WO2002077030A2 - Modified mhc molecules whose binding to cd8 or cd4 is inhibited and use thereof - Google Patents

Abstract

The invention provides a method of inhibiting the activity of T cells against a cell presenting molecules of a selected Major Histocompatibility Complex (MHC) type, the method comprising causing the cell to present modified molecules of the selected MHC type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type. Such modified molecules are also provided as are nucleic acids encoding them.

Description

Substances

The present invention relates to Major Histocompatibility Complex (MHC) molecules and to their use as inhibitors of T cell responses, e.g. for immunosuppression therapy.

MHC proteins are expressed on the surface of antigen presenting cells (APCs) and form a complex with peptide antigens so that the peptides are presented on the surface of the APCs. MHC-peptide antigen complexes are recognised by T cells via T cell receptors (TCRs) and a coreceptor expressed on the surface of the T cell. Binding of the MHC-peptide complex with the TCR and coreceptor transduces signals in the T cell that activate the cell, leading to a cellular immune response.

In humans, MHC molecules are known as Human Leukocyte Antigens (HLA) and are divided into HLA Class I and HLA Class II. The former require the CD8 coreceptor for T cell activation, and the latter require the CD4 coreceptor for T cell activation.

Class I HLA is a dimeric protein complex consisting of a variable heavy chain and a constant light chain, β₂-microglobulin (β₂m). Class I HLA molecules present peptides which are processed intracellularly, loaded into a binding cleft in the HLA molecule, and transported to the cell surface where the complex is anchored in the membrane by the HLA heavy chain. Peptides are usually 8-11 amino acids in length, depending on the degree of arching introduced in the peptide when bound in the HLA molecule. The binding cleft, which is formed by the membrane distal l and α2 domains of the HLA heavy chain, has "closed" ends, imposing quite tight restrictions on the length of peptide which can be bound.

β₂m is a polypeptide found free in serum, which is non-covalently associated with HLA Class I molecules at the cell surface and which can exchange in the HLA complex with other free β₂m molecules (Bernabeu, et al. Nature 308: 642-5 (1984); Cook, et al. J Immunol 157: 2256-61 (1996); Horig, et al. Proc Natl Acad Sci USA 94: 13826-31 (1997); Hyafil & Strominger, Proc Natl Acad Sci U SA 16: 5834-8^' (1979); Luscher, et al. J Immunol 153: 5068-81 (1994); Parker, et al. J Immunol 149: 1896-904 (1992); Smith, et al. Proc Natl Acad Sci U S A 89: 7767-71 (1992)).

CD8 is expressed as either a αα homodimer or an αβ heterodimer protein consisting of extracellular immunoglobulin, membrane-proximal stalk, transmembrane and cytoplasmic domains. The native dimers have a molecular weight of 45 & 47 kDa respectively (The Leucocyte Antigen Factsbook, 2^nd Ed., Barclay et al, (1997) Pub: Academic Press, Harcourt Brace & Company).

Class II HLA is a membrane-bound 61-65kDa αβ heterodimeric protein complex consisting of two similar non-covalently associated chains (The Leucocyte Antigen Factsbook, 2^nd Ed., Barclay et al, (1997) Pub: Academic Press, Harcourt Brace & Company). Class II HLA molecules present peptides which are processed intracellularly, loaded into a binding cleft in the HLA molecule, and transported to the cell surface where the complex is anchored in the membrane. The peptides presented by Class II HLA molecules are 12-24 amino acids in length. The binding cleft is formed by the membrane distal αl and βl domains of the MHC chains (Marsh et al, The HLA Factbook, Academic Press, 2000).

Suppressors of the cellular arm of the immune system, such as suppressors of CD4⁺ or CD8⁺ T cells, are urgently needed for the treatment of auto-immune disorders, such as rheumatoid arthritis, lupus erthymatosus, psoriasis vulgaris, ankylosing spondylitis, Reiter's disease, post-salmonella arthritis, post-shigella arthritis, post-yersinia arthritis, post-gonococcal arthritis, uveitis, amylodosis, idiopathic hemachromatosis and my asthenia gravis, as well as the prevention of graft rejection and the treatment of graft-versus-host disease. Antibodies directed against CD4 and CD 8 have been tried (De Fazio, et al. Transplantation 61: 104-10 (1996)), but with limited success and antibodies in general are not well suited as drugs since they tend to induce secondary immune responses and are short-lived. Administration of steroids is another way of suppressing the immune system but their effect is extremely indirect and associated with severe side-effects. Attempts have been made to suppress the immune system by modulating the binding of CD8 to MHC class I molecules, although these have generally involved the use of CD8-derived molecules rather than modified MHC molecules. One previous attempt involved the in vitro use of peptides derived from HLA sequences thought to interact with CD8 (Clayberger, et al. J Immunol 153: 946-51 (1994)). Two CD8 derived peptides that were also tested were found to be incapable of suppressing the differentiation of human CTL precursors into active effector cells and unable to inhibit the subsequent action of these effector cells.

Another attempt to modulate CTL responses using free CD8 derived peptides (Choksi, et al. Nature Medicinέ 4: 309-314 (1998)) showed that one peptide in particular, " CSSHNKPC" , could inhibit both the differentiation and effector stages of CTL response. However, a very high concentration of peptide (> 100 μg/ml) was required to bring about this inhibition (> 50%).

CTL inhibition has also been observed with soluble CD8αα (WO 99/21576 and Sewell, etal. Nature Medicine 5: 399-404 (1999)). The inhibitory effect of the soluble CD8 molecule was more dramatic than that observed with an anti-CD8 monoclonal antibody.

In certain aspects, the present invention aims to prevent or inhibit T cell responses by preventing or inhibiting the binding of CD8 or CD4 coreceptor to the MHC/peptide complex.

According to a first aspect of the present invention, there is provided a method of inhibiting the activity of T cells against a cell presenting molecules of a selected MHC type, the method comprising causing the cell to present modified molecules of the selected MHC type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type. In a second aspect, the invention provides a modified MHC molecule of a selected type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, or a nucleic acid molecule encoding such a modified MHC molecule, for use in medicine.

In a third aspect, the invention provides the use of a modified MHC molecule of a selected type, whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, in the manufacture of a medicament for inhibiting T cell response. A pharmaceutical composition for inhibiting T cell response, which contains a modified MHC molecule of a selected type, whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type is also provided.

In a fourth aspect, the invention provides the use of a nucleic acid molecule encoding a modified MHC molecule of a selected type, whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, in the manufacture of a medicament for inhibiting T cell response. A pharmaceutical composition for inhibiting T cell response, which contains a nucleic acid encoding a modified MHC molecule of a selected type, whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type is also provided.

In a fifth aspect, the invention provides a method for the treatment of an autoimmune disorder (which may be due to endogenous or exogenous aetiology), graft-versus-host disease or graft rejection, comprising administering to a patient a modified MHC of a selected type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, or a nucleic acid encoding such a modified MHC.

In a sixth aspect of the present invention, there is provided a cell which presents (i) molecules of a selected MHC type, and (ii) modified molecules of the selected MHC subtype whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type.

The invention will be described further with reference to the accompanying drawings in which:

Figures la and lb are diagrams illustrating the putative principle of the present invention;

Figure 2a illustrates the conservation of the 110-130 region in the α2 domain of Class I HLA molecules, and Figure 2b shows possible mutations to this region;

Figure 3a illustrates the conservation of the 210-250 region in the α3 domain of Class I HLA molecules, and Figure 3b shows possible mutations to this region;

Figure 4 illustrates preferred mutations in the 110-130 region in the α2 domain of Class I HLA molecules;

Figure 5 illustrates preferred mutations in the 210-250 region in the α3 domain of Class I HLA molecules;

Figure 6 illustrates further preferred mutations in the 210-250 region of Class I HLA molecules;

Figure 7 shows the allelic variation in the 110-130 region in the α2 domain of all known Class I HLA- A subtypes;

Figure 8 lists all of the known Class I HLA-A subtypes classified according to the variation described in Figure 7; Figure 9 shows the allelic variation in the 210-250 region in the α3 domain of all known Class I HLA- A subtypes;

Figure 10 shows all known Class I HLA-A subtypes classified according to the variation described in Figure 9;

Figure 11 shows the allelic variation in the 110-130 region in the α2 domain of all known Class HLA-B subtypes;

Figure 12 shows all of the known Class HLA-B subtypes classified according to the variation described in Figure 11;

Figure 13 shows the allelic variation in the 210-250 region in -the α3 domain of all known Class HLA-B subtypes;

Figure 14 shows all of the known Class I HLA-B subtypes classified according to the variation described in Figure 13;

Figure 15 shows the allelic variation in the 110-130 region in the α2 domain of all known Class I HLA-C subtypes;

Figure 16 shows all of the known Class HLA-C subtypes classified according to the variation described in Figure 15;

Figure 17 shows the allelic variation in the 210-250 region in the α3 domain of all known Class I HLA-C subtypes;

Figure 18 shows all of the known Class I HLA-C subtypes classified according to the variation described in Figure 17; Figure 19a shows an amino acid motif (with allelic variations compared to consensus sequence DQB 1*05011) within the β2 domain of the β chain of Class II HLA molecules in which it is preferred to make mutations, and Figure 19b shows all possible mutations that could be made in that motif;

Figure 20a shows an amino acid motif (with allelic variations compared to consensus sequence DRA*0101) within the α2 domain of the α chain of Class LT HLA molecules in which it is preferred to make mutations, and Figure 20b shows all possible mutations that could be made in that motif;

Figure 21 illustrates preferred substitution mutations which can be made in the motif shown in Figure 19a;

Figure 22 illustrates preferred substitution mutations which can be made in the motif shown in Figure 20a;

Figure 23 illustrates the most preferred substitution mutations which can be made in the motif shown in Figure 19a;

Figure 24 illustrates the most preferred substitution mutations which can be made in the motif shown in Figure 20a;

Figure 25 lists the amino acid sequences for the amino acids in the motif identified in Figure 19a for all sequenced Class II HLA-DPB, -DQB and -DRB subtypes;

Figure 26 lists the amino acid sequences for the amino acids in the motif identified in Figure 20a for all sequenced Class II -DPA, -DQA and -DRA subtypes;

Figure 27 shows the DNA sequence of a vector pEX060 containing the wild-type HLA-A*0201 gene;

Figure 28 is a graph illustrating the results of Europium release T cell assay; and Figure 29 is a graph illustrating the results of Europium release T cell assay.

Although the present invention relates to the use of modified MHC molecules in . general for inhibitors of T cell responses (for example, in immune suppression in animals), its major utility lies in the use of modified HLA molecules for such inhibition (for immune suppression in man). Therefore, for convenience and without limitation, the present invention will be described further with reference to modified HLA molecules. Nevertheless, it is to be noted that MHC molecules are highly conserved among various mammals. Therefore, the present invention is also useful for the treatment of such mammals.

The terms "type" and "subtype" as used herein with reference to classifying MHC molecules are in accordance with the definitions of these terms proposed by the 10^th International HLA Workshop (1987, Princeton, NJ, USA) and described in the HLA Factsbook (Marsh et al, 2000, Academic Press). The following examples illustrate how these terms are used in relation to both class I and class II HLA molecules.

HLA-A*2402102 is a class I HLA. "A" denotes the genetic loci in which the DNA encoding the HLA molecule is located. "Classical" class I HLA molecules are derived from genes in the A, B or C loci, and "non-classical" class I HLA molecules are derived from genes in the E, F, or G loci. The asterisk is present solely as a " spacer" .

The first two digits ("24") specify the HLA type, usually based on serological antigen carried by the HLA molecule. However, a newly-described HLA molecule may be given the same first two digits as an existing group of HLA molecules based on structural similarity. This occurs when classifying the new HLA molecule by its serological antigen would place it in a structurally disparate type. The third and forth digits ("02") denote the HLA sub-type. The numbers indicate when the particular HLA sequence was published (lower numbers showing earlier publication).

The fifth digit (" 1") denotes a sub-type that has a "silent" mutation in the coding region of the DNA sequence. These do not result in a change to the amino acid sequence of the HLA molecule as expressed on the surface of the cell. Finally, the sixth and seventh digits ("02") denote a sub-type that has a mutation in the non-coding intron regions of the DNA sequence or the 5' or 3' untranslated regions.

Class II HLA molecules follow a similar nomenclature to that used for class I HLA molecules. The digits are used in exactly the same manner as described above. However, as mentioned previously, Class II HLA molecules have two membrane- bound chains and the nomenclature is applied to both of these chains.

HLA-DRA*0101 is a class II α chain and HLA-DRB1*0101 is a class II β chain. "DR" denotes the genetic loci in which the DNA encoding the HLA molecule is located: the known Class II HLA loci are DM, DO, DP, DQ, DR. Each complete class II HLA consists of an α and β chain, both taken from the same loci. The following "A" or "B" denote the α or β chain of an HLA molecule respectively. The remaining numbers follow the same rules as discussed for class I above.

Class II disease associations are produced by antigen peptide specificity which can be induced by the α or β chain of a given HLA molecule. By convention, these disease associations are listed by reference to only the particular class II HLA chain associated to the disease, with no mention of the other chain.

For the purposes of the present invention, modified molecules of a selected HLA type are caused to be presented by a cell. These modified molecules can be of a different subtype, i.e. there can variations in the third and fourth digits, provided of course that they still possess the same or substantially the same peptide-presenting activity as the unmodified HLA type. Furthermore, there can be variations in the fifth, sixth or seventh digits because the amino acid sequence of any HLA molecule can be defined without recourse to these digits.

As used herein "inhibited", when used in the context of the binding of one entity to another, means that the binding is prevented altogether or reduced to such a level that the normal physiological results of binding cannot be observed or are not significant. Even low levels of binding inhibition can have severe physiological effects. For example, inhibition of T-cell activation has been shown to be occur when soluble CD8 occupies only 2% of the available MHC sites (WO 99/21576 and Sewell, et al. Nature Medicine 5: 399-404 (1999).

Without wishing to be bound by theory, the putative principle behind the present invention wϊϋ he described with reference to Figures la and lb. Figure la illustrates a normal immune synapse in which an antigen binding cell bearing HLA peptide complexes binds to a T cell bearing T cell receptors and co-receptors (CD8 in the case of Class I and CD4 in the case of Class II). It can be seen that the binding of the HLA peptide complexes with the T cell receptors transduces signals through the T cell receptors and the co-receptors, causing signal initiation in the T cell, and consequently an immune response. In Figure lb, the antigen presenting cell has been modified such that one of the HLA molecules cannot bind the co-receptor, but can still present peptide for T cell recognition. As a result, although the T cell receptor can bind the HLA peptide complex, the nature of the signal received in the T cell is such that T cell activation is not initiated, and there is no immune response.

By modifying a specific HLA type in such a way that either the binding of CD4 or CD8 coreceptor to it is inhibited, it is possible to inhibit cellular immune responses to antigens presented by the same HLA type. In accordance with the present invention, the activation of T cells by specific HLA types can be inhibited, irrespective of the peptide presented by the HLA type. Accordingly, the present invention can be used to treat conditions associated with one or more HLA types, such as autoimmune diseases and allergies, regardless of whether the peptide associated with the condition presented by the HLA type has been identified. It is within the scope of the present mvention to cause a cell to present modified versions of more than one HLA type so as to inhibit cellular immune responses to antigens presented by each HLA type. The antigens may be for the same or different conditions.

Sewell, et al. Nature Medicine 5: 399-404 (1999) state that "some T cells are exquisitely sensitive to interference with co-receptor function, a finding that may prove a powerful tool for therapeutic modulation of T-cell activity in the treatment of autoimmune disease or the prevention of graft rejection." However, this statement is made in the context of the finding that soluble CD8 (a TCR co-receptor) dramatically inhibits Class I MHC-mediated T cell immune response. The skilled person is therefore not taught by this statement that co-receptor function should be interfered with by modifying the MHC molecule such that it cannot bind its co-receptor. Furthermore, the finding that soluble CD8 inhibits Class I MHC-mediated T cell immune response does not provide any suggestion that T cell-mediated immune response can be suppressed in only those T cells which are restricted by a specific HLA, thereby leaving the patient with a largely intact cellular immune system, as is possible in the present invention.

In the present invention, certain T cell responses against an antigen presenting cell can be inhibited by providing the cell with a sub-population of HLA molecules which have been modified such that their binding to CD 8 or CD4 is impaired or inhibited. Surprisingly, it is expected that only a relatively small number of modified HLA molecules of a specified type (compared to wild-type or non-modified HLA molecules of the specified type) need to be presented on a cell in order to inhibit a T cell response to that cell.

For example, it is known that the Class I HLA molecule HLA-B*27 is associated with Anky losing spondylitis. The peptide antigens involved in triggering the autoimmune T cell responses causing the disease are not known. However, by expressing a modified HLA-B*27 protein that cannot be bound by CD8 on the surface of the antigen presenting cell, T cell activation to any antigen presented by HLA-B*27 molecule can be inhibited. Thus, cellular immune responses can be inhibited in an HLA type specific manner without affecting immune responses to antigens presented by other HLA molecules. The expression of a modified HLA-B *27 in an APC will result in a mixed population of wild type and modified HLA-B*27 being present on the cell surface. Surprisingly, even if the wild type HLA-B*27 greatly out-numbers the mutants on the cell surface, a strong inhibition of HLA-B *27 restricted T cell activity will occur. This is due to the delicately-balanced nature of T cell activation and demonstrates the system's extreme sensitivity to disruption.

The type of cell which presents a particular antigen depends upon how and where the antigen first encounters cells of the immune system. APCs include the interdigitating dendritic cells found in the T cell areas of the lymph nodes and spleen in large numbers; Langerhans cells in the skin; follicular dendritic cells in B cell areas of the lymphoid tissue; monocytes, macrophages and other cells of the monocyte/macrophage lineage; B cells and T cells; and a variety of other cells such as endothelial cells and fibroblasts which are not classical APCs but can act in the manner of an APC. The present invention provides that any of these cells can be caused, preferably by genetic engineering techniques, to present modified molecules of a selected MHC type whose binding to CD8 or CD4 is inhibited, but which can present the same peptide or peptides as unmodified molecules of the MHC type.

The inhibition of CD8 or CD4 binding to Class I HLA or Class II HLA molecules respectively may be achieved by providing mutant Class I HLA or Class II HLA molecules in which substitution, deletion and/or insertion mutations cause the binding of CD8 or CD4 respectively to be inhibited.

The mutated residues, for instance, may sterically and/or electrostatically prevent or impair the binding of CD8 or CD4 to HLA Class I or Class II complexes, respectively, and/or alter the degree of hydrophobicity of the local environment such that binding is prevented or impaired. Thus it is preferred if the or each mutation is in, or near, the CD8 or CD4 binding site or sites of Class I or Class II HLA molecule, respectively. The binding sites in Class I and Class II MHC molecules for CD8 and CD4, respectively, share a degree of homology. A small, exposed loop comprised of mainly negatively-charged amino acids forms part of the co-receptor binding site in both Class I and Class II HLA molecules. This loop is located at amino acids 223-229 in the α3 'domain of Class I HLA molecules and the homologous loop in the β2 domain of Class II HLA molecules occurs at amino acids 137-143 (Konig et αl (1992) Nature. 356: 796-798). A further study has identified a broadly similar binding site in the α2 domain of the α chain in Class II HLA molecules between amino acids 125-133 (Konig et al (1995) J Exp Med, 182: 119-181). These homologous loops form only a part of the entire co-receptor binding sites in MHC molecules, as studies have shown that other regions and amino acid residues are required for co-receptor binding. Gao et al. (1991, Nature 387: 630-634) have presented a crystal structure of the complex between human CD8αα and HLA-A2 that highlights a region in the α2 domain region of HLA-A2 involved in contacting CD8. Mutation studies in this region have been carried out (Sun et al., 1995, J. Exp. Med. 182: 1275-1280) which demonstrate that amino acids 115, 122 & 128 of HLA-A2 are critical for CD8 binding. These amino acids are associated with a cavity formed between the underside of the αl and α2 peptide binding domains, the α3 domain and part of β₂m.

The skilled person can determine whether a particular mutation in an HLA molecule has the effect of inhibiting CD 8 or CD4 binding. For example, APCs expressing the modified or mutant HLA molecules can be contacted with T cells, and activation (or non-activation) of the T cells detected using the ELISA-based MlP-lβ assay described in Examples 8 and 9 herein.

Substitution mutations are preferred as they create minimal disruption to the structure of HLA. Substitution mutations that are likely to interfere with the native interactions between an HLA molecule and CD8 or CD4 can be considered in categories as follows. • Mutations which inhibit interactions via steric hindrance. For example, the substitution of relatively small native amino acids such as glycine or serine with a relatively larger residues such as tyrosine or tryptophan, or vice versa.

• Mutations which abrogate hydrogen bond formation. For example, the substitution of native amino acids such as arginine or lysine which can form hydrogen bonds with residues such as alanine or valine which cannot form hydrogen bonds.

• Mutations which replace a hydrophobic native amino acid with a hydrophilic residue. For example, the substitution of tyrosine or leucine with arginine or lysine or vice versa. • Mutations which inhibit interaction via electrostatic repulsion. For example, the substitution of positively-charged native amino acids, such as arginine or lysine, • with negatively charged residues, such as aspartate or glutamate, or vice versa.

The specific mutations employed will depend on the native amino acids being replaced, and aim to prevent or reduce the contribution that the specific native amino acid makes to co-receptor binding. The person skilled in the art will be aware that substitutions which alter the size and/or the charge of the amino acid present are likely to cause inhibition of binding.

As mentioned above, mutants of Class I HLA are known, and have elucidated which regions and amino acids of Class I HLA molecules are required for CD8 binding.

For example, in Salter et al (Nature 345: 41-46, 1990), 48 separate single point substitution mutants of the α^ α₂ & α₃ domains of HLA-A2.1 were produced. The mutants were transfected into HLA-A,B-negative cells to assess their effect on CD8 binding in cell-cell adhesion assays. From these mutants, three clusters of amino acids in the α₃ domain of HLA-A2 were identified that were particularly important for CD8 binding. Cluster I is amino acids 221-223, 227, 229 & 232; cluster II is amino acids 233 & 235; and cluster III is amino acids 245 & 247. Mutations in Cluster I caused the greatest inhibition of T-cell mediated cytolysis, followed by Cluster III and Cluster II. Salter et al (Nature 338: 345-347, 1989) also describe cell adhesion assays between B cells transfected with 17 HLA types and CD8⁺ CHO cells. All HLA types demonstrated specific cell binding except HLA-Aw68.1 and HLA-A 68.2. These HLA types contained a valine at amino acid 245 instead of the wild type alanine. The ability of these 245V HLA's to be lysed by CTL's during in-vitro assays was also reduced. Site-directed mutagenesis was used to reverse this mutation and led to a renewal of CD8 binding.

In a further study (Shen et al (1996) J. Exp. Med. 184: 1671-1683), the modes of interaction between CD8 and Class I MHC were investigated using a Glu227-»Lys substitution mutant of HLA. Studies were conducted using immobilised wild type (WT) and mutant MHC which were contacted with the appropriate T cells. The results generated show that this mutation results in a large reduction in the ability of CTLs to initiate cell lysis. The Glu227-»Lys mutant functioned as a poor co-receptor, requiring 8-16 fold higher densities of the immobilised mutant HLA to elicit the same level of cell binding and response observed with immobilised WT HLA.

Cell-cell adhesion assays have been used to investigate the effect of mutations in the α2 and α3 regions of Class I MHC (HLA-A2010) on CD8-mediated binding (Sun et al (1995) J. Exp. Med. 182: 1275-1280). Three alanine substitution mutations to the α2 regions of MHC Class I (Q115, D122 and E128) were identified that showed no specific binding to CD8.

The binding of soluble CD8 to bound mutant HLA molecules has been studied directly using a Surface Plasmon Resonance (SPR)-based assay (Gao et al. , 2000, J. Biol.Chem. 275 (20): 15232-15238). The binding of soluble CD8 to MHC Class I molecules attached to a BIAcore chip results in a signal response. This response can be interpreted to provide kinetic information on the interaction under investigation. This study demonstrated the ability of a number of single and triple mutations in the α3 domain of various class I HLA molecules to cause inhibition of CD8 binding (amino acid residues 219, 223, 224 & 245). These mutations lie either within an exposed loop in the α3 domain of Class I MHC molecules (223, 224) or at amino acids crucial for the functional conformation of this loop (219, 245).

Point mutations have been used to investigate the nature of amino acids required for CD8 interaction in and around residue 227 in the α3 domain of MHC class I molecules (Connolly J et al, 1990, Proc. Natl. Acad. Sci USA. 87: 2137-2141). It was shown that residues 222, 223, 227 & 229 must be acidic in nature (glutamic/aspartic acid) to support CD8 recognition. A range of tests, including monoclonal antibody (mAb) staining and the use of non-CD8 dependent MHC molecules, were used to demonstrate that the effects caused were not due to allosteric changes in the αl or α2 domains responsible for antigen binding by MHC class 1 molecules. The mutants that were not capable of recognising CD8 failed to elicit a primary CTL response in CD8 dependent T cells. However the same mutants could elicit a secondary CTL response in CD8 independent T cells. Therefore, the antigen binding regions of the MHC molecules were functionally unaffected by the mutations.

These known MHC Class I molecule mutations and the effect that they had on CD8 binding are summarised in Table 1 below.

Table 1 - Known Mutations to MHC Class I molecules

* Mutant was expressed at <40% of level of wild type.

Any of the known "Non-Binding" modified HLA molecules mentioned in Table 1 may be used in the present invention. In addition, the present invention may utilise hitherto unknown modified Class I HLA molecules which cannot bind CD8. Studies suggest that the binding of Class I HLA molecules with CD8 is dependent on amino acid residues 115 to 262 of Class I HLA molecules.

In the following, references to the numbering of Class I HLA amino acids is based on the amino acid numbering described in Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1-1137.

Thus, in a further aspect of the invention, there is provided a modified class I MHC molecule of a selected MHC type whose binding to CD 8 is inhibited, but which can present the same peptide or peptides as unmodified molecules of the MHC type, excluding human HLA-A2, 245A-»T; human HLA-A2, 219R→Q; human HLA-A2, 223D-»G; human HLA-A2, 224Q→H; human HLA-A2.1, 37D- Y; human HLA- A2.1 , 210P→S; human HLA A2.1 , 215L→A; human HLA-A2.1 , 217W→A; human HLA-A2.1, 223D- A; human HLA-A2.1, 224Q→E; human HLA-A2.1, 225T→D; human HLA-A2.1, 226Q→A; human HLA-A2.1, 227D->A; human HLA-A2.1, 227D→K; human HLA-A2.1, 228T-»A; human HLA-A2.1, 228T→E; human HLA- A2.1, 229E-»A; human HLA-A2.1, 233T→I; human HLA-A2.1, 235P→A; human HLA-A2.1, 242Q->K; human HLA-A2.1, 244W→A; human HLA-A2.1, 245A→S; human HLA-A2.1, 228T-»A; human HLA-A2.1, 228T→E; human HLA-A2.1, 245A- S; human HLA-A2.1, 246A→V; human HLA-A2.1, 247V-»A; human HLA- A2.1 , 245A→V; human HLA-A2, 227E→K; human HLA-A2, 245 A→V; human HLA-A2, 115Q→A; human HLA-A2, 122D→A; human HLA-A2, 128E->A; murine H-2D^d, 227E→A; murine H-2D^d, 227E→K; murine H-2D^d, 227E→R; murine H-2D^d, 227E→H; murine H-2D^d, 227E→Y; murine H-2D^d, 227E→L; murine H-2D^d, 227 E→P; murine H-2D^d, 227E→F; murine H-2D^d, 222E→K; murine H-2D^d, 223E- K; murine H-2D^d, 229E→K.

In a preferred embodiment of this aspect of the invention, the modified class I MHC molecule whose binding to CD8 is inhibited excludes any of the above-mentioned mutants of any species, i.e. HLA-A2, 245A-»T; HLA-A2, 219R-»Q; HLA-A2, 223D→G; HLA-A2, 224Q→H; HLA-A2.1 , 37D→Y; HLA-A2.1 , 210P- S; HLA- A2.1, 215L→A; HLA-A2.1, 217W→A; HLA-A2.1, 223D→A; HLA-A2.1, 224Q→E; HLA-A2.1, 225T- D; HLA-A2.1, 226Q→A; HLA-A2.1, 227D- A; HLA-A2.1 , 227D-»K; HLA-A2.1 , 228T→A; HLA-A2.1 , 228T→E; HLA-A2.1 , 229E- A; HLA-A2.1, 233T→I; HLA-A2.1, 235P→A; HLA-A2.1, 242Q→K; HLA- A2.1, 244W→A; HLA-A2.1, 245A→S; HLA-A2.1, 228T-»A; HLA-A2.1, 228T→E; HLA-A2.1, 24 A- S; HLA-A2.1, 246A→V; HLA-A2.1, 247V→A; HLA-A2.1, 245A→V; HLA-A2, 227E- K; HLA-A2, 245 A→V; HLA-A2, 115Q→A; HLA-A2, 122D→A; HLA-A2, 128E→A; H-2D^d, 227E→A; H-2D^d, 227E→K; H-2D^d, 227E→R; H-2D^d, 227E→H; H-2D^d, 227E→Y; H-2D^d, 227E- L; H-2D^d, 227 E→P; H-2D^d, 227E→F; H-2D^d, 222E→K; H-2D^d, 223E→K; H-2D^d, 229E- K.

It is preferred if the modified class I MHC molecule is derived from human class I MHC molecules. The sequences of many of these proteins are known, for example from The HLA Factsbook, Marsh et al 2000, Academic Press, and the sources referenced therein. It is preferred if one or more of amino acid residues 105 to 262 is/are mutated. More preferably, the or each mutated residue is in residues 110-130 and/or 210-250. These regions are involved with CD8 binding and are highly conserved between different Class I HLA types.

Figure 2a illustrates the conservation of the 110-130 region in Class I HLA molecules. The first line shows the residues in HLA-A*01011 as the consensus sequence, with the subsequent lines showing the known allelic variations in these residues in known Class I HLA molecules. In Figure 2b, all possible substitution mutations that can be made to each of the residues 110-130 are shown. The present invention encompasses modified Class I HLA molecules incorporating one, two, three, four, five, or more of these mutations.

Figure 3a illustrates the conservation of the 210-250 region in Class I HLA molecules. The first line shows the residues in HLA-A*01011 as the consensus sequence, with the subsequent lines showing the known allelic variations in these residues in known Class I HLA molecules. In Figure 3b, all possible substitution mutations that can be made to each of the residues 210-250 are shown. The present invention encompasses modified Class I HLA molecules incorporating one, two, three, four, five or more of these mutations.

Figures 4 and 5 illustrate the preferred substitution mutations which can specifically disrupt the contribution of each amino acid of the respective 110-130 and 210-250 regions to CD8 binding, whilst maintaining the overall conformation of HLA molecule. For example, 110L is preferably mutated to D, E, R, H, K, S, T, Y, N, G or Q. The present invention encompasses modified HLA molecules in which one, two, three, four, five or more of residues 110-130 are mutated to any one of the amino acids indicated in Figure 4, and/or one, two, three, four, five or more of residues 210- 250 are mutated to any one of the amino acids indicated in Figure 5.

Most preferably, the mutated residue is one or more of 115, 122 and 128 and/or the one or two amino acids adjacent these residues. This is because residues 115, 122 and 128 are the amino acids associated with the docking of CD 8 into a cavity in Class I MHC molecules formed between the underside of the αl and α2 peptide binding domains, the α3 domain and part of β2m. These residues can be mutated to any of the amino acids shown in Figure 4 (these residues are shaded).

Equally preferably, the mutated residue is one or more of 219, 223-229 and 233, 235, 245 and 247 and/or the one or two amino acids adjacent these residues. This is because these amino acids are either (i) in an exposed loop in the α3 domain of Class I MHC molecules, (ii) essential for the functional conformation of this loop, and/or (iii) directly associated with CD8 docking. These residues can be mutated to any of the amino acids shown in Figure 5 (these residues are shaded).

There are certain wild-type class I HLA molecules which have a low affinity for CD8. These include HLA-A*6801, HLA-B*4801, HLA-B*81 and HLA-E*0101 (Gao et al, 2000, J. Biol. Chem. 275(20): 15232-15238). Accordingly, in one embodiment of the invention, the selected class I HLA type is modified so that the 210-250 region thereof resembles, e.g. has the same sequence and/or conformation and/or charge and/or steric attributes, as the 210-250 region of HLA-A*68, HLA-B*48, HLA-B*81 or HLA-E. Because these HLA types are present in humans, the modified HLA molecule should be non-immunogenic, i.e. because the part of the HLA molecule which is modified is recognised as a native HLA molecule by the immune system. Class I HLA sub-types which have a low affinity for CD8 include HLA-A*68011, HLA-A*68012, HLA- A*6802, HLA-A*68031 HLA-A*6808, HLA-A*6813, HLA-A*6817 and HLA- B*8101.

In a preferred embodiment, the modified molecules of the selected HLA type are modified so that they resemble the 210-250 region of HLA-E, preferably HLA-E*01. In particular, one, two or all three of residues 219, 223 and 224 may be mutated to Q, G and H, respectively. These residues are associated with the non-binding of CD8 in wild-type HLA-E*01. Additionally, one or more of residues 183, 268, 270 and 275 may be mutated to E, E, V and K respectively. Although these residues are not likely to be essential for the modified HLA to have inhibited CD8 binding, they help the modified HLA to "appear" like HLA-E to the immune system.

Examples of such modified class I HLA molecules are known. In Gao et al , 2000, J. Biol. Chem. 275 (20): 15232-15238, the binding of soluble CD8 to bound mutant HLA was studied directly using a Surface Plasmon Resonance (SPR)-based assay. The binding of soluble CD8 to MHC Class I molecules attached to a BIAcore chip results in a signal response. This response can be interpreted to provide kinetic information on the interaction under investigation. In this study, residues 219, 223 and 224 of HLA-A2 were mutated from R, D and Q to Q, G and H, respectively so that the resulting molecule had the same 210-250 region as HLA-E*01. The resulting molecule was unable to bind CD8. In addition, an HLA-E molecule was mutated "in the other direction", i.e. so that residues 219, 223 and 224 were mutated from Q, G and H to R, D and Q, respectively. The resulting molecule was able to bind CD8 as its 210-250 region resembled that of HLA-A2.

Alternatively, residue 245 may be mutated to V or T so that the modified HLA molecule resembles HLA-A*68 or HLA-B *48, respectively.

These mutations are illustrated in Figure 6, which shows partial amino acid sequences for residues 210-250 of certain HLA molecules. The 250-210 region of HLA-E is underlined, and the residues of interest are shaded.

The invention also provides a modified class I MHC molecule of a selected MHC type whose binding to CD8 is inhibited because the 210-250 region thereof has been modified to resemble the 210-250 region of HLA-E, but which can present the same peptide or peptides as unmodified molecules of the MHC type, excluding: human HLA-A2,.219R→Q, 223D→G, and 224Q->H. In this aspect of the invention, the modified class I MHC whose binding to CD8 is inhibited excludes any of the above- mentioned mutants of any species, i.e. HLA-A2, 219R→Q, 223D-»G, and 224Q-»H. Nucleic acids encoding such molecules are also provided. It is preferred if the modified class I MHC molecule is derived from a human class I MHC moiecule. The sequences of many of these proteins are known, for example from The HLA Factsbook, Marsh et al, Academic Press and the sources referenced therein. In the modified class I MHC molecules of this aspect of the invention, it is preferred if one, two or all three of residues 219, 223 and 224 are mutated to Q, G and H, respectively. These residues are associated with the non-binding of CD8 in wild-type HLA-E. Additionally, one or more of residues 183, 268, 270 and 275 may be mutated to E, E, V and K respectively.

Although the above-described mutations are preferred, those skilled in the art will appreciate that it is possible to alter a selected Class I HLA molecule in other ways so that the 210-250 region thereof resembles the corresponding region in HLA-E. For example, residues other than the ones described above can be can be changed, and other residues added or deleted, which alter the 210-250 region of a selected HLA type so that it resembles the conformation, charge or steric attributes of HLA-E.

Figures 7-18 list the amino acid sequences for amino acids 110-130 and 210-250 for all sequenced Class I HLA-A, -B and -C subtypes. Figure 7 shows the variation in the 110-130 region of all known HLA-A subtypes, with Figure 8 showing all of the known HLA-A subtypes classified according to the variation described in Figure 7. Figure 9 shows the allelic variation in the 210-250 region of all known HLA-A subtypes, with Figure 10 showing all of the known HLA-A subtypes classified according to the ' variation described in Figure 9. Figure 11 shows the allelic variation in the 110-130 region of all known HLA-B subtypes, with Figure 12 showing all of the known HLA- B subtypes classified accordmg to the variation described in Figure 11. Figure 13 shows the allelic variation in the 210-250 region of all known HLA-B subtypes, with Figures 14 a, b and c showing all of the known HLA-B subtypes classified according to the variation described in Figure 13. Figure 15 shows the allelic variation in the 110-130 region of all known HLA-C subtypes, with Figure 16 showing all of the known HLA-C subtypes classified according to the variation described in Figure 15. Figure 17 shows the allelic variation in the 210-250 region of all known HLA-C subtypes, with Figure 18 showing all of the known HLA-C subtypes classified according to the variation described in Figure 17.

As mentioned previously, mutants of Class II MHC molecules are known, although only in murine MHC molecules. For example, point substitution mutations of mouse MHC Class II molecules have been carried out (Konig et al, 1992, Nature 356: 796-798) in which the corresponding amino acids from human MHC class II molecules were introduced into the murine MHC molecule (it is known that mouse CD4 will not react effectively with human MHC class II molecules). The effect of the individual point mutations on CD4/MHC binding was assessed via cell assays using T cells and COS7 cells expressing the mutant MHC molecules. The amino acids mutated were from the β2 domain of the β chain of MHC class II molecules (amino acids 95-147). Substitutions at amino acids 110 and 137 were particularly effective at reducing CD4/MHC class II molecule interactions. A similar study (Konig et al, 1995, J Exp Med 182: 779-787) utilised mutations in a broadly homologous region in the α2 domain of the α chain of murine Class II MHC molecule. A number of mutations were identified which caused a decrease in T cell activity. The substitution mutations which elicited the greatest reduction in murine CD4 function were those at amino acid residues 125, 129, 131, 137 and 142.

The exact role of CD4 in T helper cell development and activation is unclear. Two studies (Mostaghel et al, 1998, J. Immunol. 161: 6559-6566) and (Riberdy et al, 1998, Proc. Natl. Acad. Sci USA 95: 4493-4499) have shown that, in the absence of MHC/CD4 binding, a reduced subset of functional CD4 independent T cell activation can occur.

The known MHC Class II molecule mutations and the affect on CD4 binding caused are summarised in Table 2 below.

Table 2 -Known mutations to MHC Class II molecules

Any of the known "Nόn-Binding" modified HLA molecules mentioned in Table 2 may be used in the present invention. In addition, the present invention may utilise hitherto unknown modified Class II HLA molecules which cannot bind CD4 but which can present the same peptide or peptides as the unmodified Class II HLA molecule. Thus, in a further aspect of the invention, there is provided a modified class II MHC molecule whose binding to CD4 is inhibited but which can present the same peptide or peptides as the unmodified Class II MHC molecule, excluding the following murine mutants: βllON→Q; βl37E→A; β 140V→A; βl41G→A; βl42V- A; βl37E->A; 142V→A; αl25S→G; αl25S→A; α 129T→A; αl29T→N; αl31G→A; αl27S→N; αl29T-»A; βl37E- A; βl42V→A.

In a preferred embodiment of this aspect of the invention, the modified class II MHC molecule is a human Class II HLA molecule, whose binding to CD4 is inhibited but which can present the same peptide or peptides as the unmodified Class II HLA molecule. The sequences of many of these proteins are known, for example from The HLA Factsbook, Marsh et al, 2000, Academic Press and the sources mentioned therein.

The inventors have identified two highly conserved regions in Class II HLA molecules - one in the α chain and one in the β chain - which appear to comprise the CD4 binding regions. It is preferred that any modifications or mutations be made in one or both of these regions.

The region in the β chain has the motif:

(Q/N/H/E) [gap of 23 amino acids] N(D/S/G) Q E E (T/K) (A/T) G (V/M) V S T (P/G/N) L I (R/H/Q) N G D

The motif is shown in Figure 19a where the first line shows the residues in HLA- DQB 1*05011 as the consensus sequence, with the subsequent lines showing the known allelic variations in these residues in known Class II HLA molecules. Below these in Figure 19b are shown all possible substitution mutations that can be made to each of the residues in the β chain conserved motif. The numbering follows that described in Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1-1137.

The region in the α chain has the motif:

(F/I) (T/F) P P V (V/L) N (V/I) T W L (R/S/C) N G (K/Q/E/H) (P/S/L/A) V T (T/E) G V (S/A) E (T/S) (V/S/L) F L (P/S) (R/K) (E/S/T) D (H/Y) (L/S) F (RF/H) K (F/I/) (H/S) Y L (P/T) F (L/V)

The motif is shown in Figure 20a where the first line shows the residues in HLA DRA*0101 as the consensus sequence, with the subsequent lines showing the known allelic variations in these residues in known Class II HLA molecules. Below these in Figure 20b are shown all possible substitution mutations that can be made to each of the residues in the α chain conserved motif. The numbering follows that described in Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1-1137.

Figures 21 and 22 illustrate preferred substitution mutations which can specifically disrupt the contribution of each amino acid of the respective β chain and α chain conserved regions to CD4 binding, whilst maintaining the overall conformation of the HLA molecule. The present invention encompasses modified HLA molecules in which one, two, three, four, five or more of the residues in the β chain conserved region are mutated to any one of the amino acids indicated in Figure 21, and/or one, two, three, four, five or more of residues in the α chain conserved region are mutated to any one of the amino acids indicated in Figure 22.

The residues in the β chain conserved region which are most preferably mutated are shown in Figure 23, together with the preferred mutations of these residues, and the residues in the α chain conserved region which are most preferably mutated are shown in Figure 24, together with the preferred mutations of these residues. Figures 25 and 26 respectively list the amino acid sequences for the amino acids in the motifs identified above for all sequenced Class II HLA-DPB, -DQB -DRB, -DPA, - DQA and -DRA subtypes. In Figure 25, the allelic variation in the motif identified in the β chain is shown for all known HLA Class II subtypes, together - in the cases of - DQB and -DRB - with all known subtypes. In Figure 26, the allelic variation in the motif identified in the α chain is shown for all known HLA Class II subtypes, together with all known subtypes.

A modified Class I or Class II MHC molecule (which is preferably human) of the present invention may be provided in substantially pure form. For example, it may be provided in a form which is substantially free of other proteins.

The skilled person will appreciate that homologues or derivatives of modified MHC proteins of the invention will also find use in the context of the present invention, i.e. in inhibiting the CD8⁺ or CD4⁺ T cell responses. Thus, for instance proteins which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar "type" . For instance, replacing one hydrophobic amino acid with another. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate arnino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention.

In the case of homologues and derivatives, the degree of identity with a protein as described herein is less important than that the homologue or derivative should not be able to bind CD8 or CD4. However, suitably, homologues or derivatives having at least 60% similarity (as discussed above) with the proteins or polypeptides described herein are provided. Preferably, homologues or derivatives having at least 70% similarity, more preferably at least 80% similarity are provided. Most preferably, homologues or derivatives having at least 90% or even 95% similarity are provided.

The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g. , gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e. , % identity = number of identical positions/total number of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength =• 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. BioscL, 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

In an alternative approach, the homologues or derivatives could be fusion proteins, incorporating moieties which render expression on the cell surface easier, for example by effectively tagging the desired protein or polypeptide.

The modified MHC proteins of the present invention can be provided alone, as a purified or isolated preparation. They may be provided as part of a mixture with one or more other proteins of the invention.

Gene cloning techniques may be used to provide a modified MHC protein of the invention in substantially pure form. These techniques are disclosed, for example, in J. Sambrook et al Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Thus, in a further aspect, the present invention provides a nucleic acid molecule comprising a sequence encoding a modified MHC protein of the present invention, or a complementary sequence thereto.

The skilled person will appreciate that the present invention can include novel variants of the nucleic acid molecules. For example, additions, substitutions and/or deletions are included. Thus, synthetic or non-naturally occurring variants are also included within the scope of the invention.

When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity, one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package). BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention compare when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length.

Preferably, sequences which have substantial identity have at least 50% sequence identity, desirably at least 75% sequence identity and more desirably at least 90 or at least 95% sequence identity with said sequences. In some cases, the sequence identity may be 99% or above.

Desirably, the term "substantial identity" indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art nucleic acid sequences.

It should however be noted that, where a nucleic acid sequence of the present invention codes for at least part of a novel gene product, the present invention includes within its scope all^' possible sequence coding for the gene product or for a novel part thereof.

The nucleic acid molecule may be in isolated or recombinant form. It may be incorporated into a vector and the vector may be incorporated into a host. Such vectors and suitable hosts form yet further aspects of the present invention.

In the present invention, it is preferred that the modified HLA molecule be expressed on the antigen presenting cell surface by administration of a nucleic acid comprising a sequence encoding a mutant HLA molecule, i.e. by way of gene therapy. Gene therapy refers to administration to a subject of an expressed or expressible nucleic acid.

Any of the methods for gene therapy available in the art can be used according to the present invention. For example, US Patent No. 5750102 describes the co-expression of HLA types on cells (in this instance for increasing the immune reaction to those cells). Other exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5): 155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a further aspect of the invention, there is provided a compound which comprises a nucleic acid encoding a modified HLA molecule or fragment or chimeric protein thereof, said nucleic acid being part of an expression vector that expresses a modified HLA molecule or fragment or chimeric protein thereof in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the modified HLA coding region, said promoter being inducible or constitutive (and, optionally, tissue- specific). Thus, for human gene therapy, the promoter, which term includes not only the sequence necessary to direct RNA polymerase to the transcriptional start site, but also, if appropriate, other operating or controlling sequences including enhancers, is preferably a human promoter sequence from a human gene, or from a gene which is typically expressed in humans, such as the promoter from human cytomegalo virus (CMV). Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late

SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.

In another particular embodiment, a nucleic acid molecule is used in which the modified HLA coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the modified HLA nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector; this approach is known as in vivo gene therapy. Alternatively, delivery of the nucleic acid into the patient may be indirect, in which case cells are first transformed with the nucleic acid in vitro and then transplanted into the patient; this approach is known as ex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4,980,286); by direct injection of naked DNA; by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); by coating with lipids, cell-surface receptors or transfecting agents; by encapsulation in liposomes, microparticles or microcapsules; by administering it in linkage to a peptide which is known to enter the nucleus; or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu & Wu, 1987, J. Biol. Chem. 262:4429- 4432), which can be used to target cell types specifically expressing the receptors. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysόsomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g. , WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller & Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). A great variety of expression vectors can be used to express modified HLA molecule for use in the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adeno viruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express a polypeptide in a host may be used for expression in this regard.

In a specific embodiment, a viral vector that contains a nucleic acid encoding a modified HLA molecule is used. For example, a retro viral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The nucleic acid encoding the modified HLA molecule to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al. , 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83: 1467-1473; Salmons & Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman & Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110- 114.

Adeno viruses are other viral vectors that can be used in gene therapy. Adeno viruses are especially attractive vehicles for delivering genes to respiratory epithelia.

Adeno viruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adeno viruses have the advantage of being capable of infecting non-dividing cells. Kozarsky & Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et ah, 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al, 1992, Cell 68:143-155; Mastrangeli et al, 1993, J. Clin. Invest. 91:225-234; WO94/12649; and Wang, et al, 1995, Gene Therapy 2:775-783.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al, 1993, Proc. Soc. Exp. Biol Med. 204:289-300;_, U.S. Patent No. 5,436,146).

The vector may also include transcriptional control signals, situated 3' to the modified HLA molecule encoding sequence, and also polyadenylation signals, recognisable in the subject to be treated, such as, for example, the corresponding sequences from viruses such as, for human treatment, the SV40 virus. Other transcriptional controlling sequences are well known in the art and may be used.

Generally, vectors for expressing a modified HLA polypeptide for use in the invention comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

The following vectors, which are commercially available, are provided by way of example: pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors, which can be used for in situ expression, are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for expression of a polypeptide for use in the therapy of the invention may be used in this aspect of the invention.

Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.

Polynucleotides for use in the therapy of the invention, encoding the mutant or modified HLA polypeptide generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5' to a ribosome binding site. The ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.

Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiation codon. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal in constructs for use in eukaryotic hosts. A transcription termination signal appropriately disposed at the 3' end of the transcribed region may also be included in the polynucleotide construct.

A wide range of intron sequences, known to those skilled in the art, can be used to influence the level of in vivo expression that occurs. These work by adding to the processability of the transgenic DNA and are thereby included in the present invention.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide when recombinantly synfhesised. These signals may be endogenous to the polypeptide or they may be heterologous signals. Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expression.

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection; or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler & Behr, 1993, Meth. Enzymol 217:599-618; Cohen et al, 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, epithelial cells are injected, e.g., subcutaneously. In another embodiment, recombinant skin cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to neuronal cells, glial cells (e.g., oligodendrocytes or astrocytes), epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, Langerhan's cells, Mast cells hepatocytes; blood cells such as T lymphocytes, B lymphocytes, Natural Killer cells, monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood or foetal liver.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, a nucleic acid encoding a mutant HLA molecule is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem or progenitor cells which can be isolated and maintained in vitro can be used in accordance with this embodiment of the present invention (see e.g. WO 94/08598; Stemple & Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow & Scott, 1986, Mayo Clinic Proc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

Direct injection of a DNA coding for a modified HLA molecule may also be performed according to, for example, the techniques described in United States Patent No. 5,589,466. These techniques involve the injection of "naked DNA", i.e., isolated DNA molecules in the absence of liposomes, cells, or any other material besides a suitable carrier. The injection of DNA encoding a protein and operably linked to a suitable promoter results in the production of the protein in cells near the site of injection. In a preferred embodiment, naked DNA comprising (a) DNA encoding a modified HLA molecule and (b) a promoter and appropriate control sequences are injected into a subject.

Medicaments in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient).

It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions)

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions oils (e.g. vegetable oils) may be used to provide oil-in-water or water-in-oil suspensions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dόse or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. The dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

In many cases, the particular antigens involved in causing, for instance, autoimmune diseases, are not known. However, substantial information is available concerning the link between HLA type and disease. For example, significant HLA associations have been noted for renal, neurological, endocrine, gastrointestinal, respiratory, eye, dermatological, neurological and infectious diseases (Lechler et al. , 2000, HLA in Health and Disease. 2^nd Ed. Academic Press). An impressive body of data has been accumulated which links specific HLA antigens with particular disease states (this is summarised in Table 3). The relationships are influenced by linkage disequilibrium, a state where closely linked genes on a chromosome tend to remain associated rather than undergo genetic randomisation in a given population, so that the frequency of a pair of alleles occurring together is greater than the product of the individual gene frequencies. This could result from natural selection favouring a particular haplotype or from insufficient time elapsing since the first appearance of closely located alleles to allow to become randomly distributed throughout the population.

With the odd exception, such as idiopathic hemochromatosis and congenital adrenal hyperplasia resulting from a 21-hydroxylase deficiency, HLA-linked diseases are intimately bound up with immunological processes. The HLA-D related disorders are largely autoimmune with a tendency for DR3 to be associated with organ-specific diseases involving cell surface receptors. A popular model of MHC and disease association is that efficient binding of autoantigens by disease-associated MHC molecules leads to a T cell-mediated immune response and the resultant autoimmune sequelae. Alternative models have also been put forward; for example, Ridgway and Fathman (Clin Immunol Immunopathol 86(1):3-10 (1998)) suggest that the association of MHC with autoimmunity results from "altered" thymic selection in which high- affinity self-reactive (potentially autoreactive) T cells escape negative selection.

Table 3 - Association of HLA with disease Disease HLA Relative allele risk

(a) Class II associated

Hashimoto's disease DR5 3.2

Rheumatoid arthritis DR4 5.8

Dermatitis herpetiformis DR3 56.4

Chronic active hepatitis (autoimmune) DR3 13.9

Pemphigus vulgaris DR4 14

Systemic lupus erythermatosus DR3 6

Myasthemia gravis DR3 3

Coeliac disease DR3 10.8

Sjogren's syndrome DR3 9.7

Addison's disease (adrenal) DR3 6.3

Insulin-dependent diabetes DR3 5.0

DR4 6.8

DR3/4 14.3

DR2 0.2

Thyrotoxicosis (Grave's) DR3 3.7

Primary myxedema DR3 5.7

Goodpasture's syndrome DR2 13.1

Tuberculoid leprosy DR2 8.1

Multiple sclerosis DR2 4.8

(b) Class I, HLA-27 associated

Ankylosing spondylitis B27 87.4

Reiter's disease B27 37.0

Post-salmonella arthritis B27 29.7

Post-shigella arthritis B27 20.7

Post-yersinia arthritis B27 17.6

Post-gonococcal arthritis B27 14.0

Uveitis B27 14.6

Amyloidosis in rheumatoid arthritis B27 8.2

(c) Other Class I associations

Subacute thyroiditis Bw35 13.7

Psoriasis vulgaris Cw6 13.3

Idiopathic hemochromatosis A3 8.2

Myasthenia gravis B8 4.4

(Data from Ryder et al. HLA and disease Registry 1979. Tissue Antigens, supplement 1979 & Marsh et al. The HLA FactsBook 2000. Pubs. Academic Press )

Class II associations

A number of diseases have been linked to HLA Class II alleles, particularly DR2, DR3 and DR4. The most significant association appears to be that of dermatitis herpetiformis (coeliac disease of the skin), although associations have also been reported for coeliac disease itself, rheumatoid arthritis, insulin-dependent diabetes and multiple sclerosis. Other less common diseases with relatively high associations with HLA type are chronic active hepatitis, Sjogren's syndrome, Addison's disease and Goodpasture's syndrome.

The genetic contribution to the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis is a chronic inflammatory disease that primarily affects the joints and surrounding tissues. Although the cause of rheumatoid arthritis is unknown, infectious, genetic, and endocrine factors may play a role. The disease can occur at any age, but the peak incidence of disease onset is between the ages of 25 and 55. Women are affected 3 times more often than men and incidence increases with age. Approximately 3 % of the population is affected. The onset of the disease is usually slow, with fatigue, loss of appetite, weakness, and vague muscular symptoms. Eventually, joint pain appears, with warmth, swelling, tenderness, and stiffness after inactivity of the joint. After having the disease for 10 to 15 years, about 20 percent of people will have had remission. Only 50% to 70% will remain capable of full-time employment and after 15 to 20 years, 10% of patients are invalids. The average life expectancy may be shortened by 3 to 7 years; factors contributing to death may be infection, gastrointestinal bleeding, and drug side effects. There is no known cure for rheumatoid arthritis and the disease usually requires life-long treatment. Current treatment includes various medications (including nonsteroidal anti-inflammatory drugs, gold compounds, immunosuppressive drugs), physical therapy, education, and possibly surgery aimed at relieving the signs and symptoms of the disease.

The association of HLA-DR4 or other HIA-DRBl alleles encoding the shared (or rheumatoid) epitope has now been established in nearly every population. Similarly, the fact that the presence and gene dosage of HLA-DRBl alleles affect the course and outcome of rheumatoid arthritis has likewise been seen in most (although not all) studies. Susceptibility to develop rheumatoid arthritis maps to a highly conserved amino acid motif expressed in the third hypervariable region of different HLA-DRBl alleles. This motif, namely QKRAA, QRRAA or RRRAA helps the development of rheumatoid arthritis by an unknown mechanism. However, it has been established that the shared epitope can shape the T cell repertoire and interact with 70 kDa heat shock proteins (Reveille, Curr Opin Rheumatol 10(3): 187-200 (1998)).

Coeliac disease and dermatitis herpetiformis

Coeliac disease is one of the most common gastrointestinal disorders, affecting between 1:90 to 1:600 persons in Europe. The disease is a permanent intolerance to ingested, gluten that results in immunologically mediated inflammatory damage to the small-intestinal mucosa. Coeliac disease is associated with HLA and non-HLA genes and with other immune disorders, notably juvenile diabetes and thyroid disease. The classic sprue syndrome of steatorrhea and malnutrition coupled with multiple deficiency states may be less common than more subtle and often monosymptomatic presentations of the disease. Diverse problems such as dental anomalies, short stature, osteopenic bone disease, lactose intolerance, infertility, and nonspecific abdominal pain among many others may be the only manifestations of coeliac disease. The treatment of coeliac disease is lifelong avoidance of dietary gluten.

Recent studies using human genome screening in families with multiple siblings suffering from coeliac disease have suggested the presence of at least four different chromosomes in the predisposition to suffer from coeliac disease. Other studies based on cytokine gene polymorphisms have found a strong association with a particular haplotype in the TNF locus; this haplotype carries a gene for a high secretor phenotype of TNFα. In addition to the strong association of coeliac disease with HLA-DR3, there is also evidence for an association with HLA-DQ. Both HLA-DQ2 and HLA- DQ8 restricted gliadin-specific T cells have been shown to produce IFNγ, which appears to be an indispensable cytokine in the damage to enterocytes encountered in the small intestine, since the histological changes can be blocked by anti-IFNγ antibodies in vitro ^"(Pena^'et αl, Scαnd J Gαstroenterol Suppl 225:56-8 (1998)).

Dermatitis herpetiformis (DH) is a pruritic, papulovesicular skin disease characterised in part by the presence of granular deposits of IgA at the dermal-epidermal junction, an associated gluten sensitive enteropathy, and a strong association with specific HLA types. Dermatitis herpetiformis is fairly uncommon, affecting around 1/10,000 persons in Europe and the US. Initial investigations revealed that 60% to 70% of patients with dermatitis herpetiformis expressed the HLA antigen B8 (normal subjects = 21 %). Further investigation of the HLA associations seen in patients with dermatitis herpetiformis has revealed an even higher frequency of the HLA class II antigens HLA-DR3 (DH = 95%; normal = 23%), HLA-DQw2 (DH = 100%; normal = 40%), and HLA-DPwl (DH = 42%; normal = 11 %) (Hall and Otley, Semin Dermαtol 10(3):240-5 (1991)). The association of the HLA-B8, HLA-DR3, HLA-DQw2 haplotype with Sjogren's syndrome, chronic hepatitis, Graves' disease, and other presumably immunologically mediated diseases, as well as the evidence that some normal HLA-B8, HLA-DR3 individuals have an abnormal in vitro lymphocyte response to wheat protein and mitogens and have abnormal Fc-IgG receptor-mediated functions, suggests that this HLA haplotype or genes linked closely to it may confer a generalized state of immune susceptibility on its carrier, the exact phenotypic expression of which depends on other genetic or environmental determinants.

Genetic susceptibility factors in insulin-dependent diabetes mellitus

Diabetes mellitus is a disease of metabolic dysfunction, most notably dysregulation of glucose metabolism, accompanied by characteristic long-term vascular and neurological complications. Diabetes has several clinical forms, each of which has a distinct etiology, clinical presentation and course. Insulin-dependent diabetes mellitus (type I diabetes; IDDM) is a relatively rare disease (compared with non-insulin- dependent diabetes mellitus, NIDDM), affecting one in 250 individuals in the US where there are approximately 10,000 to 15,000 new cases reported each year. The highest prevalence of IDDM is found in northern Europe, where more than 1 in every 150 Finns develop IDDM by the age of 15. In contrast, IDDM is less common in black and Asian populations where the frequency is less than half that among the white population.

IDDM is characterised by absolute insulin deficiency, making patients dependent on exogenous insulin for survival. Prior to the acute clinical onset of IDDM with symptoms of hyperglycemia there is a long asymptomatic preclinical period, during which insulin-producing beta cells are progressively destroyed. The autoimmune destruction of beta cells is associated with lymphocytic infiltration. In addition, abnormalities in the presentation of MHC Class I antigens on the cell surface have been identified in both animal models and in human diabetes. This immune abnormality may explain why humans become intolerant of self-antigens although it is not clear why only beta cells are preferentially destroyed.

The genetics of IDDM is complex, but a number of genes have been identified that are associated with the development of IDDM. Some HLA loci (in particular DR3 and DR4) are associated with an increased risk of developing IDDM, whereas other loci appear to be protective. Substitution of alanine, valine or serine for the more usual aspartic acid residue at position 57 of the β-chain encoded by the HLA-DQ locus has also been found to be closely associated with the increased risk of developing IDDM, although different combinations of DQA1 and DQB1 genes confer disease risk to differing degrees (Zamani and Cassiman, Am J Med Genet 76(2): 183-94 (1998)).

Genetics of multiple sclerosis

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the nervous system that is the most common cause of chronic neurological disability among young adults. MS is characterised by discrete demyelinating lesions throughout the CNS.

The random nature of these lesions results in a wide variety of clinical features such as loss of sensations, muscle weakness, visual loss, cognitive impairment and fatigue.

The mean age of onset is 30 years and females are more susceptible to MS than males by a factor that approaches 2:1. MS afflicts people almost worldwide, although there is epidemiologic variation in incidence and prevalence rates. The prevalence varies with latitude, affecting primarily northern Caucasian populations (e.g., 10 per 100,000 in southern USA, 300 per 100,000 in the Orkneys). Approximately 300,000 people are afflicted with MS in the US and 400,000 in Europe. In North European populations, MS has been linked with Class I HLA alleles A3 and B7 and with Class II HLA alleles DR2, DQwl, DQA1 and DQB Particular HLA alleles (especially DR2) are considered to be risk factors for MS, and not simply genetic markers for the population of origin. However, this relationship is not universal and MS is linked to alleles other than DR2 in some populations (e.g.,

Jordanian Arabs and Japanese). This suggests that there is some heterogeneity in the contribution of HLA polymorphisms to MS susceptibility. Although particular alleles increase the risk for MS, no specific allele has yet been identified that is necessary for the development of MS. Overall, the contribution of the MHC to MS risk is believed to be fairly minor (Ebers and Dyment, Semin Neurol 18(3):295-9 (1998)).

Class I associations

The best known association of Class I HLA types with disease is that of HLA-B27 with anklyosing spondylitis and the related group of spondylarthropathies. Of the other Class I associations, the most important is probably that of HLA-Cw6 with psoriasis, although associations have also been reported for subacute thyroiditis, idiopathic hemochromatosis and myasthenia gravis.

HLA-B27 and the seronegative spondylarthropathies The seronegative spondylarthropathies include anky losing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, arthritis associated with ulcerative colitis and Crohn's disease, plus other forms which do not meet the criteria for definite categories and are called undifferentiated. Seronegative spondylarthropathies have common clinical and radiologic manifestations: inflammatory spinal pain, sacroilitis, chest wall pain, peripheral arthritis, peripheral enthesitis, dactylitis, lesions of the lung apices, conjunctivitis, uveitis and aortic incompetence together with conduction disturbances.

In the 25 years since^" the initial reports of the association of HLA-B27 with ankylosing spondylitis and subsequently with Reiter's syndrome/reactive arthritis, psoriatic spondylitis, and the spondylitis of inflammatory bowel disease, the association of HLA-B27 with the seronegative spondyloarthropathies has remained one of the best examples of a disease association with a hereditary marker. The association of HLA- 27 with in ankylosing spondylitis is quite remarkable, where up to 95% of patients are of B27 phenotype as compared to around 5% in controls. The prevalence of spondylarthropathies is directly correlated with the prevalence of the HLA-B27 antigen in the population. The highest prevalence of ahkylosing spondylitis (4.5%) has been found in Canadian Haida Indians, where 50% of the population is B27 positive. Among Europeans, the frequency of the B27 antigen in the general population ranges from 3 to 13 % and the prevalence of ankylosing spondylitis is estimated to be 0.1- 0.23% (Olivieri et al Eur J Radiol 27 Suppl l:S3-6 (1998)).

Experimental evidence from humans and transgenic rodents suggests that HLA-B27 itself may be involved in the pathogenesis of the spondyloarthropafhies, and population and peptide-specificity analysis of HLA-B27 suggest it has a pathogenic function related to antigen presentation. In Reiter's syndrome (reactive arthritis) and ankylosing spondylitis putative roles for infectious agents have been proposed.

However, the mechanism by which HLA-B27 and bacteria interact to cause arthritis is not clear and there are no clear correlations between peptide sequence, differential binding to B27 subtypes and recognition by peptide-specific T cell receptors (Lopez- Larrea et al. Mol Med Today 4(12):540-9 (1998)).

HLA-B27 and uveitis

Uveitis involves inflammation of the uveal tract which includes the iris, ciliary body, and the choroid of the eye. Causes of uveitis can include allergy, infection, chemical exposure, trauma, or the cause may be unknown. The most common form of uveitis is anterior uveitis which affects the iris. The inflammation is associated with autoimmune diseases such as rheumatoid arthritis or ankylosing spondylitis. The disorder may affect only one eye and is most common in young and middle-aged people. Posterior uveitis affects the back portion of the uveal tract and may involve the choroid cell layer or the retinal cell layer or both. Inflammation causes spotty areas of scarring that correspond to areas with vision loss. The degree of vision loss depends on the amount and location of scarring. In a recent study, Tay-Kearney et al (Am J Ophthalmol 121(l):47-56 (1996)) reviewed the records of 148 patients with HLA-B27-associated uveitis. There were 127 (86%) white and 21 (14%) nonwhite patients, and a male-to-female ratio of 1.5:1. Acute anterior uveitis was noted in 129 patients (87%), and nonacute inflammation was noted in 19 (13 %). An HLA-B27-associated systemic disorder was present in 83 patients (58%), 30 of whom were women, and it was diagnosed in 43 of the 83 patients as a result of the ophthalmologic consultation. Thirty-four (30%) of 112 patients had a family history of a spondyloarthropathy.

The genetics of psoriasis

Psoriasis is a disease characterised by uncontrolled proliferation of keratinocytes and recruitment of T cells into the skin. The disease affects approximately 1-2% of the Caucasian population and can occur in association with other inflammatory diseases such as Crohn's disease and in association with human immunodeficiency virus infection. Non-pustular psoriasis consists of two disease subtypes, type I and type II, which demonstrate distinct characteristics. Firstly the disease presents in different decades of life, in type I before the age of 40 years and later in type II. Secondly, contrasting frequencies of HLA alleles are found: type I patients express predominantly HLA-Cw6, HLA-B57 and HLA-DR7, whereas in type H patients HLA- Cw2 is over-represented. Finally, familial inheritance is found in type I but not in type II psoriasis. The study of concomitant diseases in psoriasis contributes to deciphering the distinct patterns of the disease. Defence against invading microorganisms seems better developed in psoriatics than in controls. This evolutionary benefit may have caused the overall high incidence of psoriasis of 2% (Henseler. Arch Dermatol Res 290(9) :463-76 (1998)).

Despite the HLA component, psoriasis in some families is inherited as an autosomal dominant trait with high penetrance. Susceptibility loci on other chromosomes have been identified following genome- wide linkage scans of large, multiply affected families although the extent of genetic heterogeneity and the role of environmental triggers and modifier genes is still not clear. The precise role of HLA also still needs to be defined. The isolation of novel susceptibility genes will provide insights into the precise biochemical pathways that control this disease. Such pathways will also reveal additional candidate genes that can be tested for molecular alterations resulting in disease susceptibility.

Thus, it can be seen that the association between certain HLA types and particular diseases has been well established. The best known of these is the association between the Class I molecule HLA-B27 and the spondylarthropathies, in particular ankylosing spondylitis. Despite the gene frequency of HLA-B27 being relatively high in Caucasians (3-13 %), this group of diseases is not common and the overall significance of the association is therefore somewhat reduced. Similarly, the HLA-DR3 allele

(present in approximately 11% of the Caucasian population) is associated with a high risk (56.4) for the development of dermatitis herpetiformis, a relatively rare (1/10,000) skin disorder. However, there are associations between HLA types and more prevalent diseases with greater socio-economic impact. For example, the relative risk of an individual with an HLA-DR4 allele developing rheumatoid arthritis is 5.8. Although this association is less than that between HLA-B27 and ankylosing spondylitis, rheumatoid arthritis affects approximately 3 % of the population and the HLA-DR4 allele has a gene frequency of nearly 17% in Caucasian Americans. Similarly,: although coelic disease has a relatively low risk associated with the presence of HLA-DR3 (10.8), this is a common haplotype and coelic disease is a prevalent gastrointestinal disorder.

In summary, there are a number of clinical diseases where there is an association with a particular HLA type (or types). The diseases with the most significant association with HLA type tend to be somewhat uncommon. However, there are a number of examples where the prevalence of the disease combined with the frequency of the HLA allele in the population make the association more significant, even if the risk associated with the particular HLA type is relatively low.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law. The invention will now be described further in the following non-limiting examples.

Examples

Primer design

In the following examples, the sequence variation that occurs within HLA molecules has been allowed for by the primer design. A number of ambiguity codes that designate positions at which one of two or three bases should be used have been included in the primers. The designation of these codes follows that described in Biochemical Nomenclatore and Related Documents. (1992) 2^nd Edition, Portland Press. The following table summarises the ambiguity codes used in the examples:

Example 1. Construction of plasmids for cellular expression of HLA-A wild type and mutant genes.

To enable the testing, in cell culture, of the inhibitory effect of expression of class I HLA mutants, DNA expression plasmids are constructed for HLA-A wild type and mutant genes.

HLA-A genes are amplified from cDNA isolated from the blood of a healthy human subject, using the polymerase chain reaction (PCR), with the following synthetic DNA primer pairs: Kba I HLA-AF d: 5'- CCC CCC TCT AGA ATG GCC RTC ATG SCK CCC CG -3'

HindiII HLA-ARe : 5' - CCC CCC AAG CTT TCA CAC TTT ACA AGC TGT GRG AG -3 '

The resulting PCR product is digested with the restriction enzymes Xbα I and Hind III, the recognition sites of which are indicated in the primer sequences above. Following digestion, the fragment is ligated with T4 DNA ligase and ATP into the corresponding restriction sites of vector pcDNA3. l/Hygro(-) (Invitrogen, Groningen, The

Netherlands). The ligated DNA is then transformed into an E.coli strain and amplified from a single colony. DNA manipulations and cloning described above are carried out as described in Sambrook, J et αl, (1989). Molecular Cloning - A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press, USA.

Mutations designed to decrease the suitability of the HLA-A protein as a binding site for the T cell co-receptor CD 8, are introduced using one of the following primer pairs:

HLA-A-245VFwd: 5-GR ACC TTC CAG AAG TGG GTG KCT GTG GTG GTR CCT TCT-3'

HLA-A-245VRev: 5' -AGA AGG YAC CAC CAC AGM CAC CCA CTT CTG GAA GGT YC-3'

These primer pairs replace an alanine with a valine in the HLA molecule at amino acid position 245.

HLA-A-245TF d:5' -GR ACC TTC CAG AAG TGG ACG KCT GTG GTG GTR CCT TCT-3'

HLA-A-2 5TRev:5' - AGA AGG YAC CAC CAC AGM CGT CCA CTT CTG GAA GGT YC-3'

These primer pairs replace an alanine with a threonine in the HLA molecule at amino acid position 245. Plasmid DNA is purified on a Qiagen™ mini-prep column according to the manufacturer's instructions, and the sequence verified by automated sequencing at the sequencing facility of Department of Biochemistry, Oxford University.

Example 2. Construction of plasmids for cellular expression of HLA-B wild type and mutant genes.

To enable the testing, in cell culture, of the inhibitory effect of expression of class I HLA mutants, DNA expression plasmids are constructed for HLA-B wild type and mutant genes. The HLA-B gene is amplified and inserted in a vector in the same manner as described in Example 1, except that the following synthetic DNA primer pair is used:

Xba I HLA-BFwd: 5 ' - CCC CCC TCT AGA ATG CKG GTC AYG GMG CCC CG -3 '

HindiII HLA-BRev: 5'- CCC CCC AAG CTT TCA AGC TGT GAG AGA CAC ATC AG -3'

Mutations designed to decrease the suitability of the HLA- B protein as a binding site for the T cell co-receptor CD8 are introduced using the one of the following primer pairs:

HLA B-245VFwd: 5'-GA ACC TTC CAG AAG TGG GTA GCT GTG GTG GTG CYT TCT-3'

HLA-B-245VRev:

5' -AGA ARG CAC CAC CAC AGC TAC CCA CTT CTG GAA GGT TC-3'

These primer pairs replace an alanine with a valine in the HLA molecule at amino acid position 245.

HLA-B-24'5TF d:

5'-GA ACC TTC CAG AAG TGG ACA GCT GTG GTG GTG CYT TCT-3' HLA-B-245TRev :

5' -AGA ARG CAC CAC CAC AGC TGT CCA CTT CTG GAA GGT TC-3 '

These primer pairs replace an alanine with a threonine in the HLA molecule at amino acid position 245.

Plasmid purification and sequencing is carried out as in Example 1.

Example 3. Construction of plasmids for cellular expression of HLA-DRA and HLA- DRB wild type and mutant genes.

To enable the testing, in cell culture, of the inhibitory effect of expression of class II HLA mutants, DNA expression plasmids are constructed for HLA-DRA wild type and mutant genes. HLA-DRA genes are amplified and inserted in a vector as described in Example 1, except that the following synthetic DNA primer pair is used:

DRAFwd:

Xba l 5'- CCC CCC TCT AGA ATG GCC ATA AGT GGA GTC CCT GTG -3'

DRARe :

H dlll

5 ' - CCC CCC AAG CTT TTA CAG AGG CCC CCT GCG TTC -3 '

Mutations designed to decrease the suitability of the DRA protein as a binding site for the T cell coreceptor CD4, will be introduced using one of the following primer pairs:

DRA-129A-Fwd:

5'- CTT CGA AAT GGA AAA CCT GTC GCC ACA GGA GTG TCA GAG ACA GTC -3'

DRA-129A-Rev:

5'- GAC TGT CTC TGA CAC TCC TGT GGC GAC AGG TTT TCC ATT TCG AAG -3'

These primers replace a threonine with an alanine at amino acid position 129 in all published HLA-DRA molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1- 1137) - for homology to human sequences refer to Figure 20.

DRA-131A-F d:

5'- GGA AAA CCT GTC ACC ACA GCA GTG TCA GAG ACA GTC TTC -3'

DRA-G131-A-Rev: ,

5'- GAA GAC TGT CTC TGA CAC TGC TGT GGT GAC AGG TTT TCC -3'

This primer pair replaces a gly cine with an alanine at amino acid position 131 in all published HLA-DRA molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1- 1137) - for homology to human sequences refer to Figure 19.

Plasmid purification and sequencing is carried out as in Example 1.

Example 4. Construction of plasmids for cellular expression of HLA-DRB wild type and mutant genes.

To enable the testing, in cell culture, of the inhibitory effect of expression of class II HLA mutants, DNA expression plasmids are constructed for HLA-DRB wild type and mutant genes. HLA-DRB genes are amplified and inserted in a vector in the same manner as described in Example 1, except that the following synthetic DNA primer pair is used:

DRBFwd:

Xba l 5 ' - CCC CCC TCT AGA ATG GTG TGT CTG RG TC CCT G -3 '

DRBRe :

Hind III

5 ' - CCC CCC AAG CTT TCA GCT CAV GAR TCC TST TGG -3 ' Mutations designed to decrease the suitability of HLA-DRB protein as a binding site for the T cell coreceptor CD4, are introduced using the one of the following primer pairs:

DRB-140A-141A-Fwd

5'- AAY RGC CAG GAA GAG AAG GCT GCG RTG GTG TCC ACR GGC CTG -3'

DRB-1 0A-141A-Rev

5'- CAG GCC YGT GGA CAC CAY CGC AGC CTT CTC TTC CTG GCY RTT -3'

These primers substitute amino acid positions 140 and 141 for alanine in all published HLA-DRB molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1-1137) - for homology to human sequences, refer to Figure 19.

Plasmid purification and sequencing is carried out as in Example 1.

Example 5. Construction of plasmids for cellular expression ofHLA-DQA wild type and mutant genes.

HLA-DQA genes are amplified and inserted in a vector in the same manner as described in Example 1, except that the following synthetic DNA primers are used:

DQAF d:

Xba l

5 ' - CCC CCC TCT AGA ATG GTC CTA AAC AAA GCT CTG MTG CTG G -3 '

DQARe : Hmdffl

5 ' - CCC CCC AAG CTT TCA CAA KGG CCC TTG GTG TCT -3 '

Mutations designed to decrease the suitability of the DQA protein as a binding site for the T cell co-receptor CD4 are introduced using one of the following primer pairs: DQA-129A-Fwd :

5'- G AGC AAT GGG CAS KCA GTC GCA GAA GGT GTT TCT GAG AC -3'

DQA-129A-Rev:

5'- GT CTC AGA AAC ACC TTC TGC GAC TGM STG CCC ATT GCT C -3'

These primers replace a threonine with an alanine at amino acid position 129 in all published HLA-DQA molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1- 1137) - for homology to human sequences, refer to Figure 20.

DQA-131A-Fwd: 5'- GGG CAS KCA GTC ACA GAA GCT GTT TCT GAG ACC AGC TTC CTC -3'

DQA-131A-Rev:

5'- GAG GAA GCT GGT CTC AGA AAC AGC TTC TGT GAC TGM STG CCC -3'

These primers replace a gly cine with an alanine at amino acid position 131 in all published HLA-DQA molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1- 1137) - for homology to human sequences, refer to Figure 19.

Plasmid purification and sequencing is carried out as in Example 1.

Example 6. Construction of plasmids for cellular expression of HLA-DQB wild type and mutant genes.

HLA-DQB genes are amplified and inserted in a vector in the same manner as described in Example 1, except the following synthetic DNA primers are used:

DQBFwd Xba l 5'- CCC CCC TCT AGA ATG GCT TGG AAR AAG KCT TTG CGG -3'

DQBRev ffindlll 5'- CCC CCC AAG CTT TCA GTG CAG YAG CCC TTT CYG AC -3'

These primers amplify all published DQB molecules except DQB 1*05031 and

DQB 1*06011 which have a 24bp insertion within the DQBRev primer binding region.

Mutations designed to decrease the suitability of HLA-DQB protein as a binding site for the T cell co-receptor CD4 are introduced using one of the following primer pairs:

DQB-140A-Fwd

5'- CGG AAT GRY CAG GAR GAG GCA RCY GGC GTT GTG TCC A -3'

DQB-140A-Rev

5'- T GGA CAC AAC GCC RGY TGC CTC YTC CTG RYC ATT CCG -3'

These primers replace a threonine with an alanine at amino acid position 140 in all published HLA-DQB molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1- 1137) - for homology to human sequences, refer to Figure 19.

DQB-141A-Fwd

5 ' - GRY CAG GAR GAG ACA RCY GCC GTT GTG TCC ACC CCC CTY ATT -3'

DQB-141A-Rev

5'- AAT RAG GGG GGT GGA CAC AAC GGC RGY TGT CTC YTC CTG RYC -3'

These primers replace a glycine with an alanine at amino acid position 141 in all published HLA-DQB molecules. Amino acid numbering is based on the mouse sequence (Kabat, 1991, Sequences of Proteins of Immunological Interest, 5^th edition, US Dept of Health & Human Services, Public Health Service, NIH, Bethesda, MD 1- 1137) - for homology to human sequences, refer to Figure 20. Plasmid purification and sequencing is carried out as in Example 1.

Example 7. Transfection of mammalian cells for in-vitro T cell assays.

The mammalian expression plasmids constructed in accordance with Examples 1-6 are stably or transiently transfected into mammalian cells using the detailed protocols provided by the manufacturer of pcDNA3.1/Hygro(-) (Invitrogen, Groningen, The Netherlands). Alternatively, transfection methods as described in Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) or Sambrook et al. ,

MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y (1989) can be used.

The target mammalian cells used for transfection are those that do not express the HLA type of interest, such as CIR, T293 or HeLa cells, such that the cells can be transfected to express wild type (wfHLA) and/or mutant HLA (mHLA) on their surface.

Antibody-based tests are used to ensure the cells are expressing wtHLA or mHLA molecules on their surfaces. Immunofluorescent cell-surface staining followed by fluorescence activated cell sorter (FACS) is employed to confirm transfection by detecting the presence of HLA (mutant or wild type) on the target cells. FACS data is displayed as graphs shown with fluorescence intensity against cell numbers. Alternatively, immunocytochemistry is used. This involves fixing the transformed cells to a glass microscope slide and using an enzyme-linked antibody to detect cell surface-expressed HLA molecules. The following publications give detailed descriptions of such methods: Immunocytochemical Methods and Protocols, 2nd. ed., Lorette C. Javois, Ed. 1998, ISBN 0-89603-570-0; Orfao & Ruiz-Arguelles, Clin Biochem 1996 29:1 5-9; Herzenberg & De Rosa mmunol Today 2000 21:8 383-90;, Bosman, Acta Histochem Suppl 1988 35: 27-32; Poulter et al J Immunol Methods 1987 Apr 16 98:2 227-34. Monoclonal antibodies directed against specific HLA molecules can be obtained from a number of sources, including: Research Diagnostics Inc, Pleasant Hill Road Flanders NJ 07836; Chromaprobe, Inc. , 897 Independence Avenue, Building 4C Mountain View, Califonia 94043; One Lambda, Inc., 21001 Kittridge Street Canoga Park, CA 91303-2801.

The cell types produced can be used to express the following HLA molecules on their cell surfaces in either a stable or transient manner:

• Transient: wtHLA

• Transient: mHLA

• Transient: both wtHLA and mHLA

• Stable: wt HLA

• Stable : mHLA

• Stable: wtHLA with transient expression of mHLA

• Stable: mHLA with transient expression of wtHLA

• Stable: both wtHLA and mHLA

• Control cell s exDressine neither wtHLA or mHLA

Example 8 - Assessing the ability of mutant Class I HLA molecules to inhibit T cell activation

Target cells are grown in RPMI culture medium containing 10% human serum for 5 days. These cells are incubated in RMPI medium containing lμM peptide for 2 hours. The target cells are placed into microtitre plates with CTL (cyto-toxic lymphocytes) at a range of Effector : Target cell (E:T) ratios. Supernatants are harvested after 2 hours.

Example combinations of Class I HLA molecules and their respective T cells:

Experimental Design

Negative control Target cells which do not normally express the Class I HLA type of interest transformed to express only mutant Class I HLA molecules.

Positive control Antigen presenting target cells expressing wild-type Class I HLA molecules. Test Samples - Antigen presenting target cells transformed to co-express mutant

HLA molecules and native Class I HLA molecules of the same type, capable of binding the same peptide.

A standard kit-based MlP-lβ assay (Quantikine^® - Human MlP-lβ Immunoassay, Cat No: DMBOO, R&D Systems Europe, Abingdon UK) is carried out on the supernatant in accordance with the manufacturers instructions. MlP-lβ (macrophage inflammatory protein - lβ) is a chemokine cell activation marker expressed by a range of cells, including CTL. Therefore, any reduction of MlP-lβ concentration observed on expression of the mutant HLA molecules, compared to that seen with target cells expressing only wild-type HLA molecules, indicates that the CTLs are being inhibited.

Example 9 - Assessing the ability of mutant Class II HLA molecules to inhibit T cell activation

T cell activation assays are carried out as described in Example 9. Example combinations of Class II HLA molecules and their respective T cells:

Example 10 - Production of vectors encoding HLA-A*020I mutants

200ng of vector pEX060 (see Figure 27 for DNA sequence) was digested with 7.5units Xbal and 4units Notl. The 1. lkb fragment containing the wild-type HLA-A*0201 gene was gel-extracted following electrophoresis on a 1 % agarose gel. The Wild-type HLA-A*0201 gene was then ligated into pBluescript II KS- (Strategene, 11011 North Torrey Pines Road, La Jolla, CA 92037 USA) which was digested with 7.5units Xbal and 4units Notl and gel-extracted following electrophoresis on a 1 % agarose gel. The resulting clone, pEX076a, was mutated using the QuikChange™ site-directed mutagenesis kit (Strategene, 11011 North Torrey Pines Road, La Jolla, CA 92037 USA) as per the manufacturer's instructions with the following alterations - 25ng of DNA was used per mutagenesis with 125ng of each primer using an extension time of lOminutes at 68C for the PCR reaction.

The following primers were used to generate the HLA-A *0201 E-mutant clone (219R^Q, 223D- G, 224Q^H) (pEX079d):

A2+B8-HLAEfwd

5 ' - GAT CAC ACT GAC CTG GCA GCA GGA TGG GGA GGG CCA TAG CCA GGA CAC GGA GCT -3'

A2+B8-HLAErev 5 ' - ACG AGC TCC GTG TCC TGG GTA TGG CCC TCC CCA TCC TGC TGC CAG GTC AGT GTG ATC -3'

The following primers were used to generate the HLA-A*0201 245V mutant clone (pEX080d):

A2-245VF d

5'- GAA CCT TCC AGA AGT GGG TGG CTG TGG TGG TGC CTT CT -3'

A2-245Rev 5'- AGA AGG CAC CAC CAC AGC CAC CCA CTT CTG GAA GGT TC -3'

The following primers were used to generate the HLA-A*0201 245T mutant clone (pEX081d):

A2-245TFwd

5'- GAA CCT TCC AGA AGT GGA CGG CTG TGG TGG TGC CTT CT -3'

A2-245Trev 5'- AGA AGG CAC CAC CAC AGC CGT CCA CTT CTG GAA GGT TC -3'

Example 11 - Production of vectors encoding Green Fluorescent Protein (GFP) fusions with HLA-A*0201 mutants

The following primers were used to amplify the HLA-A*0201 wild type, HLA- A*0201 E-mutant, HLA-A*0201 245V mutant and HLA-A*0201 245T mutant from pEX076a, pEX079d, pEX080d and pEX081d respectively.

A2-SSN-fwd

5'- CC CCC CCG CGG GTC GAC GCT AGC ATG GCC GTC ATG GCG CCC CGA ACC -3'

A2-EB-rev

5' - CCC GAA TTC TCA AGA TCT CAC TTT ACA AGC TGT GAG AGA CAC -3'

The PCR products were then digested with 5u N and lOu BglΩ., gel extracted following electrophoresis on an agarose gel and ligated into the vector pGFP²-Ν2 (BioSignal Packard, 1744 William, Suite 600, Montreal, Canada H3J 1R4) which was digested with 5units NM and lOunits BamRl and gel extracted following electrophoresis on a 1% agarose gel. The resulting clones pEX193, pEX194, pEX195 and pEX196 contained the GFP sequence fused to the 3' end of the HLA-A*0201 wild type, HLA-A*0201 E-mutant, HLA-A*0201 245V mutant and HLA-A*0201 245T mutant sequences respectively.

The 78 Ibp Xbal - Stul fragment from ρEX076a was replaced by the Xbal - Stul fragment from an expression construct containing the HLA-A *0201 227D→K/228T→A mutant gene (Purbhoo et al. (2001) J Biol Chem. 276 (35) 32786- 32792). The 586bp Kpnl - Sapl fragment from the resulting clone pEX604, containing the 227/228 mutation, was then used to replace the corresponding HLA- A*0201 wild type sequence in pEX193. The resulting construct pEX605 contains the GFP sequence fused to the 3' end of the HLA-A*0201 227/228 mutant gene. The integrity of all the fusion constructs were confirmed by DNA sequencing.

Example 12 - Transfection of antigen producing cells with mutant HLA-A*0201 constructs

In the following example, two CIR (Zemmour et al, (1992) J. Immunol 148 (6) 1941- 8) derived cell lines were used. The first had been transfected to express Wild-type HLA-A*0201 and the second had been transfected to express the 227D->K/228T-»A HLA-A*0201 mutant. These two cell lines will be referred to as SHI and SH2 respectively.

Each of the following HLA-A*0201 mutant constructs (ρEX194, ρEX195, ρEX196 and pEX605) were electroporated into SHI cells using the following two protocols.

A. lOg of Pvullll linearised endotoxin-free DNA was added to IO⁷ cells, washed twice in PBS, resuspended in 0.5ml PBS in a 0.4cm cuvette and pulsed at 250V, 800F with a Gene Pulser (BioRad, Richmond, CA, USA). The cells were incubated at room temperature for 5 minutes after which the transfectants were cultured overnight (37°C, 5% CO₂) in RPMI containing 10% Foetal bovine serum, 1% glutamine and 1 % Penicillin/streptomycin (R10).

B. lg of PvuIIR linearised endotoxin-free DNA was added to 10° cells, washed twice in serum-free RPMI medium, resuspended in 0.5ml serum-free RPMI medium in a 0.4cm cuvette and pulsed at 500V, 100F with a Gene Pulser (BioRad, Richmond, CA, USA). The cells were incubated at room temperature for 10 minutes after which the transfectants were cultured overnight (37°C, 5 % CO₂) in R10 medium Cultures from each of the electroporation protocols for each mutant were combined and maintained in RIO medium containing O.lmg/ml zeocin for 3 -4 weeks. (37°C, 5% CO₂) A stable cell line (NJ8) was selected on GFP expression by flow cytometry. The combined expression of HLA-A*0201 and GFP in NJ8 was confirmed by FACS analysis.

The following cell lines were then used for T cell assays:

SHI - expressing only Wild-type HLA-A *0201 SH2 - expressing only HLA-A*0201 227D→K/228T→A mutant.

NJ8 - expressing Wild-type and mutant HLA-A*0201 227D→K/228T→A mutant.

Example 13 - Cytoxicity Assay

Cytoxicity assays were done using the Delfia EuTDA cytoxicity reagents (PerkinElmer Life Sciences - Wallac Oy, PO Box 10, FIN-20101, TURKU, Finland). The targets used were SHI, SH2 and NJ8. The polyclonal effector T cell line used for the assay was isolated from Peripheral blood mononucleate cells (PBMC) from an HLA-A*0201 positive donor by a single initial stimulation by the HLA-A*0201 binding Epstein-Bar virus (EBV) BMLFI/280-288 peptide GLCTLVAML, IL-2 and IL7. The cells were then propagated for two weeks with additional IL-2 stimulation every four days. The cells were starved of IL-2 stimulation for 5 days prior to the assay being carried out.

The assays were carried out as per the manufacturer's instructions with the following changes.

• 0.25 x 10⁶ target cells were incubated for 30 minutes in RIO media with or without IO^"6 M GLCTLVAML peptide and then labelled with 2.51 of the fluorescence enhancing ligand (Delfia BATDA) for a further 30 minutes. • The target cells were then washed twice with RIO containing 100M - mercaptoethanol (ME) and resuspended in AB media (RPMI containing 10% AB serum, 1 % glutamine and 1 % Penicillin/streptomycin) containing 150M ME to a concentration of 1 x 10⁵cells /ml.

• 501 of effectors (5,000 cells) and 501 target cells (40,000 cells) were used at an E:T ratio of 1:8. • The lysis buffer used was 0.25 % Triton instead of digitonin.

• 501 of AB media was added to 501 of targets in the Spontaneous release, Maximum release and Background wells.

• 1801 of Europium solution was added to 201 of cell supernatant to the flat bottom plate and fluorescence measured after shaking for 15 minutes on a Wallac Victor 2 Fluor imeter (PerkinElmer Life Sciences - Wallac Oy, PO Box

10, FIN-20101, TURKU, Finland).

The level of T cell killing is represented by the specific release of europium chelate and is calculated using the formula:

% Specific release = Experimental release (counts') -Spontaneous release (counts^') xlOO Maximum release (counts) - Spontaneous release (counts)

The cytotoxicity assay was carried out twice using two different polyclonal T cell lines. The europium release data generated by these assays (see Figures 28 and 29) indicates that the presence of the mutated HLA-A *0201 molecules (227D-»K/228T-»A mutant) on the surface of antigen presenting cells inhibits T cell killing by approximately 30% in the presence of Wild-type HLA-A*0201.

Claims

Claims

1. A method of inhibiting the activity of T cells against a cell presenting molecules of a selected Major Histocompatibility Complex (MHC) type, the method comprising causing the cell to present modified molecules of the selected MHC type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type.

2. A method as claimed in claim 1 wherein the MHC is a Human Leucocyte Antigen (HLA).

3. A method as claimed in claim 1 or claim 2, wherein the modified MHC molecules have one or more substitution, deletion and/or insertion mutations which cause the binding of CD8 or CD4 to be inhibited.

4. A method as claimed in claim 3, wherein the or each substituted or inserted residue sterically and/or electrostatically prevents or impairs the binding of CD8 or CD4, and/or alters the degree of hydrophobicity of the local environment such that binding is prevented or impaired.

5. A method as claimed in claim 4, wherein the or each mutated residue is in, or near, the CD8 or CD4 binding site of Class I or Class II MHC molecules, respectively.

6. A method as claimed in any preceding claim, wherein the modified MHC molecules are modified Class I MHC molecules.

7. A method as claimed in claim 6, wherein one or more of amino acid residues 105 to 262 of Class I MHC is/are mutated.

8. A method as claimed in claim 7, wherein the or each mutated residue is in residues 110-130 and/or 210-250 of Class I MHC.

9. A method as claimed in claim 8, wherein one or more of residues 115, 122 and 128 and/όr the one or two amino acids adjacent these residues is/are mutated.

10. A method as claimed in claim 8 or claim 9, wherein one or more of residues 219, 223-229 and 233, 235, 245 and 247 and/or the one or two amino acids adjacent these residues is/are mutated.

11. A method as claimed in claim 8, wherein the 210-250 region of the modified MHC molecule has been modified to resemble the 210-250 region of HLA-E.

12. A method as claimed in claim 11, wherein the modified MHC molecule has one, two or all three of residues 219, 223 and 224 mutated to Q, G and H, respectively.

13. A method as claimed in claim 12, wherein the modified MHC molecule has one or more of residues 183, 268, 270 and 275^'mutated to E, E, V and K respectively.

14. A method as claimed in any one of claims 1 to 5, wherein the modified MHC molecules are modified Class II MHC molecules.

15. A method as claimed in claim 14, wherein the modified Class II MHC molecules have one or more mutations in a region in the β chain having the motif:

(Q/B/H/E) [gap of 23 amino acids] N (D/S/G) Q E E (T/K) (A/T) G (V/M) V S T (P/G/N) L I (R/H/Q) N G D

16. A method as claimed in claim 14 or claim 15, wherein the modified Class U MHC molecules have one or more mutations in a region in the α chain having the motif: (F/I) (T/F) P P V (V/L) N (V/I) T W L (R/S/C) N G (K/Q/E/H) (P/S/L/A) V T (T/E) G V (S/A) E (T/S) (V/S/L) F L (P/S) (R/K) (E/S/T) D (H/Y) (L/S) F (RF/H) K (F/I/) (H/S) Y L (P/T) F (L/V)

17. A method as claimed in claim 15 or claim 16, wherein one or more of the highlighted amino acids is/are mutated.

18. A modified MHC molecule of a selected type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, or a nucleic acid molecule encoding such a modified MHC molecule, for use in medicine.

19. A modified MHC molecule as claimed in claim 18, modified by the features of one or more of claims 2 to 17.

20. The use of a modified MHC molecule of a selected type, whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, in the manufacture of a medicament for inhibiting T cell response.

21. The use of a nucleic acid molecule encoding a modified MHC molecule of a selected type, whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, in the manufacture of a medicament for inhibiting T cell response.

22. The use as claimed in claim 20 or claim 21 modified by the features of one or more of claims 2 to 17.

23. A method for the treatment of an autoimmune disorder (which may be due to andogenous or exogenous aetiology), graft-versus-host disease or graft rejection, comprising administering to a patient a modified MHC molecule of a selected type whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type, or a nucleic acid encoding such a modified MHC molecule.

24. A method as claimed in claim 23 modified by the features of one or more of claims 2 to 17.

25. A cell which presents (i) molecules of a selected MHC type, and (ii) modified molecules of the selected MHC subtype whose binding to CD8 or CD4 is inhibited but which can present the same peptide or peptides as unmodified molecules of the MHC type.

26. A cell as claimed in claim 25 modified by the features of one or more of claims 2 to 17.

27. A modified class I MHC molecule of a selected MHC type whose binding to CD8 is inhibited, but which can present the same peptide or peptides as unmodified molecules of the MHC type, excluding human HLA-A2, 245A-»T; human HLA-A2, 219R→Q; human HLA-A2, 223D→G; human HLA-A2, 224Q-»H; human HLA- A2.1, 37D→Y; human HLA- A2.1, 210P→S; human HLA- A2.1, 215L→A; human HLA-A2.1, 217W→A; human HLA-A2.1, 223D→A; human HLA-A2.1, 224Q→E; human HLA-A2.1, 225T→D; human HLA-A2.1, 226Q→A; human HLA-A2.1, 227D→A; human HLA-A2.1, 227D→K; human HLA-A2.1, 228T->A; human HLA- A2.1, 228T→E; human HLA-A2.1, 229E-»A; human HLA-A2.1, 233T→I; human HLA-A2.1, 235P→A; human HLA-A2.1, 242Q→K; human HLA- A2.1, 244W→A; human HLA-A2.1 , 245A- S; human HLA-A2.1 , 228T→A; human HLA-A2.1 ,

228T→E; human HLA-A2.1, 245 A→S; human HLA-A2.1, 246A→V; human HLA- A2.1, 247V→A; human HLA-A2.1, 245A→V; human HLA-A2, 227E→K; human HLA-A2, 245 A→V; human HLA- A2, 115Q- A; human HLA-A2, 122D→A; human HLA-A2, 128E→A; murine H-2D^d, 227E→A; murine H-2D^d, 227E→K; murine H- 2D^d, 227E→R; murine H-2D^d, 227E-»H; murine H-2D^d, 227E→Y; murine H-2D^d, 227E→L; murine H-2D^d, 227 E→P; murine H-2D^d, 227E→F; murine H-2D^d, 222E→K; murine H-2D^d, 223E→K; murine H-2D^d, 229E→K.

28. A modified Class I MHC as claimed in claim 27, which excludes HLA-A2, 245A→T; HLA-A2, 219R→Q; HLA-A2, 223D→G; HLA-A2, 224Q→H; HLA-

A2.1, 37D→Y; HLA-A2.1, 210P-»S; HLA-A2.1, 215L- A; HLA-A2.1, 217W→A; HLA-A2.1, 223D→A; HLA-A2.1, 224Q- E; HLA-A2.1, 225T→D; HLA-A2.1, 226Q→A; HLA-A2.1, 227D→A; HLA-A2.1, 227D- K; HLA-A2.1, 228T→A; HLA-A2.1, 228T→E; HLA-A2.1, 229E→A; HLA-A2.1, 233T- I; HLA-A2.1, 235P→A; HLA-A2.1, 242Q→K; HLA-A2.1, 244W-»A; HLA-A2.1, 245A→S; HLA-A2.1, 228T→A; HLA-A2.1, 228T→E; HLA-A2.1, 245A→S; HLA-A2.1, 246A→V; HLA-A2.1, 247V→A; HLA-A2.1, 245 A→V; HLA-A2, 227E→K; HLA- A2, 245A- V; HLA-A2, 115Q→A; HLA-A2, 122D→A; HLA-A2, 128E→A; H- 2D^d, 227E→A; H-2D^d, 227E→K; H-2D^d, 227E→R; H-2D^d, 227E->H; H-2D^d, 227E-»Y; H-2D^d, 227E-»L; H-2D^d, 227 E→P; H-2D^d, 227E→F; H-2D^d, 222E→K; H-2D^d, 223E→K; H-2D^d, 229E→K.

29. A modified Class I MHC as claimed in claim 27 or claim 28 modified by the features of one or more of claims 1 to 13.

30. A modified class II MHC molecule whose binding to CD4 is inhibited but which can present the same peptide or peptides as the unmodified Class II MHC molecule, excluding the following murine mutants βllON→Q; βl37E-»A; β 140V-»A; βl41G→A; βl42V→A; βl37E→A; 142V→A; αl25S→G; l25S->A; α 129T→A; αl29T→N; αl31G→A; αl27S→N; αl29T→A; βl37E→A; βl42V- A.

31. A modified class II MHC molecule as claimed in claim 30 which is a human Class II HLA molecule, whose binding to CD4 is inhibited but which can present the same peptide or peptides as the unmodified Class II HLA molecule.

32. A modified Class II MHC molecule as claimed in claim 30 or claim 31 modified by the features of one or more of claims 1 to 5 and 14-17.

33. A nucleic acid molecule encoding a modified MHC molecule as defined in any one of claims 27-32, or a complementary sequence thereto.

34. A vector comprising a nucleic acid molecule as claimed in claim 33.

35. A host cell including a vector as claimed in claim 34.