Identification of self and non-self antigens implicated in autoimmune diseases Number:7,084,247 from the United States Patent and Trademark Office (PTO) owispatent

Title: Identification of self and non-self antigens implicated in autoimmune diseases

Abstract: The present invention provides isolated peptides relating to the autoimmune disease pemphigus vulgaris. The peptides relating to pemphigus vulgaris are self epitopes derived from human pathogens which are implicated in the aetiology and remissions of the disease. Pharmaceutical preparations for tolerizing and/or immunizing individuals are provided as well as methods relating thereto. Methods are provided for identifying other self and non-self epitopes involved in human autoimmune disease and similar pharmaceutical preparations and methods of use for these epitopes are also provided.

Patent Number: 7,084,247 Issued on 08/01/2006 to Rasmussen,   et al.

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Inventors:	Rasmussen; James (Cambridge, MA); Yu; Bei (West Roxbury, MA)
Assignee:	Peptimmune, Inc. (Cambridge, MA)
Appl. No.:	10/799,005
Filed:	March 11, 2004

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Current U.S. Class:	530/326 ; 424/185.1
Current International Class:	A61K 38/04 (20060101)
Field of Search:	514/2 424/185.1

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References Cited [Referenced By]

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U.S. Patent Documents

	5130297		July 1992		Sharma et al.
	5194425		March 1993		Sharma et al.
	5874531		February 1999		Strominger et al.

Foreign Patent Documents

	WO 90/08161		Jul., 1990		WO
	WO 92/16234		Oct., 1992		WO
	WO 92/18150		Oct., 1992		WO
	WO 93/10813		Jun., 1993		WO
	WO 94/05303		Mar., 1994		WO
	WO 94/06828		Mar., 1994		WO
	WO 95/12313		May., 1995		WO

Other References

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Primary Examiner: Chan; Christina
Assistant Examiner: Szperka; Michael
Attorney, Agent or Firm: Fish & Neave IP Group Ropes & Gray LLP

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Claims

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We claim:

1. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or a pharmaceutically acceptable salt thereof.

2. A composition comprising a pharmaceutically acceptable carrier and the polypeptide of claim 1.

3. A composition comprising the pharmaceutically acceptable salt of the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

4. The composition of claim 3, wherein the pharmaceutically acceptable salt is an acetate salt.

5. The composition of claim 2, further comprising a pharmaceutically acceptable additive selected from the group consisting of a sugar and a surfactant.

6. The composition of claim 2, further comprising buffers and bulking agents.

7. The composition of claim 5, wherein the sugar is dextrose or mannitol.

8. The composition of claim 7, wherein the composition is formulated for administration with about 5% dextrose.

9. The composition of claim 5, wherein the surfactant is selected from the group consisting of polysorbate 20 and polysorbate 80.

10. The composition of claim 9, wherein the composition is formulated with from about 0.01% to about 5% surfactant.

11. An isolated polypeptide wherein the polypeptide has an acetyl cap at its N-terminus, an amide cap at its C-terminus or both, and consists of the amino acid sequence of SEQ ID NO:1.

12. A composition comprising the polypeptide of claim 11 and a pharmaceutically acceptable carrier.

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Description

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FIELD OF THE INVENTION

The present invention relates to the field of immunology and, in particular, to the identification of self and non-self antigens implicated in human autoimmune responses. The invention relates to methods of identifying such self antigens and provides examples of such antigens relating to pemphigus vulgaris. The invention also relates to the use of such antigens for in vitro assays, animal models, therapeutic agents and vaccines.

BACKGROUND OF THE INVENTION

Human autoimmune diseases have a striking genetic association with particular alleles of major histocompatability complex ("MHC") class I or class II genes. The field was established by the seminal discovery of HLA-B27 linked susceptibility to ankylosing spondylitis, a chronic inflammatory joint disease (Brewerton (et al., 1973; Schlosstein et al., 1973). MHC associated susceptibility has now been documented for a variety of human autoimmune diseases, including insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), pemphigus vulgaris (PV), multiple sclerosis (MS) and myasthenia gravis (MG), just to name a few (Todd et al., 1987; Ahmed et al., 1990; Ahmed et al. 1991; Lanchbury & Panayi, 1991; Spielman & Nathenson, 1982; Protti et al., 1993).

The MHC locus most commonly associated with autoimmune disease is the HLA-DRB locus (also known as DRB1), a highly polymorphic locus with over fifty known alleles. For example, a large body of epidemiological work has documented the association of rheumatoid arthritis with the DR4 (DRB1*0401, DRB1*0404) and DR1 (DRB1*0101) alleles, with the DR4 alleles conferring a higher risk than DR1 (Lanchbury & Panayi, 1991). The risk is dramatically increased when the subject is homozygous or heterozygous for DRB1*0401 and/or DRB1*0404. The observation that arthritis is associated with three DR alleles that are structurally similar led to the development of the `shared epitope` hypothesis as DRB1*0401, 0404 and 0101 share critical polymorphic residues in the DR.beta.67 71 cluster (Gregersen et al. 1987; Lanchbury & Panayi, 1991). These residues (in particular DR.beta.71) appear to be critical in defining the selectivity of peptide binding to the disease associated molecules.

Pemphigus vulgaris (PV) is an autoimmune disease of the skin in which high titer auto-antibody production to an epidermal cell adhesion molecule (desmoglein 3) results in a loss of keratinocyte adhesion (acantholysis) and severe blister formation (Amagai et al., 1991). In different ethnic groups the disease is associated either with a DR4 allele (DRB1*0402) or with a rare DQ1 allele (DQB1*05032); only a small fraction of PV patients have neither susceptibility gene (Ahmed et al., 1991; Ahmed et al., 1990; Scharf et al., 1988). The DR4 subtype associated with pemphigus differs only at three positions in the DR.beta.67 71 cluster from the DR4 subtype associated with RA. The PV associated molecule has a negative charge (Glu) at the critical position (DR.beta.71); the neighboring position (DR.beta.70) is also negatively charged. The DR4 subtype associated with PV is the only one that carries a negative charge at DR.beta.71; a positive charge (Arg) is found at DR.beta.71 in the RA associated DR4 molecules.

Efforts to identify sequence homologies between self-peptide epitopes that might be involved in autoimmunity and various bacterial and viral pathogens have therefore been made. These homology searches have focused on alignments with sequence identity. No success has been reported using such alignments in identifying epitopes from pathogens that could cross react with presumably pathogenic T cell lines from human patients with autoimmune disease (Oldstone, 1990). A sequence identity was recently found between an epitope in a Coxsackie virus protein and GAD65, suspected of being an autoantigen in diabetes. These peptides could reciprocally generate polyclonal T cell lines from mice that cross react with the other peptides (Tian, et al., 1994). No evidence, however, was provided that these peptides could stimulate clones from diabetic mice (or humans).

Recent developments in the field, in particular the identification of allele specific peptide binding motifs have transformed the field (Madden et al., 1991; Rotschke & Falk, 1991). Based on this knowledge the structural basis for MHC linked susceptibility to autoimmune diseases can be reassessed at a level of detail sufficient for solving longstanding questions in the field. Motifs for peptide binding to several MHC class I and class II molecules have been defined by sequence analysis of naturally processed peptides and by mutational analysis of known epitopes. MHC class I bound peptides were found to be short (generally 8 10 amino acids long) and to possess two dominant MHC anchor residues; MHC class II bound peptides were found to be longer and more heterogeneous in size (Madden et al., 1991; Rotschke & Falk, 1991; Jardetzky et al. 1991, Chicz et al. 1993). Due to the size heterogeneity, however, it has proven more difficult to define MHC class II binding motifs based on sequence alignments. More recently, a crystal structure for HLA-DR1 demonstrated that there is a dominant hydrophobic anchor residue close to the N-terminus of the peptide and that secondary anchor residues are found at several other peptide positions (Brown et al., 1993). Even this work, however, could not provide a detailed description of the binding pockets of HLA-DR proteins, the particular residues involved in the formation of these pockets of the structural requirements or antigens for MHC binding.

In the present disclosure, a detailed description of the HLA-DR antigen binding pockets is provided (Stem et al., 1994). With this information, together with functional information defining those amino acids of the self or non-self antigen that are needed for MHC binding and TCR contact (e.g., Wucherpfennig et al. 1994a,), binding motifs for the various HLA-DR allotypes may be developed, self epitopes involved in autoimmune disease may be identified and a method is provided for identifying bacterial and viral epitopes which may initiate a human autoimmune response.

SUMMARY OF THE INVENTION

The present invention provides, in one aspect, isolated polypeptides derived from the human desmoglein 3 protein and implicated as self epitopes in the autoimmune disease pemphigus vulgaris (PV). These polypeptides consist essentially of the amino acid sequence disclosed herein and have been designated SEQ ID NO: 1. These polypeptides consist of SEQ ID NO: 1. In particular, the invention provides isolated polypeptides which consist of these sequences, the core MHC binding residues of these sequences, or the inner core MHC binding residues of these sequences.

Compositions of the present invention comprise a pharmaceutically acceptable carrier and a polypeptide of SEQ ID NO: 1. The composition can also comprise a pharmaceutically acceptable salt of the polypeptide. A preferred pharmaceutically acceptable salt is an acetate.

Compositions of the present invention can further comprising a pharmaceutically acceptable additive, such as a sugar or a surfactant. Acceptable sugars are those such as dextrose and mannitol. In one embodiment, the composition is formulated with about 5% sugar. The composition can further comprising buffers, such as dihydrate sodium citrate and monohydrate citric acid, and bulking agents, such as mannitol. In a further embodiment, a surfactant, such as can be polysorbate 20 or polysorbate 80, can be added to the composition in an amount of from about 0.01% to about 5%. One embodiment of the present invention comprises an immunogenic composition of the polypeptide.

In one embodiment, the composition comprises a lyophilized polypeptide of SEQ ID NO: 1. In one embodiment, the lyophilized polypeptide has a reconstitution time of less than 15 minutes, more preferably, the reconstitution time is less than 10 minutes, more preferably, the reconstitution time is less than 5 minutes, and more preferably, the reconstitution time is less than 3 minutes.

In one embodiment, the purity of the peptide is greater than 90%, more preferably, the purity is greater than 93%, more preferably, the purity is greater than 95%, and more preferably, the purity is greater than 96%.

In one embodiment, the composition has bacterial endotoxin contamination of less than about 5 EU/mL, more preferably, the bacterial endotoxin contamination is less than about 3 EU/mL, more preferably, the bacterial endotoxin contamination is less than about 2 EU/mL, and more preferably, the bacterial endotoxin contamination is less than about 1.25 EU/mL.

More preferably, the composition of the present invention has the formulation as set forth in Table 3.

In another set of embodiments, the invention provides for pharmaceutical preparations for use in tolerizing individuals to auto-antigens. The preparations include a pharmaceutically acceptable carrier and an isolated human polypeptide which includes an amino acid sequence corresponding to a sequence motif for an HLA-DR protein which is associated with a human autoimmune disease. These polypeptides are capable of binding to the HLA-DR protein to form a complex which activates autoreactive T cells in subjects having the autoimmune disease. The peptides are not derived from human collagen or human myelin basic protein.

In particular embodiments, such pharmaceutical preparations are provided in which the HLA-DR protein is HLA-DR4 protein and the autoimmune disease is pemphigus vulgaris. In addition, a particular sequence motif is provided for pemphigus vulgaris and pharmaceuticals having peptides with this motif are provided. Specific embodiments of the pharmaceuticals include each of the polypeptides described above with respect to pemphigus vulgaris. Thus, methods of tolerizing an individual to a pemphigus vulgaris autoantigen are also provided.

In another aspect of the invention, pharmaceuticals are provided for vaccination against a human pathogen implicated in the aetiology of autoimmune disease. These pharmaceutical preparations include a pharmaceutically acceptable carrier and an immunogenic preparation effective to immunize against a human pathogen. The human pathogen is one which in its native form includes a polypeptide having an amino acid sequence corresponding to a sequence motif for an HLA-DR protein which is associated with the autoimmune disease. These polypeptides are capable of binding to the HLA-DR protein to form a complex which activates T cells which become autoreactive and initiate the autoimmune disease. The preparations of the present invention specifically do not include such polypeptides but, rather, include other antigens from the pathogen.

In particular embodiments, such pharmaceutical preparations are provided in which the HLA-DR protein is HLA-DR4 protein and the autoimmune disease is pemphigus vulgaris. In addition, a particular sequence motif is provided for pemphigus vulgaris and pharmaceuticals which lack peptides having this motif are provided. Specific embodiments of the pharmaceuticals include preparations lacking each of the polypeptides described above with respect to pemphigus vulgaris. Thus, methods of immunizing an individual against pathogens which may cause pemphigus vulgaris are also provided.

The present invention also provides general methods for evaluating a peptide for an ability to induce an autoimmune response. These methods involve choosing an MHC HLA-DR molecule associated with the autoimmune response, selecting at least two major MHC binding pockets of the HLA-DR molecule, identifying sets of amino acid residues which bind within each of the selected pockets, developing a sequence motif for the HLA-DR molecule in which the sets of amino acids define the allowed amino acids at the corresponding positions of the motif, and then comparing the amino acid sequence of the peptide to the sequence motif. Peptides which match the motif have a much greater likelihood of inducing the autoimmune disease. In addition, if there is a known epitope implicated in the disease, the method may further include selecting at least one TCR contact residue of the epitope, identifying a set of amino acid residues which may serve as the TCR contact, and including this set in the motif at the appropriate position. In preferred embodiments, the motifs include restrictions on the residues at positions corresponding to at least the PI MHC binding pocket and at least one of the P4 and P6 pockets.

In another embodiment of the invention, methods are provided specifically for identifying foreign antigens implicated in human autoimmune response. These methods include the same steps as the previously described methods, but further include a comparison of the resulting sequence motif to sets of human pathogens. In preferred embodiments, peptide sequences from one or more species in the normal human intestinal flora are excluded from consideration. In another preferred embodiment, sequences from one or more species of pathogen which is negatively correlated with the incidence of the disease are excluded. In a most preferred embodiment, the human pathogen peptides are searched and evaluated on a computer database using the motif as a search criterion.

The present invention provides, in one aspect, isolated peptides derived from the human desmoglein 3 (Dsg3) protein, and uses thereof. These peptides, for example, consist essentially of SEQ ID NO: 1, and bind to a HLA-DR4 protein to form a complex which activates autoreactive T cells in a subject having pemphigus vulgaris.

Certain methods of the invention comprise administering to a subject a pharmaceutically effective amount of a Dsg3 peptide or peptidomimetic, which binds to a HLA-DR protein to form a complex which activates autoreactive T cells in subjects having the autoimmune disease. Administration of the peptidomimetic results in tolerization of the subject or can be used as a vaccine.

In further aspects, the invention provides nucleic acids comprising a coding sequence for a Dsg3 peptide that binds to a HLA-DR molecule. In certain embodiments, such nucleic acids may be used, for example, to produce Dsg3 peptides, including fusion proteins. In other embodiments, such nucleic acids may be administered to a subject so as to cause production of the Dsg3 peptide in vivo.

In certain aspects, the Dsg3 therapeutic comprises a fusion protein comprising a first polypeptide and a second polypeptide wherein the first polypeptide consists essentially of a Dsg3 peptide/peptidomimetic, and wherein the second polypeptide comprises a carrier protein, a production proteins, or a stabilizing protein.

In certain embodiments, the compounds of the present invention are represented by the following motifs:

TABLE-US-00001 E P N H L N S K I A F K I V S Q E P A (SEQ ID NO:1) 1 4 6 Motif 1 1 4 6 Motif 2

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

A. The MHC Class II HLA-DR Molecular Mimicry Motif

The HLA-DR binding site is characterized by five major pockets which may bind the amino acid side chains of antigens (Stem et al., 1994, the entire disclosure of which is incorporated herein by reference). See FIG. 1. The amino acid residue of the antigen which binds in the first major pocket is designated P1. The remaining residues may then be numbered by their positions relative to P1 (with positive numbers increasing toward the carboxy terminus and negative numbers increasing toward the amino terminus):

P-i . . . P-1 P1 P2 P3 P4 . . . Pj.

Thus, the first major pocket of an HLA-DR molecule, by definition, binds the side chain of residue P1 on an antigen. The remaining major pockets bind residues P4, P6, P7 and P9. These residues are defined as the major MHC contact residues.

The amino acid side chains of residues P-1, P2, P3, P5, P8, and P11 are oriented away from the HLA-DR binding site and, therefore, are available as contact residues for a T cell receptor (TCR). All of these residues are defined as TCR contact residues.

B. The MHC Contact Residues

The first major pocket of the HLA-DR molecule is strongly hydrophobic. It is formed by a stretch of residues at about positions 85, 86, 89 and 90 of the .beta. chain, a stretch of residues at about positions 31, 32 and 34 of the .alpha. chain, and side chains from residues at about positions 7 and 43 of the .alpha. chain. For example, in HLA-DR1 (DRA, DRB1*0101), the first pocket is formed by residues .beta.85 (Val), .beta.86 (Gly), .beta.89 (Phe), .beta.90 (Thr), .alpha.31 (Ile), .alpha.32 (Phe), .alpha.34 (Phe), .alpha.7 (Ile), and .alpha.43 (Trp). The corresponding residues for other HLA-DR alleles are known in the art (see, e.g., Marsh and Bodmer, 1992, incorporated by reference herein) and are available through genetic databases.

Although most of the residues that shape the P1 pocket are from the highly conserved DR.alpha. chain, the size and nature of this pocket varies due to polymorphisms in the .beta. chain residues involved in the pocket. For the DRB1*0101 protein, the pocket is large and hydrophobic and can accommodate any of the aliphatic or aromatic residues. Polymorphism at the .beta. residues, however, may alter the binding capacity of the P1 pocket. For example, the .beta.86 residue is known to be polymorphic. Most commonly, this site is occupied by either Gly or Val. Generally, when Gly is present at .beta.86 (as in DRB1*0101), any of the aliphatic or aromatic residues may bind within the pocket. When Val is present, however, the pocket is smaller and Tyr and Trp cannot be accommodated. Thus, when .beta.86 is Gly, position P1 of the molecular mimicry motif may consist of residues chosen from V, L, I, A, M, F, Y, W and when .beta.86 is Val, position P1 of the motif may consist of residues chosen from V, L, I, A, M, F. Similar considerations apply to the other .beta. residues of the P1 pocket.

The P4 pocket of HLA-DR molecules is also a relatively large, shallow, hydrophobic pocket oriented across the antigen binding site. This pocket can bind a variety of large aliphatic side chains which can maintain hydrophobic interactions along the side and floor of the pocket. The pocket is formed by a stretch of residues at about positions 70, 71, 74 and 78 of the .beta. chain, and side chains from residues at about position 13 of the .beta. chain and about position 9 of the .alpha. chain. For example, in HLA-DR1 (DRA, DRB1*0101), the P4 pocket is formed by residues .beta.70 (Gln), .beta.71 (Arg), .beta.74 (Ala), .beta.78 (Tyr), .beta.13 (Phe), and .alpha.9 (Gln). The corresponding residues for other HLA-DR alleles are known in the art (see, e.g., Marsh and Bodmer, 1992) and are available through genetic databases.

Like the P1 pocket, the P4 pocket is largely hydrophobic but its binding capacity is affected by polymorphisms at the .beta. residues involved in the pocket. For example, different DR alleles have differently charged residues at position .beta.71: In DRB1*0404, .beta.71 is occupied by a positively charged Arg residue whereas in DRB1*0402 .beta.71 is a negatively charged Glu residue. Thus, although this pocket can generally bind a variety of aliphatic or aromatic side chains (e.g., V, L, I, A, M, F, Y, W), positively charged P4 antigen residues are disfavored when .beta.71 is positively charged and, similarly, negatively charged P4 residues are disfavored when .beta.71 is also negative. Similar considerations apply to the other .beta. residues of the P4 pocket. Note that some residues may be involved in forming each of two adjacent pockets (e.g., .beta.13 in the P4 and P6 pockets) and therefore the occupancy of one of these pockets by a particular amino acid may influence the occupancy of the other.

The P6 pocket of HLA-DR molecules is a relatively shallow pocket with a preference for smaller (e.g., A, G) P6 antigen residues. The pocket is formed by the highly conserved .alpha.11, .alpha.62, .alpha.65 and .alpha.66 residues and the highly polymorphic .beta.11 and .beta.13 residues of the HLA-DR protein. For example, in HLA-DR1 (DRA, DRB1*0101), the P6 pocket is formed by residues .alpha.11 (Glu), .alpha.62 (Asn), .alpha.65 (Val), .alpha.66 (Asp), .beta.11 (Leu) and .beta.13 (Phe). The corresponding residues for other HLA-DR alleles are known in the art (see, e.g., Marsh and Bodmer, 1992) and are available through genetic databases.

Although there are only two .beta. chain residues in the P6 pocket, they vary widely amongst the DR alleles. With a large Phe residue at .beta.13 (as in DRB1*0101), the P6 residue is preferably one of the small residues (e.g., A, G). In other DR alleles, however, .beta.13 is occupied by smaller or more polar residues such as the .beta.13 (His) of DRB1*0401. For such alleles, the P6 motif may include somewhat larger and polar residues (e.g., S, T, V) but should still avoid the largest and aromatic residues. Finally, in some alleles, .beta.11 and .beta.13 are both serine residues (e.g., DRB1*1 101) and for these cases more hydrophilic or hydrogen bonding residues may be included in the motif.

The P7 pocket of HLA-DR molecules is also a relatively shallow pocket. The pocket is formed by five residues of the .beta. chain: .alpha.8, .beta.47, .beta.61, .beta.67 and .beta.71. For example, in HLA-DR1 (DRA, DRB*0101), the P7 pocket is formed by residues .beta.28 (Glu), .beta.47 (Tyr), .beta.61 (Trp), .beta.67 (Leu) and .beta.71 (Arg). The corresponding residues for other HLA-DR alleles are known in the art (see, e.g., Marsh and Bodmer, 1992) and are available through genetic databases. This pocket does not appear to contribute greatly to the specificity of HLA-DR1 but may be important in other alleles.

The P9 pocket of HLA-DR molecules is generally a small hydrophobic pocket and, therefore, small hydrophobic residues are preferred at the P9 position of the antigen. This pocket is formed by the conserved .alpha. chain residues .alpha.69, .alpha.72, .alpha.73 and .alpha.76 and by the polymorphic .beta. chain residues .beta.9 and .beta.57. For example, in HLA-DR1 (DRA, DRB1*0101) the P9 pocket is formed by .alpha.69 (Asn), .alpha.72 (Ile), .alpha.73 (Met), .alpha.76 (Arg), .beta.9 (Trp) and .beta.57 (Asp). The corresponding residues for other HLA-DR alleles are known in the art (see, e.g., Marsh and Bodmer, 1992) and are available through genetic databases.

The P6, P7 and P9 pockets appear to be less important than the P1 and P4 pockets in binding to DR molecules but they may be more important in binding to other isotypes (e.g., the P9 pocket of DQ may be important).

C. The TCR Contact Residues

When there is no known or suspected antigen involved in an autoimmune response, the positions of the sequence motif corresponding to the TCR contact residues may be left unrestricted. That is, absent a known or suspected antigen, the TCR contact positions of the motif are preferably allowed to vary amongst all of the amino acids.

When, on the other hand, there is a known or suspected antigen involved in an autoimmune response, at least some of the motif positions corresponding to the TCR contact residues may be restricted according to the sequence of the antigen. Thus, for example, the P2 and/or P3 and/or P5 positions of the motif may be restricted to only those residues found at the corresponding positions of the antigen. Alternatively, at least some of the TCR contact residues of the motif may be restricted not just to the corresponding residues of the antigen but may be allowed to vary amongst similarly charged and/or structurally similar residues (e.g., K and R). It should be noted, however, that greater conservatism with respect to the TCR contact residues of the motif is justified by the presumably greater specificity of TCR binding relative to the known promiscuity of MHC binding.

D. Developing an HLA-DR Sequence Motif

Given the present disclosure of the HLA-DR residues involved in the formation of the P1, P4, P6, P7 and P9 MHC binding pockets, and given the nucleotide or corresponding amino acid sequence of any particular HLA-DR allele, one is now enabled to develop a sequence motif useful in evaluating or predicting the ability of peptides to bind to that MHC protein. When a particular antigen is known to (or is suspected of) binding to the MHC protein, the TCR contact residues of that antigen may also be considered in the motif.

The method first requires the selection of two or more of the MHC binding pockets for which the choice of peptide residues will be restricted at the corresponding positions of the motif. One may select all five of the major binding pockets and develop a motif in which the corresponding five positions of the motif are restricted or one may select fewer and develop a less restricted motif. As will be obvious to one of ordinary skill in the art, a more restricted motif will identify a lesser number of peptides in a database search and a less restricted motif will identify a greater number of peptides. In all instances, at least two of the major binding pockets should be selected. When fewer than all five MHC binding pockets are selected, it is preferred that at least one is P1 and that a second is chosen from P4, P6 and P9.

Either before or after the pockets to be restricted by the motif are selected, the set of amino acid side chains likely to bind within each of those pockets and, therefore, the set of amino acid residues that will define the corresponding positions of the motif, must be determined. This may be accomplished by one of ordinary skill in the art by considering the amino acid residues which form the pocket. These residues, identified in Section A above, will determine the size and nature (i.e., hydrophobic, hydrophilic, positively charged, negatively charged, uncharged) of the pocket and consequently, the side chains which may bind within the pocket. Reference may be made to FIG. 1 during these considerations but will become increasingly unnecessary as one develops familiarity with the variations of the pockets.

As a general matter, in light of the identification of the residues forming the MHC binding pockets of the HLA-DR proteins disclosed herein, one of ordinary skill in the art can easily develop a sequence binding motif for any HLA-DR protein for which these residues are known for two or more binding pockets. The major considerations are size, hydrophobicity and charge. In light of the present disclosure, each of these considerations may be addressed according to well-known principles. A baseline is disclosed herein for each pocket for the DRB1*0101 allele, and relative to this HLA-DR protein, one of ordinary skill is enabled to develop motifs for other HLA-DR alleles. Thus, substitutions which lead to larger/smaller pockets suggest that the corresponding motif positions should be restricted so as to permit smaller/larger residues. Similarly, more/less hydrophobic pockets suggest that the corresponding motif positions should be restricted to more/less hydrophobic residues. Finally, positively/negatively charged pockets suggest that positively/negatively charged residues should be excluded and negatively/positively charged residues may be included at the corresponding motif positions. As noted above, the present disclosure enables one of ordinary skill to develop motifs based upon these well-established principles.

For example, and not by means of limitation, consider the P1 pocket of the HLA-DR protein. The residues forming this pocket in the DRB1*0101 were described above. For DRB1*0101, the P1 pocket is large and hydrophobic and can accommodate any of the aliphatic or aromatic residues (e.g., V, L, I, A, M, F, Y, W). For the DRB1*1602 protein the same is true. On the other hand, in the DRB1*1501 protein, the .beta.86 position is occupied by Val instead of the Gly found in DRB1*0101 and DRB1*1602. This substitution decreases the size of the P1 pocket in this MHC protein and, as a result, the pocket cannot easily accommodate Tyr or Trp side chains. Thus, for DRB1* 1501, the sequence motif at position P1 may be restricted to residues chosen from V, L, I, A, M and F.

Similarly, in light of the present disclosure, one of ordinary skill in the art may consider each of the MHC binding pockets, or only selected pockets, and develop a sequence motif for any HLA-DR protein for which the residues involved in pocket formation are known. These residues will determine both the size and nature of the pocket and, thereby, the size and nature of the residues which may bind within it. When the pocket is relatively small, the largest amino acid residues (e.g., Y, W) may be excluded from the corresponding position of the motif and alternatively, when the pocket is charged, amino acid residues of the same charge may be excluded.

If a self or foreign epitope involved in immune response is known or suspected, and particularly if its TCR contact residues can be defined through the use of responsive T cell clones, the TCR contact residues of the epitope may also be considered in developing a sequence motif. As with the MHC contact residues, all or merely some of the TCR contact residues may be restricted in the motif. And, as with the MHC positions, the restriction of more positions (or the greater restriction of any one position) will result in the identification of fewer peptides in a database search. Unlike the MHC contact residues, for which at least two positions should be restricted in the motif, it is acceptable to omit any restrictions of TCR contact residues in the motif.

If any TCR contact residue positions are restricted in the sequence motif, it is preferred that a position selected from positions P2, P3 and P5 be chosen. Because, in contrast to the relative promiscuity of MHC binding pockets, TCR contact residues appear to have greater specificity, it is preferred that any TCR contact residue positions which are restricted in the motif be rather narrowly restricted. That is, it is preferred that such positions be restricted to just the residue found at the corresponding position of the known antigen or just to residues which are highly similar in structure and charge.

Obviously, MHC and TCR positions not selected for restriction may be represented by, in the notation of this disclosure, an X. Similarly, as shown in the examples below, several motifs may be developed with varying numbers of positions restricted to varying extents.

II. Definitions

For clarity of interpretation and to clearly and distinctly point out the subject matter of the claimed invention, the following definitions are provided for several terms used in the claims appended hereto.

"Activate" or "activation" as used herein is intended to indicate that the subject Dsg3 peptide binds to a HLA-DR protein to form a complex which activates autoreactive T cells in subjects having an autoimmune disease.

By the terms "amino acid residue" and "peptide residue" is meant an amino acid or peptide molecule without the --OH of its carboxyl group. In general, the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11: 1726 1732). For instance, Met, Ile, Leu, Ala and Gly represent "residues" of methionine, isoleucine, leucine, alanine and glycine, respectively. By the residue is meant a radical derived from the corresponding a-amino acid by eliminating the OH portion of the carboxyl group and the H-portion of the .alpha.-amino group. The term "amino acid side chain" is that part of an amino acid exclusive of the --CH--(NH.sub.2)COOH portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1996, pages 2 and 33; examples of such side chains of the common amino acids are --CH.sub.2CH.sub.2SCH.sub.3 (the side chain of methionine), --CH.sub.2 (CH.sub.3)--CH.sub.2CH.sub.3 (the side chain of isoleucine), --CH.sub.2CH(CH.sub.3).sub.2 (the side chain of leucine) or H-- (the side chain of glycine).

For the most part, the amino acids used in the application are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, and those amino acids and amino acid analogs which have been identified as constituents of peptidoglycan bacterial cell walls.

The term amino acid residue further includes analogs, derivatives and congeners of any specific amino acid referred to herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g., modified with an N-terminal or C-terminal protecting group). For example, the present invention contemplates the use of amino acid analogs wherein a side chain is lengthened or shorted while still providing a carboxyl, amino or other reactive precursor functional group for cyclization, as well as amino acid analogs having variant side chains with appropriate functional groups).

An "amino acid motif" is a sequence of amino acids, optionally a generic set of conserved amino acids, associated with a particular functional activity.

The term "binding" refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions, and including interactions such as salt bridges and water bridges.

"Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A cell to be contacted with subject Dsg3 therapeutics includes a cell in culture, a cell that is part of an organized assemblage, such as an organ or tissue, or a cell that is part of an organism.

A "chimeric protein" or "fusion protein" is a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the first amino acid sequence. A chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergenic", etc. fusion of protein structures expressed by different kinds of organisms.

The terms "compound", "test compound" and "molecule" are used herein interchangeably and are meant to include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, natural product extract libraries, and any other molecules (including, but not limited to, chemicals, metals and organometallic compounds).

The phrase "conservative amino acid substitution" refers to grouping of amino acids on the basis of certain common properties. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this manner include: (i) a charged group, consisting of Glu and Asp, Lys, Arg and His, (ii) a positively-charged group, consisting of Lys, Arg and His, (iii) a negatively-charged group, consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic nonpolar group, consisting of Val, Leu and Ile, (vii) a slightly-polar group, consisting of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue may form its own group, and the group formed by an individual amino acid may be referred to simply by the one and/or three letter abbreviation for that amino acid commonly used in the art.

A "conserved residue" is an amino acid that is relatively invariant across a range of similar proteins. Often conserved residues will vary only by being replaced with a similar amino acid, as described above for "conservative amino acid substitution".

The term "consisting essentially of" as used in reference to a peptide including one or more designated amino acid sequences indicates that no more than 20 to 30 amino acids are added to the designated amino acid sequence(s), and furthermore that these additional amino acids do not substantially alter the function of the designated amino acid sequence(s). The term "consisting essentially of" as used in reference to a peptidomimetic indicates that no more than 20 30 amino acid mimetic units are added to the designated sequence, and that these added units do not substantially alter the function of the designated sequence.

An "effective amount" of, e.g., an Dsg3 peptide or peptidomimetic, with respect to the subject methods of treatment, refers to an amount of active ingredient in a preparation which, when applied as part of a desired dosage regimen brings about, e.g., binds to a HLA-DR4 protein to form a complex which activates autoreactive T cells in subjects having pemphigus vulgaris.

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is "unrelated" or "non-homologous" shares less than 40% identity, though preferably less than 25% identity with a sequence of the present invention. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.

The term "homology" describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present invention may be used as a "query sequence" to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 10. 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 utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used (See www.ncbi.nlm.nih.gov).

As used herein, "identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods