least 14-19 amino acid residues in length.

In a particularly preferred embodiment, the present invention provides an isolated CLIP peptide, CLIP analogue or a derivative thereof which comprises a sequence of amino acids of at least 9 residues in length derived from amino acid residues 86-104 of the invariant protein Ii, which is capable of binding to an MHC class II molecule, preferably selected from the list comprising murine I-A^u, I-A\ I-A^g7 and I-A^d or human HLA-DR, HLA-DP and HLA-DQ molecules, especially HLA-DR3, wherein said CLIP peptide, CLIP analogue or derivative is further at least 70% identical to a sequence set forth in any one of SEQ ID NOs: 1-54.

The inventors of the present invention have discovered further mat particular amino acid substitutions performed at precise locations in a CLIP peptide, CLIP analogue or a derivative thereof confer haplotype-specificity on said CLIP molecule or, alternatively, alter the haplotype-specificity of a haplotype-specific CLIP molecule. Such an advance in the art provides the means for targeting specific MHC class II molecules in diagnostic or therapeutic applications. In particular, the inventors have discovered d at haplotype- specific or narrow-spectrum high affinity CLIP analogues may be produced by performing any of the amino acid substitutions set forth in Table 3.

Furthermore, haplotype-specificity may also be conferred by deleting specific amino acid residues from a CLIP peptide, CLIP analogue or derivative thereof, for example by deleting amino acid residues corresponding to residues 86-88 in combination with residues 102-104 of Ii, or residues 86-89 in combination with residues 101-104 of Ii, or residues 86-89 of Ii alone.

The present invention encompasses any amino acid substitutions or deletions from the

CLIP peptide set forth in SEQ ID NO: l wherein said substitution or deletion confers upon or alters haplotype-specificity of the CLIP analogue or derivative produced thereto. TABLE 3

Amino acid in Ii Substituent amiiio acid in CLIP analogue.

Lys-86 Ala, Dal

Pro-87 Ala, Dal

Val-88 Ala, Dal Ser-89 Ala, Dal

Met-91 Ala

Met-93 Ala, Abu

Pro-96 Ala

Leu-97 Ala Met-99 Ala, Abu

Arg- 100 Ala

Accordingly, a further aspect of the present invention is directed to an isolated haplotype- specific CLIP peptide, CLIP analogue or a derivative thereof.

The term "haplotype-specific" as used hereinafter shall be taken to refer to the ability of a CLIP peptide, CLIP analogue or a derivative thereof to bind with high affinity to a restricted set of MHC class II molecule haplotypes. Preferably, a haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof will bind with high affinity to a single MHC class II molecule haplotype and with only low affmity to other MHC class II molecule haplotypes from the same species. For example, the CLIP analogue set forth in SEQ ID NO: 12 comprises L-alanine at position 12 instead of L-leucine and binds with high affmity to the murine MHC class II haplotype I-A", but with only low affinity to the murine MHC class II haplotypes I-A and I-A^d, when compared to the binding of the CLIP peptide set forth in SEQ ID NO:l. In contrast, the CLIP analogue set forth in SEQ ID NO: 11 has enhanced affinity for the murine MHC class II haplotype I-A\ while the CLIP analogues set forth in SEQ ID NO:9 and SEQ ID NO:20 have enhanced affinity for the murine haplotypes I-A^k and I-A^u, compared to the affinity of the CLIP peptide set forth in SEQ ID NO: 1.

In a preferred embodiment of the present invention, there is provided a haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof which comprises a sequence of amino acids selected from the list of amino acid sequences comprising SEQ ID NOs:2, 3, 4, 5, 7, 9, 11 , 12, 13, 15, 16, 20, 23, 26, 27, 28 or 29, or is at least 70% identical thereto.

More preferably, said haplotype-specific CLIP peptide, CLIP analogue or derivative thereof further possesses immunomodulatory activity, for example in T-cell responses which are involved in autoimmune diseases, in humans and other mammals, for example diabetes, multiple sclerosis, EAE or insulin-dependent diabetes melitis (IDDM), amongst others.

The high-affinity haplotype-specific CLIP analogues discussed herein are also useful as diagnostic reagents for determining the contribution of specific MHC class II molecules to autoimmune diseases, or in determining an individual's MHC class II haplotype for me puφose of assessing said individual's genetic susceptibility to a particular autoimmune disease.

Accordingly, a further aspect of the present invention contemplates a method for identifying a specific MHC class II molecule haplotype in a biological sample, said mediod comprising isolating said biological sample and contacting same or purified or partially-purified a fraction thereof with a binding-effective amount of an isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof and then detecting said binding.

The biological sample may be any tissue sample which contains MHC class II molecules, such as blood, bone marrow and other lymphoid organs.

According to this aspect of the invention, said haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof is labelled with a reporter molecule which is capable of giving an identifiable signal, such as an enzyme tag (eg. horseradish peroxidase), fluorescent tag, biotinylated molecule, or a radioisotope such as ³H, ¹⁴C ^{1 i} lor³⁵ S. Methods for the detection of such reporter molecules are well-known in the art.

Preferably, said haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof comprises a sequence of amino acids which is substantially the same as, or at least 70% identical to any one of the sequences set forth in SEQ ID NOs: 2-5, ^" , 9, 11-13, 15-16, 20, 23 or 26-29.

The present invention further extends to diagnostic kits for determining the presence of an MHC class II molecule haplotype in a biological sample, comprising several first compartments, each of which is adapted to contain an isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof, optionally labelled with a reporter molecule. Preferably, said haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof comprises a sequence of amino acids which is substantially the same as, or at least 70% identical to any one of the sequences set forth in SEQ ID NOs: 2-5, 7, 9, 11-13, 15-16, 20, 23 or 26-29.

A further aspect of the invention is directed to an isolated CLIP peptide, CLIP analogue or a derivative thereof which comprises a sequence of amino acids set forth in any one of SEQ ID NOs: 1-54 or is at least 70% identical thereto.

Preferably, said CLIP peptide, CLIP analogue or derivative is capable of binding to an MHC class II molecule which originates from a mammalian species selected from the list comprising human, mouse, rat, rabbit, pig, horse, cattle, guinea pig or other domesticated mammal. More preferably, said CLIP peptide, CLIP analogue or derivative is capable of binding to said MHC class II molecule in a haplotype-specific manner.

Even more preferably, said CLIP peptide, CLIP analogue or derivative further possesses immunomodulatory activity. Still more preferably, said isolated CLIP peptide, CLIP analogue or a derivative thereof possesses immunomodulatory activity in relation to an autoimmune disease in humans or other mammals, for example diabetes, multiple sclerosis, EAE or IDDM, amongst others. Said CLIP molecules are also useful in suppressing immune responses generally, as required during and following transplantation procedures.

The CLIP peptides, CLIP analogues and derivatives thereof which are described herein and/or exemplified by any amino acid sequence set forth in SEQ ID NOs: 1-54, are useful as therapeutic agents which inhibit, delay, reduce or otherwise attenuate an immune response in a mammal, preferably a human or a domesticated mammal selected from the list comprising mouse, rat, rabbit, guinea pig, horse, cat, dog, pig or cow, amongst others. The inherent properties of a CLIP peptide, CLIP analogue or a derivative thereof described herein, in particular immunomodulatory activity and haplotype-specificity, make the subject molecules especially suitable as merapeutic agents in the treatment of autoimmune diseases in humans, for example multiple sclerosis or diabetes, or an equivalent disease in a domesticated mammal.

The present invention provides an opportunity to reduce, inhibit or otherwise attenuate an immune response in humans and animals such as domestic animals selected from the list comprising mice, rats, rabbits, dogs, cats, horses, pigs, cattle, guinea pigs or sheep, amongst others, by the administration of a CLIP peptide, CLIP analogue or a derivative mereof, in particular a CLIP peptide or CLIP analogue disclosed herein, either directly or via a genetic sequence encoding same. This is of particular importance since a CLIP peptide, CLIP analogue or derivative thereof is only weakly antigenic or preferably immunologically unreactive in which case it does not elicit a T-cell or B-cell response, when administered to a human or other mammal. The CLIP peptide, CLIP analogue or derivative thereof may be administered alone, in combination with a protectant to prevent degradation of said CLIP molecule, and/or as a fusion molecule. Administration may be via an attenuated virus, recombinant viral vector or bacterial vector or may be via administration of the CLIP peptide, CLIP analogue or derivative by, for example, injection or oral ingestion (e.g. in medicated food material).

A further aspect of the present invention extends to a pharmaceutical composition for use in a human or a domesticated animal to reduce, delay, inhibit or otherwise attenuate an immune response therein, said composition comprising an isolated CLIP peptide, CLIP analogue or a derivative thereof in combination with a pharmaceutically-acceptabie carrier, diluent or excipient.

In a preferred embodiment, the present invention provides a pharmaceutical composition for use in a human or a domesticated animal to reduce, delay, inhibit or otherwise attenuate an immune response therein, said composition comprising an isolated CLIP peptide, CLIP analogue or a derivative thereof in combination with a pharmaceutically- acceptabie carrier, diluent or excipient, wherein said CLIP peptide, CLIP analogue or derivative thereof comprises a sequence of amino acids which is substantially the same as one or more of the sequences set forth in SEQ ID NOs: 1-54 or is at least 70% identical thereto.

More preferably, said CLIP peptide, CLIP analogue or derivative CLIP molecule is haplotype-specific .

Optionally, the pharmaceutical compositions discussed supra may further comprise a protectant to protect said CLIP molecule from degradation by enzymes within the human or domesticated animal to which it is administered. Alternatively, or in addition, said CLIP molecule may be in a form to increase its half-life following ;administration to a human or domesticated animal, for example it may be encapsulated in a liposome or other protease-resistant form. It is also possible to derivatize a CLIP peptide, CLIP analogue or derivative thereof, as discussed supra for this puφose.

Preferably, where the composition comprises a synthetic peptide, analogue or derivative thereof, the pharmaceutical composition is injected or orally administered. Where the composition comprises genetic material encoding a CLIP peptide, CLIP analogue or a derivative thereof, it is administered as part of a viral vector, live viral vector or live bacterial vector.

Conditions in humans and other animals for which treatment might be required include any condition where the specific or general inhibition, reduction or attenuation of an immune response is indicated, such as in the treatment of an autoimmune disease or during or following transplantation procedures. Specific autoimmune diseases in which the present invention is likely to be administered include, but are not limited to, cancer, multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), diabetes, insulitis, or associated diseases thereto, amongst others. The pharmaceutical compositions described herein may also contain other active molecules such as antibiotics.

The present invention extends to pharmaceutical compositions comprising combinations of CLIP peptides, CLIP analogues or derivatives thereof which are more effective than single CLIP molecules in attenuating the immune response of a human or domesticated animal, in particular where a combination of haplotype-specific CLIP analogues is utilised to reduce immune responses involving multiple MHC class II molecules.

The present invention merefore provides a pharmaceutical composition comprising an immunomodulatingly effective amount of a CLIP peptide, CLIP analogue or a derivative thereof, or a genetic construct capable of expressing same, in combination with a pharmaceutically-acceptabie carrier, diluent or excipent.

The active ingredient(s) of the pharmaceutical composition is/are contemplated to exhibit excellent activity in inhibiting, delaying, reducing or otherwise attenuating an immune response in an animal species and in particular a human or domesticated animal when administered in an immunomodulatingly-effective amount, which depends on the particular case. The variation depends, for example, on the CLIP peptide, CLIP analogue or derivative CLIP molecule and, in some cases, the antigenic peptide which is involved in stimulating the immune response which is intended to be attenuated. For example, from about 0.1 μg to about 20 mg of a particular CLIP peptide, CLIP analogue or derivative thereof, which may be combined with other CLIP molecules, per kilogram of body weight per day, may be required. Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered in one or more of daily, weekly or monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The active compound may be administered by injection or by oral ingestion in any convenient manner or may be administered via a genetic sequence such as in a viral or bacterial vector.

The active compounds may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures mereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for parenteral administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ehanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures mereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecitiiin, by the maintenance of the required particle size in the case of dispersion and by die use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacte.rial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and me like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example.

Sterile injectable solutions are prepared by incoφorating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilisation. Generally, dispersions are prepared by incoφorating the various sterilised active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for die preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

Carriers and/or diluents suitable for pharmaceutical use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and me like. The use of such media and agents for pharmaceutically-active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incoφorated into the compositions. The latter is particularly contemplated as far as the present invention extends to multivalent vaccines or multi-component cytokine molecules.

A further aspect of die present invention extends to a method of delaying, inhibiting, reducing or otherwise attenuating an immune response in a human or domesticated animal comprising administration diereto of an immunomodulatingly-effective amount of a pharmaceutical composition comprising a CLIP peptide, CLIP analogue or a derivative thereof in combination with a pharmaceutically-acceptabie carrier, diluent or excipient. Preferably, the immune response which is to be attenuated by administering a CLIP peptide, CLIP analogue or derivative thereof, is associated with an autoimmune disease. In such cases, the CLIP molecule administered may be haplotype-specific, producing inhibition of antigen binding to a restricted class of MHC class II molecules in said human or animal subject. In such cases, the method of the present invention will generally be preceded by determination of the contribution of specific MHC class II molecule haplotypes to the conditions to be treated. In other cases, in particular in relation to transplantation procedures, it may be desirable to attenuate immune responses generally in a subject human or animal species. In such cases, a pharmaceutical composition comprising a non-haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof as an active ingredient or alternatively, multiple haplotype-specific CLIP molecules may be preferred.

The present invention is further described in the following Examples. The embodiments exemplified hereinafter are in no way to be taken as limiting die subject invention.

EXAMPLE 1 Cell Lines, Culture Conditions, and Peptides

B-cell lines A20 (I-A^d), CH27 (I-A^k), and M12.C3 transfected with cDNAs encoding I- A^u and α and β chains (Gautam et al, 1992) were used as APCs. An ovalbumin (Ova) 323-339 peptide-specific, I-A^d-restricted T-cell hybridoma, 3DO 5.4.8 (Buus et al , 1986), a myelin basic protein (MBP) N-acetyl 1-11 (Acl-11) peptide-specific. I-A^u- restricted T-cell hybridoma, 1934,4 (Gautam et al , 1992), and a hen egg lysozyme (HEL) 46-61 peptide-specific, I-A^k-restricted T-cell hybridoma, 3A9 (Babbitt et al , 1985), were used in this s dy. Normally cells were maintained in RPMI 1640 with 10% fetal bovine serum, streptomycin (100 μg/ml), penicillin (100 units/ml), 2mM glutamine, and 0.05 mM 2-mercaptoetiιanol. Cells transfected wim cDNA encoding I-A^u or site- specifically mutated I-A^u were cultured in medium with G418 (200 μg/ml). The drug was removed from the cells prior to assays. Peptides were synthesized by standard tert- butoxycarbonyl chemistry and by purified HPLC, amino acid composition was determined, and molecular weight was confirmed by mass spectroscopy.

EXAMPLE 2 T-cell hybridoma assays

T-cell hybridoma assays were performed as described by Gautam et al , (1992). Murine B-cell lines A20 (I-A^d), CH27 (I-A"), M12.D and 1-5.4 (the latter two derived from M12.C3 (see Gautam et al , 1992 for details) transfected with cDNA encoding I-A^d or I-A^u α and β chains respectively) were used as APCs. Antigen was presented to the appropriate peptide-specific T-cell hybridoma, 3DO.548 (Ova 323-339-specific, I-A^d restricted), 349 (HEL 46-61 -specific, I-A^k restricted) or 1934.4 (MBP Ac 1-11 -specific, I-A^u restricted) as follows: T-cell hybridomas (2.5 x 10*) wre co-incubated with APC (2.5 x 10⁴), a suboptimal dose of antigenic peptide and a dilution series of competitor CLIP peptide. After 24 hours, 50μL of supernatant was harvested from each well and tested for IL-2 activity using the IL-2-dependent cell line, HT-2. Proliferation of HT-2 was measured from ³[H]-mymidine incoφoration as previously described by Gautam et al, (1992). All cell lines were maintained at 37°C in a 5 % CO₂ atmosphere in RPMI 1640 medium supplemented with 10% FCS, 0.05 mM 2-ME. 2mM glutamine, 100 U/mL penicillin and lOOμg/mL streptomycin. Growth medium for the cell lines transfected with cDNA encoding class II genes was also supplemented with G418 (200μg/mL).

EXAMPLE 3 Cell Surface Peptide-Binding Assay

Cells (10⁵) expressing I-A! or I-A were incubated in normal culture medium with biotinylated antigenic peptide or alternative, biotinylated CLIP peptide set form in SEQ ID NO: 1, for 18-20 hr. Cells were washed tiiree times with ice-cold phosphate-buffered saline containing 0.1 % bovine serum albumin and 0.05% sodium aside. Fluorescein isothiocyanate-conjugated streptavidin (Pierce) was then added and incubated for 30-40 min on ice. After three washes, cells were analyzed by flow cytometry (Gautam et al , 1992). For inhibition of binding by competitor peptides, competitor peptides and biotinylated Ova-(322-339) were coincubated for the entire duration of the incubation. Specific binding is expressed as percentage of inhibition of control binding of biotinylated peptide in the absence of competitor (Buus et al , 1986). For inhibition of binding of CLIP peptide by D-alanine-substituted CLIP analogues, a dilution series of competitor peptides and biotinylated CLIP peptide were coincubated for the duration of the incubation.

EXAMPLE 4 Biochemical Analysis of Purified I-A⁴ Molecules

I-A^u molecules were affmity purified from 10¹⁰ cells as described (Gautum et al , 1992). Samples (10μl) of I-A^u (pH 6.35-6.5) were acidified with 3.25 μl of a pH 3 solution of glycine. Immediately after acidification, samples were exposed to 5 μl of 5 mM peptide solutions (pH 7). This treatment resulted in a final pH of 4.5-5. Reaction mixtures were incubated at 37 ^βC for 2 hr and neutralized wiui 1.5 μl of 2 M Tris* HCl (pH 8.5). Samples were run under nonreducing conditions in SDS/polyacrylamide gels at 4°C, blotted onto nitrocellulose membranes for 1-2 hr, blocked with 10% milk powder, and hybridized with I-A^u-specific antibody 10.3.6 (provided by H.O.M.) OVERNIGHT. Membranes were washed with 10% milk powder and incubated with horseradish peroxidase-linked anti-mouse IgG (Amersham) for 45 min. Membranes were developed with an enhanced chemiluminescence (ECL) kit (catalogue no. RPN 2109: Amersham) according to me standard protocol. Membranes were exposed to x-ray films for up to l hr.

EXAMPLE 5 Circular dichroism spectra of CLIP analogues

Peptides were dissolved into 50% (v:v) trifluoroemano lOmM sodium phosphate, pH 7.4 at a concentration of 50 μM and placed into cells with a 1mm path length. Circular dichroism was recorded at 25 ^βC using a Jobin Yvon CD6 (Cedex, France) with time averaging over 2 seconds per 0.2nm increment. EXAMPLE 6 Inhibition of biotinylated antigenic peptide binding by CLIP peptides and analogues

The CLIP peptide set forth in SEQ ID NO: 1 was used as a competitor peptide with antigenic peptides to determine its binding to various murine MHC class II molecules. T-cell hybridoma assays were performed as described in Example 2. As shown in figure 1 , the CLIP peptide set forth in SEQ ID NO: 1 (hereinafter referred lo as "CLIP-(86- 104)" and designated as "CLIP" in the Figures) competes with antigenic peptides for presentation by murine I-A\ I-A^d and I-A^u MHC class II molecules to antigen-specific T-cell hybridomas.

To determine the requirements for binding of CLIP-(86-104) to I-A, CLIP analogues with single amino acid substitutions with alanine at each position were synthesized and their ability to compete for antigen presentation with antigenic peptides in T-cell hybridoma assays was determined. Strikingly, peptides with substitutions of alanine for methionine in CLIP-(86-104) at positions 93 and 99 (M93A and M99A) displayed enhanced binding as measured by their ability to compete for antigen presentation with MBP-(Acl-l l), HEL-(46-61), and Ova-(323-339) peptides on cells expressing I-A^u (Fig. la), I-A^k (Fig. lb), and I-A (Fig. Ic), respectively. Despite certain haplotypic differences tiiere are clearly general similarities in the way these peptides competed for presentation by me various I-A molecules.

A similar pattern was observed when alanine-substituted CLIP analogues were tested for binding to I-A^u and I-A^d in a cell-surface flow cytometry-based assay (Gautam et al , 1992). An I-A^-binding peptide, Ova (323-339), competed at least as well as CLIP-(86- 104). Suφrisingly, the majority of the other alanine-substituted peptides bound essentially like CLIP-(86-104) in both assays (Fig. 1 and Table 4). TABLE 4

Inhibition of biotinylated Ova-(322-339) binding by alanine-substituted CLIP-(86-104)

% inhibition

Peptide I-A^u I-A^d

CLIP-(86-104) 38.6 64.0

M91A 39.1 58.3

R92A 13.6 62.0

M93A 46.0 72.7

P96A 60.8 70.1

L97A 37.5 50.6

M99A 72.7 77.9

Ova-(323-339) 52.8 58.3

Percent inhibition of cell surface class II (I-A^u or I-A^d) binding of biotinylated Ova-(322- 339) is shown in Table 4. B-cells expressing I-A^u or I-A^d were incubated with biotinylated Ova-(322-339) (15 μM) either alone or with competitor peptides (100 μM). Peptides with critical substitutions in CLIP-(86-104) are shown. Control binding (as measured by mean fluorescence) of biotinylated Ova-(322-339) to I-A^u and I-A^d was 106 with a background of 5.98 and 53.5 with a background of 13.2, respectively. All peptides have been tested in mis assay at least five times, competition with only ken CLIP analogues is shown because all otiier alanine-substituted peptides competed essentially like wild-type CLIP-(86-104).

EXAMPLE 7 Effects of substitutions at positions 93 and 99 in CLIP analogues

To investigate die enhanced binding to I-A observed when methionine-93 and methionine-99 were replaced by alanine and to determine its implications for CLIP binding, sets of CLIP-(86-104) peptides with multiple substitutions of hydrophobic residues at these positions were synthesized. If the presence of methionine at 93 and 99 modulates binding due to steric occlusion, and higher binding affinity results when this repulsion is obviated by alanine substitutions, men incoφoration of residues with intermediate steric bulk might result in intermediate binding properties. In this case, one can argue that the CLIPs of Ii have evolved to have an "intermediate" affinity, so tfiat tiiey can be more easily displaced by peptides generated in endosomes. Thus, peptides with norleucine (Nle), norvaline (Nva), and α-aminobutyric acid (Abu) at positions 93 and 99 in the CLIP sequence were synthesized. These substitution'} provide residues approximating to the side-chain length of methionine decreasing progressively by a single memylene group to that one greater than alanine. Fig. 2 shows completion for presentation by three different cell lines expressing I-A^u, I-A\ or I-A^d. In each case, there appeared to be reduced binding as residues with longer side chains were used. For example, peptides widi Nle at 93 and 99 in place of methionine (m93Nle and M99Nle) competed like the wild type CLIP-(86-104) for all I-A molecules used in presentation assays. On the other hand, peptides with Abu residues at 93 and 99 (M93Abu and M99Abu) competed for presentation essentially like M93A and M99A. These results indicated diat steric hindrance by bulky residues, such as methionines, at positions 93 and 99 may modulated die interaction wim I-A molecules. Moreover, these data provide evidence tiiat CLIPs with methionine residues at 93 and 99 bind with an "intermediate", or submaximal affinity. Three omer me ionine residues in me CLIP-(86- 104) sequence have no general effect on I-A binding.

The striking pattern of these same residues (metiιionine-93 and -99) conferring parallel properties of CLIP binding to various MHC class II molecules (Fig. 3), argue that CLIPs bind in a distinct manner from antigen-derived peptides. This is furdier substantiated by our biochemical analysis on purified I-A^u class II molecules, which showed that Ova- (323-339) prevents SDS-stable high molecular mass (110-120 kDa) class II aggregation whereas CLIP-(86-104) does not (Fig. 3). EXAMPLE 8 Competition for presentation by CLHM86-104) on site-specific I-A^u mutants

We next asked whemer CLIP-(86-104) interacts at distinct locations or residues in I-A molecules. The binding region in class II molecules is not known for the Ii or CLIP association. To address this, we have utilized several sets of site-specific mutants in I-A^u class II molecules. Aspartic acid, a conserved residue at position 59 in the α chain, was mutated to either lysine (D59K) or alanine (D59A). Threonine, a nonconserved residue at position 86 in the β chain, was mutated to either serine (T86S) or leucine (T86L) (Fig. 4a). All the mutants present MBP-(Acl 11) peptide essentially like wild-type I-A^u. Therefore, we asked whether CLIP-(86-104) would compete similarly for binding to these mutants. If CLIP-(86-104) failed to compete for binding to any of the mutants, this would suggest that the mutated residue in I-A^u was involved in CLIP-(86-104) binding. The results obtained employing me does of CLIP-(86-104) that competed most effectively with MBP-(Acl-l 1) in a T-cell hybridoma assay are shown in Fig. 4. CLIP-(86-104) inhibited me T-cell hybridoma response by up to 90%. The same does of CLIP-(86-104) peptide, when used as a competitor on the D59K mutant, failed to compete with MBP- (Acl-11). At the same time, CLIP-(86-104) competed well on D59A mutants, although not as well as on wild-type I- A" (Fig. Ab). In a separate experiment, CLIP-(86-104) competed with MBP-(Acl-l 1) very poorly on me T86L mutants but competed normally on the T86S mutant (Fig. Ac). These results indicated that CLIP-(86-104) interacts in proximity to aspartic acid-59 in die α chain and tiιreonine-86 in β chain of I-A^u. Since the site-specific mutants present MBP-(Acl-ll) normally, different amino acids in I-A^u are involved in binding to CLIP-(86-104) and MBP-(Acl-l 1). Mutations at two other conserved positions in I-A^u (leucine to glutamic acid at position 55 in the α chain and tyrosine to phenylalanine at position 60 in die β chain) did not affect the binding of CLIP-(86-104) or MBP-(Acl-l 1) (data not shown).

Interestingly, crystal structures of MHC class II molecules (Brown et al , 1993; Stern et al. , 1994) predict aspartic acid at 59 in the α chain to be at the back of die α-helix and not in me binding groove. Therefore, it is not surprising that me presentation of MBP- (Acl-11) peptide is not affected by mutation at mis position. However, it is possible mat substitutions at 59 in the α chain perturb me local conformation in the class II molecules such mat it affects only the CLIP binding. On me other hand, threonine at position 86 in the β chain is predicted to be on d e penultimate mm of the α-helix and may form the outermost region of the peptide-binding groove. Since T86L mutation does not affect the presentation of MBP-(Acl-l l) but affects me ability of CLIP-(86-104) to compete, it is conceivable that CLIPs bind in proximity to this threonine residue in me β chain, thereby blocking premature entry of antigenic peptide into me binding groove of MHC class II molecules. Perhaps it is also significant mat bo aspartic acid at 59 h the α chain and threonine at 86 in the β chain are located on the opposite sides of the binding groove. A crystal structure of a class II wim Ii or CLIPs is required to confirm the nature of this interaction unequivocally.

EXAMPLE 9 Binding to MHC class II molecules by D-alanine-substituted CLIP analogues

In order to examine the general pattern of CLIP binding to class II and to determine die specific effect of backbone stereochemistry on the CLIP-class II interaction, CLIP 86-104 peptides wim single D-alanine amino acid substitutions were syndiesized. The ability of these peptides to compete widi antigen peptides for antigen presentation in T-cell hybridoma assays was determined and compared to Uiat of side chain- substituted CLIP peptides (Fig. 5). A striking feature in tiiese assays was the loss of competitive ability against all I-A haplotypes tested when D-alanine substitutions fell in the central region of the peptide (approximately between residues 90-100 allowing for haplotypic differences) (Fig. 5a, b, c). This trend was not apparent in me assay series of the control L-alanine-substituted peptides (Fig. 5d, e.fand Example 6), indicating a negligible effect of specific side chains on these observations.

Similar data were obtained using a CLIP analogue to compete for binding of biotinylated CLIP-(86-104) peptide (SEQ ID NO: 1) in a flow cytometry-based cell surface peptide binding assay, as described in Example 3 (see Fig. 6). The substitution by D-stereoisomers within a peptide sequence has been established to lead to a disruption of secondary structure. The difference in me binding capacity of D- alanine-substituted CLIP analogues versus those substituted wim L-alanine thus may arise due to a disruption of native backbone structure. To substantiate tiiis idea, circular dichroism (CD) scans of the single D-alanine substituted CLIP peptides were performed. These CD curves show that D-alanine substitutions disrupt the structure of the peptide (Fig. la), particularly between residues 90-100. Under strong α-helix-promoting conditions, the difference CD reveals a marked weakening of features in the A94D-Ala peptide characteristic of α-helical conformation (Fig. lb). The pattern of secondary structure abrogation in the set of configurationally-substimted peptides correlates well with the disruption of binding ability to class II molecules and suggests that CLIP may acquire a periodic conformation with some form of helicity upon binding to class II.

EXAMPLE 10 Binding of truncated CLIP peptides to MHC class II molecules

To characterise further the positive biding determinants in CLIP, a set of truncated peptides were synmesised from which residues were sequentially removed from each end (SEQ ID NOs: 45-49). These truncated peptides were then examined for d eir binding ability to I-A molecules using die competitive cell surface binding assay (Fig. 8a). The shorter CLIP analogues set form in SEQ ID NOs: 45-46 consistently compete better tiian CLIP-(86-104) for cell surface binding to I-A^u and I-A^k molecules, suggesting mat ese shorter peptides have a higher affmity for two of me diree I-A molecules tested. However, when die length of die CLIP analogue was truncated to 12 amino acids or below, me ability of these peptides to compete for binding was greatly reduced. This is best illustrated by die inability to detect any binding of me CLIP analogue set forth in SEQ ID NO: 49 to I-A^u and I-A^d molecules and very little to I-A^k.

Substitution of several native N-terminal CLIP residues wim D-alanine or truncation increases uie binding of CLIP analogues to I-A molecules as shown in Fig. 5a, b, c, Fig.

6 and Fig. 8α. By contrast, substitution of die last tiiree C-terminal native residues with D-alanine has no marked effect on CLIP binding. Therefore, it is conceivable that die native N-terminal residues are important in making mis peptide of a binding affinity suitable for easy release when the class II molecules encounter potential antigen-derived peptides in die acidic endosomal compartments. To examine mis more precisely, a set of 15 residue CLIP analogues was synthesised (SEQ ID NOs: 50-54). This particular lengm was chosen since it represented the minimum length within the native CLIP sequence which bound efficiently and competed well wim the antigenic peptide. Competitive cell surface binding assay (Fig. 8b) revealed that those peptides containing all or part of the original N-terminal sequence (SEQ ID NOs: 50-52 inclusive). Possibly, these N-terminal residues serve to distinguish CLIP binding from that of the antigenic peptide, for example in terms of facile peptide displacement in the endosomes and the general inability of die CLIP-class II complexes to form SDS-stable dimers.

In this study we have shown mat interactions determined by the backbone ramer than those of specific side chains dominate the binding properties of CLIP to I-A class II molecules. The notion tiiat CLIP adopts a structure of periodic disposition of side chains for binding to class II correlates wim data available for the antigenic peptide (Stern et al , 1994) and is compatible with bodi peptides vying for the same or similar binding site within me MHC class II molecule. The X-ray crystallographic structure of influenza virus haemagglutinin peptide (306-318) widiin me peptide binding groove of HLA-DR 1 reveals that die bound peptide assumes a periodic structure in die form of a type II polyproline helix (Stern et al , 1994). Without being bound by any theory or mode of action, CLIP may bind promiscuously to I-A allelic variants by adopting a conformation with minimal deleterious contacts of side chains with residues of die peptide binding groove. In tiiis instance it is attractive to speculate tiiat it would be advantageous to project every second or tiiird side chain out of the groove to reduce me possibility of such conflicts while, per contra, maximising potential for backbone contacts. If an extended β-sheet-Iike conformation was assumed, only every alternate side cltain could project, increasing die overall steric interactions between die residues of CLIP and peptide- binding groove while minimising backbone contacts. In consideration of this, a periodic pattern where every tiiird or fourth residue displays either increased or decreased binding is visible for each haplotype when the native CLIP residues were singly substituted with L-alanine (Fig. 5d, e, f).

EXAMPLE 11 Use of CLIP analogues in the treatment of diabetes

Insulin-dependent diabetes mellitus (IDDM) is widely believe to be an autoimmune disease that affects 0.5% of Caucasians. IDDM results from autoimmune destruction of the insulin-secreting B-cells of die pancreas. In addition to lymphocytic infiltration (insulitis), patients wim IDDM have autoantibodies directed against the components of the islet cells. Several B-cell proteins have been identified as candidate autoantigens. The non-obese diabetic (NOD) mouse is a murine model for spontaneous IDDM.

Having determined me binding of CLIP to NOD-linked MHC class II molecules and also its immunogenicity, newborn NOD mice are administered widi a selected dose of CLIP analogues in me presence of a pharmaceutically-acceptabie carrier, diluent or excipient. Effect of mis neonatal treatment on adult diabetes is then monitored.

EXAMPLE 12 Use of CLIP analogues in the treatment of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) occurring in (Pl/J x SJL/J) Fi mice is a useful animal model for studying human multiple sclerosis. The EAE model of human MS (discussed above) is used to determine whemer CLIP and related peptides can treat or/and block me onset of EAE. EAE in mice is induced by immunizing mice with myelin basic protein (MBP). MBP is constitutively expressed in the brain and has been implicated as one of die self antigens in causing EAE and even MS . Like diabetes, MS is also a genetic disease. Self-reactive T-cells (and presumably B-cells), specific for self antigens of central nervous system (CNS) origin have been implicated in causing MS. Self MS class II molecules present myelin or brain antigens to these auto-aggressive T- cells. In order to test whether CLIP peptides and CLIP analogues are effective against EAE, mice are pretreated wim these peptides, administered in a pharmaceutically-acceptabie carrier, diluent or excipient, for various lengths of time. Mice are then immunised with MBP for me induction of EAE. Effect of pretreatment with CLIP peptides is then monitored.

Furthermore, mice that have already developed EAE are treated with various doses of CLIP peptides and CLIP analogues in saline or in a pharmaceutically-acceptabie carrier, adjuvant or excipient as described above for NOD diabetes. Mice are monitored to determine the effect of this treatment on the disease process.

Those skilled in the art will appreciate that die invention described herein is susceptible to variations and modifications otiier tiian those specifically described. It is to be understood tiiat die invention includes all such variations and modifications. The invention also includes all of the steps, features compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

REFERENCES:

1. Anderson, M.S. , Swier, K., Arneson, L. & Miller, J.J. exp. Med. 178, 1959- 1969 (1993).

2. Babbitt, B.P., Allen, P.M., Matsueda, G., Haber, E. & Unanue, E.R. Nature (London) 317, 359-362.

3. Blum, J.S. & Cresswell, P. Proc. natl. Acad. Sci. U.S.A. 91, 2171-2175 (1988).

4. Brown, J.H., Jardetzky, T., Gorga, J.C., Stern, L.J. , Urban, R.G., Strominger, J.L. & Wiley, D.C. Nature (London) 364, 33-39 (1993).

5. Buus, S. , Sette, A., Colon, S.M., Jenis, D.M. & Grey, H.M. Cell 47, 1071- 1077 (1986).

6. Cresswell, P. Annu. Rev. Immunol 12, 259-293 (1994).

7. Gautam, A.M., Pearson, C.I., Smilek, D.E., Steinmann, L. & McDevitt, H.O.J. exp. Med. 176, 605-609 (1992).

8. Riberdy, J.M., Newcomb, J.R., Suman, M.J., Barbosa, J.A. & Cresswell, P. Nature 360, 474-476 (1992).

9. Roche, P.A. & Creswell, P. Proc. natl Acad. Sci. U.S.A. 88, 3150-3154 (1991).

10. Sant, A.J. & Miller, J. Current Opinion in Immunol 6, 57-63 (1994).

11. Sette, A. , Ceman, S., Kubo, R.T., Sakaguchi, K., Appella, E., Hunt, D.F., Davis, T.A., Michel, H., Shabanowitx, J., Rudersdorf, R. , Grey, H.M. & -44- DeMars, R. Science 258, 1801-1804 (1992).

12. Stern, L.J., Brown, J.H., Jardetzky, T.S., Gorga, J.C, Urban, R.G., Strominger, J.L. & Wiley, D.C. Nature 368, 215-221 (1994).

13. Teyton, L., O'SuUivan, D., Dickson, P.W., Lotteau, V., Sette, A., Fink, P. & Peterson, P.A. Nature 348, 39-44 (1990).

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The Australian National University

(ii) TITLE OF INVENTION: CLIP ANALOGUES AND AUTOIMMUNE DISEASE

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Lys Pro Dal Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15 Met Ser Met

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Lys Pro Val Dal Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO-.30:

Lys Pro Val Ser Dal Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Ser Met (2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Lys Pro Val Ser Gin Dal Arg Met Ala Thr Pro Leu Leu Met Arg Pro

1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Lys Pro Val Ser Gin Met Dal Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Lys Pro Val Ser Gin Met Arg Dal Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Lys Pro Val Ser Gin Met Arg Met Dal Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: Lys Pro Val Ser Gin Met Arg Met Ala Dal Pro Leu Leu Met Arg Pro

1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Dal Leu Leu Met Arg Pro 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Dal Leu Met Arg Pro 1 5 10 15

Met Ser Met (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Dal Met Arg Pro 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Dal Arg Pro

1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Dal Pro

1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Dal 1 5 10 15

Met Ser Met

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Dal Ser Met

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Dal Met

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro

1 5 10 15

Met Ser Dal

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

Met Ser

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Val Ser Gin Met Arg Met Ala Thr Pro Lβu Leu Met Arg Pro Met 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro

1 5 10

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

Met Arg Met Ala Thr Pro Leu Leu Met 1 5

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Lys Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg

1 5 10 15

( 2 ) INFORMATION FOR SEQ ID NO : 51 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 15 amino acids

(B) TYPE : amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

Pro Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Val Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro Met 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Ser Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro Met Ser 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

Gin Met Arg Met Ala Thr Pro Leu Leu Met Arg Pro Met Ser Met 1 5 10 15

Claims

CLAIMS:

1. An isolated CLIP peptide, CLIP analogue or a derivative thereof comprising a sequence of at least 9 amino acid residues derived from amino acid residues 86-104 of the invariant protein Ii.

2. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 1 wherein the sequence comprises at least 10-13 amino acid residues in length.

3. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 1 or 2 wherein the sequence comprises at least 14-19 amino acid residues in length.

4. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of claims 1 to 3, wherein the sequence of amino acid residues is at least 70% identical to one or more of the amino acid sequences set forth in SEQ ID NOs: 1-54.

5. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 4 wherein the CLIP peptide, CLIP analogue or a derivative comprises a sequence of amino acids substantially as set forth in any one of SEQ ID Nos: 45 to 49.

6. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 4 wherein the CLIP peptide, CLIP analogue or a derivative comprises a sequence of amino acids substantially as set forth in any one of SEQ TD Nos: 1 to 25.

7. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 4 wherein at least one amino acid residue is a non-conventional amino acid selected from the list set forth in Table 2.

8. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 7 wherein the non-conventional amino acid residue is a D-stereoisomer of a naturally-occurring amino acid.

9. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 8 wherein the D-stereoisomer of a naturally-occurring amino acid is D-alanine.

10. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 9 wherein the CLIP peptide, CLIP analogue or a derivative comprises a sequence of amino acids substantially as set forth in any one of SEQ ID Nos: 26 to 44.

11. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of claims 1 to 10 wherein said CLIP peptide, CLIP analogue or a derivative is further capable of binding to at least one MHC class II molecule derived from a mammal.

12. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 11 , wherein said CLIP peptide, CLIP analogue or a derivative is further capable of inhibiting the binding of an antigenic peptide to the MHC class II molecule.

13. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 6, wherein said CLIP peptide, CLIP analogue or a derivative further possesses immunomodulatory activity as hereinbefore defined.

14. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 13 wherein the immunomodulatory activity is in relation to an autoimmune disease in humans or animals.

15. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 14 wherein said autoimmune disease is selected from the list comprising diabetes, multiple sclerosis, EAE or IDDM.

16. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 14 wherein the immunomodulatory activity is in relation to transplantation procedures.

17. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of claims 11 to 16 wherein the mammal from which the MHC class II molecule is derived is selected from the list comprising human, mouse, rat, pig, horse, cattle, guinea pig or other domesticated animal.

18. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of claims 11 to 16 wherein the mammal from which the MHC class II molecule is derived is a human.

19. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 18 wherein the MHC class II molecule is selected from the list comprising human haplotypes HLA-DR, HLA-DP and HLA-DQ molecules.

20. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 19 wherein the haplotype of HLA-DR is the HLA-DR3 molecuie.

21. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of claims 11 to 16 wherein the mammal from which the MHC II molecule is derived a mouse.

22. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to claim 21, wherein the murine MHC class II molecule is selected from the list comprising I-A^u, I-A , I-A«⁷ and I-A^d.

23. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of claims 12 to 22, wherein the antigenic peptide is selected from the list comprising MBP-(Acl-l l), HEL-(46-61) or Ova-(323-339).

24. The isolated CLIP peptide, CLIP analogue or a derivative thereof according to any one of ckiims 11 to 23 wherein the binding of said CLIP peptide, CLIP analogue or a derivative to the MHC class II molecule is haplotype-specific.

25. A derivatized CLIP peptide comprising a sequence of amino acids of at least 9 residues in length derived from amino acid residues 86-104 of the invariant protein Ii wherein at least one amino acid residue is a D-stereoisomer of a naturally-occurring amino acid and wherein said derivatized CLIP peptide is capable of binding with higher affinity than the corresponding un-derivatized CLIP peptide to an MHC class II molecule derived from a mammal.

26. The derivatized CLIP peptide according to claim 25 wherein the mammal is selected from the list comprising human, mouse, rat, rabbit, pig, horse, cattle, guinea pig, or other domesticated mammal.

27. The derivatized CLIP peptide according to claim 25 or 26 wherein the D- stereoisomer of a naturally-occurring amino acid is D-alanine.

28. The derivatized CLIP peptide according to any one of claims 25 to 27 wherein the MHC class II molecule is selected from the list comprising murine I-A^u, I-A , I-A^g7 and I-A^d or human haplotypes HLA-DR, HLA-DP and HLA-DQ.

29. The derivatized CLIP peptide according to claim 28 wherein the haplotype of HLA-DR is the HLA-DR3 molecule.

30. The derivatized CLIP peptide according to any one of claims 25 to 29 wherein the derivatized CLIP peptide further comprises a sequence substantially as set forth in any one of SEQ ID Nos: 1-54 or is at least 70% identical thereto.

31. The derivatized CLIP peptide according to any one of claims 25 to 30 wherein the binding to the MHC class II molecule is haplotype-specific.

32. The derivatized CLIP peptide according to claim 31 wherein the sequence of amino acids is substantially identical to any one of SEQ ID NOs: 2-5, 7, 9, 11-13, 15-

16, 20, 23 or 26-29.

33. The derivatized CLIP peptide according to any one of claims 25 to 32, wherein said derivatized CLIP peptide further possesses immunomodulatory activity.

34. The derivatized CLIP peptide according to claim 33, wherein the immunomodulatory activity is in relation to an autoimmune disease in humans or animals.

35. The derivatized CLIP peptide according to claim 34, wherein the autoimmune disease is selected from the list comprising diabetes, multiple sclerosis, EAE, or IDDM.

36. The derivatized CLIP peptide according to claim 3 i, wherein the immunomodulatory activity is in relation to transplantation procedures.

37. A method of identifying a specific MHC class II molecule haplotype in a biological sample, said method comprising contacting said biological sample or a purified or partially-purified fraction thereof with a binding effective amount of an isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof and then detecting said binding.

38. The method according to claim 37, wherein the biological sample comprises or is at least derived from blood, bone marrow or other lymphoid organs.

39. The method according to claim 37 or 38 wherein the isolated haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof is labelled with a reporter molecule.

40. The method according to claim 39 wherein the reporter molecule is selected from the list comprising an enzyme, fluorescent tag, biotinylated molecule or radioisotope molecule such as ³⁵S, Η, ^,4C or ^l2il.

41. The method according to claim 40 wherein the haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof comprises a sequence of amino acids selected from the list comprising SEQ ID NOs: 2-5, 7, 9, 11-13, 15-16, 20, 23 or 26-29 or is at least 70% identical thereto.

42. A diagnostic kit for determining the presence of an MHC class II molecule haplotype in a biological sample, said kit comprising one or more first compartments adapted to contain an isolated haplotype-specific derivatized CLIP peptide according to any one of claims 31 to 36 or a CLIP peptide, CLIP analogue or a derivative thereof according to claim 24.

43. The diagnostic kit according to claim 42 wherein the haplotype-specific CLIP peptide, CLIP analogue or a derivative thereof is labelled with a reporter molecule.

44. The diagnostic kit according to claim 36 wherein the reporter molecule is selected from the list comprising an enzyme, fluorescent tag, biotinylated molecule or radioisotope molecule such as ³ⁱS, ³H, ¹⁴C or ^l25I.

45. A pharmaceutical composition for use in a human or domesticated animal to reduce, delay, inhibit or otherwise attenuate an immune response when administered thereto, said composition comprising one or more isolated CLIP peptides, CLIP analogues or derivatives thereof according to any one of claims 1 to 24 in combination with a pharmaceutically-acceptabie carrier, diluent or excipient.

46. A pharmaceutical composition for use in a human or domesticated animal to reduce, delay, inhibit or otherwise attenuate an immune response when administered thereto, said composition comprising one or more derivatized CLIP peptides according to any one of claims 25 to 36 in combination with a pharmaceutically-acceptabie carrier, diluent or excipient.

47. The pharmaceutical composition according to claim 45 or 46, wherein said composition further comprises a protectant to prevent degradation of said CLIP peptide,

CLIP analogue or a derivative thereof within the human or domesticated animal to which it is administered.

48. The pharmaceutical composition according to any one of claims 45 to 47 when used to treat an autoimmune disease.

49. The pharmaceutical composition according to claim 48 wherein the autoimmune disease is selected from the list comprising cancer, diabetes, multiple sclerosis, EAE, insulitis or associated diseases thereto.

50. The pharmaceutical composition according to any one of claims 45 to 47 when used during or following transplantation procedures.

51. A method of delaying, inhibiting, reducing or otherwise attenuating an immune response in a human or a domesticated animal comprising administration thereto of an immunomodulatingly-effective amount of the pharmaceutical composition according to any one of claims 45 to 50.

52. The method according to claim 51 preceded by me step of determining the contribution of specific MHC class II molecule haplotypes to said immune response.

53. The method according to claim 51 or 52 wherein the immune response is associated with an autoimmune disease.

54. The method according to claim 53 wherein the autoimmune disease is selected from the list comprising diabetes, multiple sclerosis, EAE, insulitis or associated diseases thereto.

55. Use of the pharmaceutical composition according to any one of claims 45 to 50 to delay, inhibit, reduce or otherwise attenuate an immune response; in a human or a domesticated animal.

56. The use according to claim 55 wherein the immune response is associated with an autoimmune disease.

57. The use according to claim 56 wherein the autoimmune disease is selected from the list comprising diabetes, multiple sclerosis, EAE, insulitis or associated diseases thereto.