GB2251186A - Polypeptide for use in treatment of autoimmune disease - Google Patents

Abstract

The use of a polypeptide comprising an amino acid sequence not homologous to a sequence synthesised by the cells of the patient, for the manufacture of a medicament for the treatment of an autoimmune disease is described.

Description

Auto immune Disease Treatment This invention relates to the treatment of auto immune diseases, and especially the prophylactic treatment of such diseases.

Stress of a varied nature, induced as a result of heat shock, nutrient deprivation, oxygen radicals and other forms of metabolic disruption, including infection by certain viruses, bacteria and protozoans, as well as certain cases of cellular transformation, all lead to the increased synthesis of a family of proteins collectively known as stress proteins or heat shock proteins.

These stress proteins are among the most highly conserved and abundant proteins found in nature.

Further these proteins have been shown to be among the dominant antigens recognised in immune responses to a broad spectrum of pathogens. A review of the interrelationships between stress proteins, infection and immune surveillance has recently appeared, in which a clear analysis of these relationships is provided (13).

It has become apparent in recent years that a relationship exists between so-called stress or heat shock proteins and certain immune responses to infection and to the development of autoimmunity. As an example, the analysis of cell-mediated and humoral responses to a variety of bacterial and parasitic pathogens has shown that heat shock proteins are often strongly immunogenic during infection (1-8).

Proteins involved in immune responses to certain parasitic diseases such as malaria, shistosomiasis, leishmanisis, trypanosomiasis and filariasis, have been identified as members of the hsp 70 and 90 gene families. Further antigens related to hsp 70 and GroEL families have been shown to play a role in T cell and B cell recognition during bacterial infections including leprosy, tuberculosis and Q. fever. The mycobacterial GroEL stress protein has been identified as the target of a T cell clone capable of causing autoimmune disease in a rat model of adjuvant-induced arthritis (9). Similar results have been obtained as concerns the small heat shock proteins, since an immunologically important 19 Kd protein antigen of Mycobacterium leprae has been sequenced, and shown to have considerable amino acid sequence homology to the soybean l9Kd heat shock protein.

Elevated responses to the GroEL stress protein have been found by testing T cells from synovial infiltrates of rheumatoid arthritis patients (10). Autoantibodies to hsp 90 have been reported in systemic lupus erythrematosus (SLE) (11). In addition, elevated antibody responses to hsp70 and GroEL stress proteins have been found in SLE and in rheumatoid arthritis (12).

The stress proteins are remarkable in their evolutionary conservation: hsp90, hsp70, and hsp60 proteins are found in all prokaryotes and eukaryotes.

In fact comparison of almost any two hsp70 proteins from two different organisms indicates an amino acid homology of around 50%. The major stress proteins occur at low levels in normal, unstressed cells, but accumulate to very high levels in cells undergoing stress. A striking example is the case of E. coli hsp60, which accounts for 1.6% of total cell protein under normal growth conditions, and can accumulate to 15% of total cell protein after heat shock (14).

Stress proteins appear to fulfil vital roles in cells, both in the absence and in the presence of stress.

They appear to be involved in the assembly and disassembly of protein complexes, and hsp70 proteins are important for the translocation of certain proteins through cellular membranes (15). Stress proteins appear to interact with many different proteins, for example, hsp90 has been found to interact with steroid hormone receptors and with viral and cellular kinases. Hsp70 proteins bind to DNA replication complexes, clathrin baskets, the cellular tumour antigen p53, and immunoglobulin heavy chains.

Plant hsp60 interacts with Rubisco, which fixes C02 in chloroplasts, and may be the most abundant protein in the biosphere (16). The interaction of stress proteins with multiple proteins may provide an explication for the evolutionary constraints imposed on their amino acid sequences.

Stress proteins have an almost certain role in protecting cells and organisms from the deleterious effects of heat and other stresses.

It seems clear that the tight sequence regulation imposed on many heat shock protein sequences throughout evolution has led to such retained sequences between those of the host and those of the infectious agent having a significant degree of identity. Clearly the reaction of the host immune system against antigens of the infecting organism could lead to the raising of antibodies against heat shock proteins. The sequence homology within the heat shock protein family thus points to conserved sub-sequences of heat shock proteins as being serious candidates for inducing an immune response that can have specificity against self sequences, with the consequence of inducing an autoimmune reaction and the associated disease states.

The reports referenced above indicate that stress proteins, such as the heat shock proteins, provide particularly attractive targets for immune recognition.

An analysis the cross reactivity of T cell responses to stress proteins has been published recently (17), wherein the presence of human T cells was demonstrated that were capable of immune recognition of conserved sequence determinants. These authors have proposed a model in which immune responses to stress proteins provide a link between infectious and auto immune diseases.

Although models of the role of stress proteins in autoimmune diseases have been proposed, no-one has yet suggested possible treatment for autoimmune diseases.

In accordance with a first aspect of the present invention a method of treating an auto immune disease in a patient comprises introducing a compound, comprising an amino acid sequence of a protein which is not homologous with amino acid sequences synthesised by cells of the patient, into the patient.

In accordance with another aspect of the present invention there is provided use of a compound comprising an amino acid sequence of a protein for the treatment of an autoimmune disease in a patient, wherein the amino acid sequence is not homologous with amino acid sequences synthesised by cells of the patient.

Further, the invention provides a composition for treatment of an autoimmune disease in a patient, comprising a compound which comprises an amino acid sequence of a protein which is not homologous with amino acid sequences synthesised by cells of the patient, in combination with a pharmaceutical carrier.

Still further, the invention provides the use of a compound comprising an amino acid sequence of a protein which is not homologous with amino acid sequences synthesised by the cells of a patient for the manufacture of a medicament for the treatment of an autoimmune disease in the patient.

Preferably, the compound comprises a peptide which comprises the amino acid sequence and typically, the protein is a stress or heat shock protein.

Preferably, the treatment is prophylactic.

Typically, the compound could be introduced into a patient by incorporation in a cream or ointment, in a soluble glass, in slow release capsules, transdermal patches, injected, or even administered orally or in suppository form.

Preferably, the amino acid sequence has antigenic properties.

The amino acid sequence could be naturally occurring or be synthesised. If the amino acid sequence is synthesised then the peptide could comprise a number of different amino acid sequences and/or multiples of the same amino acid sequence.

The invention described here is based on the above-detailed conservation of heat shock sequences and their implication in autoimmune diseases. Contrary to the identity of certain conserved sequences, this invention, is based on the hypervariable sequences of stress proteins. Prior immunisation with natural or synthetic peptides representing such non-conserved, variable or hypervariable stress protein sequences of origin from infectious agents of bacterial and other parasitic pathogens, induces antibody responses against the stress proteins of the infecting organism, and these specifically induced antibodies are incapable of recognising self stress protein sequences.The rapid recognition of infectious agent - specific stress proteins by specific pre-existing antibodies raised against non-homologous peptides from invading stress proteins should allow the elimination of these stress proteins before they are able to elicit potentially autoimmune responses.

This invention concerns the immune recognition of peptide epitopes of specific heat shock or stress proteins, and the development of peptide-based therapy or prevention based on such epitopes.

Examples of the invention will now be described.

1. Analysis of stress protein peptide sequences In order to practice the preventive/therapeutic approach described in this invention, it is necessary to examine in detail the amino acid sequences of human heat shock proteins, and of those of organisms infecting human beings with whom correlations of immune diseases exist.

Our initial approach was to assemble a table of certain of the known sequences of stress proteins from human and infectious agent sources. A selection of these sequences are presented in Appendix 1. A thorough analysis of sequence homology between members of each of the stress protein families indicates that for each of the principle stress protein families, hsp70, hsp90 and hsp27, certain sequences have been highly conserved throughout evolution, whereas parts of the stress proteins contain amino acid sequences that are highly differentiated. One assumes that the conservational pressures concerning the retained sequences are associated with critical structural or functional aspects of these important proteins.The variable regions are presumably of less critical structural or functional importance, thus escaping from the conservative pressure/selection activities prevailing in evolving organisms.

2. Selection of candidate peptide vaccines The selection of useful candidate peptides capable of eliciting an immune response specifically against the stress proteins of the infectious agent is based on two major criteria: i) The non-identity of selected peptide sequences, and their lack of resemblance to highly, or partially conserved stress protein sequences, common to human and infectious agent proteins. The selection of such non-conserved sequences is derived from a reverse analysis of amino acid sequence homologies, in other words, concentrating on the non-homologous sequences evident from homology analyses such as those shown in (1) and in appendix 2.

For a thorough selection of sequence differences versus sequence homology, it is instructive to, in addition to amino acid identity, to look at replacements by highly conserved amino acids. Examples of such substitutions are the following groups: (aspartic acid and glutamic acid), (lysine and arginine), (serine and threonine), (phenylalanine and tyrosine), and (isoleucine, leucine, valine and methionine).

ii) An analysis of the antigenic potential of selected peptide sequences. Where information is available, peptide epitopes that conform to the criteria of both points i) and ii), and which can be demonstrated to be immunodominant, are preferred examples of the preventive/therapeutic peptides described in this invention.

Examples of the amino acid sequences of some selected peptides that reply to the criteria of point i) are presented in appendix 2.

Examples of group i) peptides that are expected to have considerable immunogenic potential have been selected on the basis of presently accepted criteria of immunological potential. Examples of certain peptides with pronounced antigenicity are shown in appendix 3.

Non-homologous sequence comparison of the known stress protein and related antigen sequences from humans and from infectious agents has been performed. In the case of Plasmodium falciparum, in addition to regions of extensive homology of amino acid sequence between the two proteins, clear regions of extensive lack of homology are also detectable, and the following sequence fragments, depicted using the one and three-letter amino acid abbreviations derived from the IUPAC-IUB Commission on Biochemical Nomenclature (see Table 1), illustrate this example:: ALIGNMENT OF RESIDUES 133 TO 254 OF 75KDa antigen of P Falciparum TO RESIDUES 357 TO 635 OF HSP70 HUMAN ENYCYGVKSSLEDKIKEKLQPAEIETCMKTITTILEWLEKNQLAGKDEYE ------------ KNALES-Y-AFNMKSA- VEDEG LKGKIS-E AKQKEAESVCAPIMSKIY-QDAA-GAAGGMPGGM-P-GGMPGGMP GGMNF ADKKKVLDKCQEVIS- WLDANTLA EKDEFEHKRKELEQVCNPIISGL-Y pG-GMpG-AGMpGNAp---AGSGpTVEEVV QGAGGPGPGGFGAQGPKGGSGSGPT---- Examples of non-homologous peptides are shown in bold letters. The second peptide of HSP70 human shown in bold above, denoted "Peptide example 1", has been compared to the sequence of the corresponding antigen of Mycobacterium tuberculosis and its highly unique sequence has little or no counterpart in the sequence of tubercular origin.

ALIGNMENT OF RESIDUES 8 TO 11 OF PEPTIDE 1 TO RESIDUES 1 TO 127 OF 71KDa antigen M.tuberculosis K----R--K-- E --------------------------------'---- KEDIDRMIKDAEAHAEEDRKRREEADVRNGAETLVYNTEKFVKEQREGG Clearly other peptide sequences unique to an infectious agent antigen exist and will have value in the applications described in this invention. In order to identify such sequences, extensive cloning, expression and sequence analysis of infectious agent antigens will be required. Such research, although technically arduous, is quite within the realms of existing technology. Similarly, once new sequences are established, the presence or absence of amino acid sequence homologies can be determined either visually, or through the use of any number of amateur or commercial sequence analysis software programs. Our intention here is to demonstrate the general procedure for identifying, and applying both specific non-homologous and specific homologous stress and infectious agent antigen peptide sequences to the vaccination, therapeutic and cosmetic applications described herein.

3 The Rational Design of Synthetic Peptides This invention is not limited to naturally occurring variant sequences within stress proteins, nor is it limited to the selection and use of a single variant epitope. For example, synthetic peptides could be used. In addition, the peptide could be synthesised to have combinations of different variant sequences or multiples of variant sequences. By synthesising peptides comprising different variant sequences and/or multiples of the same variant sequence it may be possible to design peptides having a stronger immune response against stress proteins of infectious organisms but which do not recognise human stress epitopes.

A recent analysis of variant peptide epitopes of myelin basis protein (MBP), and their influence on the incidence of experimental autoimmune encephalomyelitis (EAE) has indicated that synthetic variants of an N-terminal MBP peptide can have greatly altered properties of binding to cell surface glycoproteins encoded by the major histocompatibility complex (MHC) (18). In other words, the efficacy of the complex interactions associated with the elicitation of an effective immune response against peptide antigens, can be altered and improved in some cases, by the use of synthetic variants of natural antigens. The subject of this invention comprises those variant peptide sequence approaches that are taught by the authors of reference 18, amongst others.

An efficient mapping procedure for identifying protein antigenic determinants has been described that would be of use in the selection of useful antigenic determinants for the applications taught in this invention (19). Clearly classical chemical, enzymatic and combined synthetic procedures can be utilised to produce candidate peptides, once identified and selected, for the vaccine applications described here.

A naturally expected limitation of the peptide vaccines that can be produced using this described procedure derives from the fact that about one third of monoclonal and polyclonal antibodies made by immunising with native protein react with assembled topographic sites (20). These assembled determinants may not form the appropriate structure outside of a proteins native environment. This limitation is not expected to significantly limit the practical use of this invention.

Studies concerning T Cell recognition and activation have indicated that it may be possible to design peptides with predictable and advantageous properties (21). These authors have described two approaches for immunomodulation that could be useful for the design of therapeutic strategies against autoimmune encephalomyelitis. The first approach consists of a thorough molecular characterisation of an encephalitogenic epitope, and the subsequent design of peptide analogs that retain normal or increased major histocompatibility complex binding properties, and that fail to activate disease-inducing T cells. Secondly, novel properties of a heterocyclic peptide have been described, with the result that the peptide is highly antigenic in vitro, while being non-immunogenic in vivo.These authors have been able to demonstrate the feasibility of immune intervention in an immune disease through the use of a synthetic peptide. These results are complementary to the procedure we describe here, but are not identical, nor do they in any way predict the approach that we describe.

4 Applications of the stress protein peptides described herein The basic tenant that we have developed herein is based on the multiple observations that certain infectious agent antigens are closely related in amino acid sequence to human stress proteins, and that immune reactions against such antigens can cross react with the human proteins, leading to the possibility of developing autoimmune disease. Our invention describes the selection of stress protein peptide sequences from infectious agent antigens related to human stress proteins, but which have little or no sequence homology within such human stress proteins. The injection of such non-homologous peptides into human beings, for instance in an emulsification with Freunds complete adjuvant, would provide a route of effective vaccination against subsequent auto immune disease induced as mentioned above.The antibodies raised through such vaccination are specific to the selected infectious agent antigen from which the vaccinating peptide was derived. Such induced antibodies are specific te infectious agent antigens, thus explaining their efficacy in the application of this invention.

Further, since the vaccinating agent is a small peptide, instead of a large, complex protein such as human factor VIII, or factor IX, it is not compulsory to use an injection as a means of delivering the peptide to a human subject. We thus reserve in our application the administration of the kinds of peptides described by transdermal applications, a number of which are presently commercialised with considerable success.

Further still, since certain major diseases that are thought to have their origin in autoimmune diseases, such as arthritis and rheumatism, the peptides of this invention can be applied externally, in both local and cosmetic application to painful joints and articulations resulting from these prevalent diseases.

For example, the peptides could be administered to a patient by incorporation in a cream or ointment, in a soluable glass, in slow release capsules, transdermal patches, injected, or even administered orally or in suppository form.

In addition, due to the nature of amino acid sequences it is unlikely that treatment using these substances will produce the unpleasant side effects which are normally associates with drugs.

APPENDIX 1 NON-HOMOLOGOUS SEQUENCES WHICH ARE ALSO KNOWN ANTIGENICS ARE DENOTED BY UNDERLINING AND NON-HOMOLOGOUS ONLY SEQUENCES ARE DENOTED BY BOLD LETTERING SEQUENCE OF HUMAN STRESS PROTEINS A) Sequence HSP90 Human Rebbe N F, Ware J, Bertina M, Modrich P, Stafford D W Gene 53:235-245(1987) EMBL; M16660; HSHSP90 KW Heat Shock. Sequence 724 AA; 83293 MW MPEEVHHGEE EVETFAFQAE IAQLMSLIIN TFYSNKEIFL 40 RELISNASDA LDKIRYESLT DPSKLDSGKE LKIDIIPNPQ 80 ERTLTLVDTG IGMTKADLIN NLGTIAKSGT KAFMEALQAG 120 ADISMIGQFG VGFYSAYLVA EKVVVIRKHN DDEQYAWESS 160 AGGSFTVRAD HGEPIGMGTK VILHLKEDQT EYLEERRVKE 200 VVKKHSQFIG YPITLYLEKE REKEISDDEA EEEKGEKEEE 240 DKDDEEKPKI EDVGSDEEDD SGKDKKKKTK KIKEKYIDQE 280 ELNKTKPIWT RNPDDITQEE YGEFYKSLTN DWEDHLAVKH 320 FSVEGQLEFR ALLFIPRRAP FDLFENKKKK NNIKLYVRRV 360 FIMDSCDELI PEYLNFIRGV VDSEDLPLNI SREMLQQSKI 400 LKVIRKNIVK KCLELFSELA EDKENYKKFY EAFSKNLKLG 440 IHEDSTNRRR LSELLRYHTS QSGDEMTSLS EYVSRMKETQ 480 KSIYYITGES KEQVANSAFV ERVRKRGFEV VYMTEPIDEY 520 CVQQLKEFDG KSLVSVTKEG LELPEDEEEK KKMEESKAKF 560 ENLCKLMKEI LDKKVEKVTI SNRLVSSPCC IVTSTYGWTA 600 NMERIMKAQA LRDNSTMGYM MAKKHLEINP DHPIVETLRQ 640 KAEADKNDKA VKDLVVLLFE TALLSSGFSL EDPQTHSNRI 680 TYMIKLGLGI DEDEVAAEEP NAAVPDEIPP LEGDEDASRM 720 EEVD 724 B) Sequence HSP70 Human [1] Hunt C, Morimoto R I; Proc Natl Acad Sci USA 82:6455-6459(1985) EMBL; M11236; HSHSP701 EMBL; MII717; HSHSP70D KW Heat Shock Sequence 640AA; 69867 MW MAKAAAVGID LGTTYSCVGV FQHGKVEIIA NDQGNRTTPS 40 YVAFTDTERL IGDAAKNQVA LNPQNTVFDA KRLIGRKFGD 80 PVVQSDMKHW PFQVINDGDK PKVQVSYKGE TKAFYPEEIS 120 SMVLTKMKEI AEAYLGYPVT NAVITVPAYF NDSQRQATKD 160 AGVIAGLNVL RIINEPTAAA IAYGLDRTGK GERNVLIFDL 200 GGGTFDVSIL TIDDGIFEVK ATAGDTHLGG EDFDNRLVNH 240 (3) FVEEFKRKHK KDISQNKRAV RRLRTACERF EGIDFYTSIT 280 RARFEELAKR TLSSSTOASL EIDSLCSDLF RSTLEPVEKA 320 (4) LRDAKLDKAQ IHDLVLVGGS TRIPKVQKLL QDFFNGRDLN 360 KSINPDEAVG YGAAVQAAIL MGDKSENVQD LLLLDVAPLS 400 LGLETAGGVM TALIKRNSTI PTKQTQIFTT YSDNQPGVLI 440 QVYEGERAHT KDNNLLGRFE LSGIPPAPGV PQIEVTFDID 480 (1) ANGILNVTAT DKSTGKANKI TITNDKGRLS KEEIERI4VQE 520 AEKYKAEDEV QRERVSAKNA LESYAFNMKS AVEDEGLKGK 560 (2) ISEADKKKVL DKCQEVISWL DANTLAEKDE FEHKRKELEQ 600 VCNPIISGLY QGAGGPGPGG FGAQGPKGGS GSGPTIEEVD 640 C) Sequence Human HSP27 Hickey E, Brandon S E, Potter R, Stein G, Stein J, Weber L A; Nucl.Acids Res 14:4127-4145(1986) EMBL;X03900; HSHSP27 KW: HEAT SHOCK SEQUENCE 199 AA; 22327 MW; MTERRVPFSL LRGPSWDPFR DWYPHSRLFD QAFGLPRLPE 40 EWSQWLGGSS WPGYVRPLPP AAIESPAVAA PAYSRALSRQ 80 LSSGVSEIRH TADRWRVSLD VNHFAPDELT VKTKDGVVEI 120 TGKHEERQDE HGYISRCFTR KYTLPPGVDP TQVSSSLSPE 160 GTLTVEAPMP KLATQSNEIT IPVTFESRAQ LGGRSCKIR 200 D) Sequence Human HSP60 Sequence not yet available, submitted for publication: Gupta R S, Jinal S, Harley C B and Dudani A K(1989) SEQUENCE OF HSP60 YEAST Reading D S, Hallberg R L and Myers A M (1989).Nature 337 655 MLRSSVVRSR ATLRPLLRRA YSSHKILKFG VIGRASLLKG 40 VETLAIAVAA TLGPKGRNVL IEQPFGPPKI TKDGVTVAKS 80 IVLKDKFINM GAKLLQIVAS KTNIAAGDGT TSATVLGRAI 120 FTISVKNVAA GCNPMDLRRG SQVAVIKVIL FLSANKKEIT 160 TSEEIAQVAT ISANGDSHVG KLLASAMEKV GKEGVITIRE 200 GRITLEDELE VTEGMRFDRG FISPYFITDP KSSKVEFEKP 240 LLLLSEKKIS SIQDILPALE ISNQSRRPLL IIAEDVDGEA 280 LAACILNKLR GQVKVCAVKA PGFGDNRKNT IGDIAVLTGG 320 TVFTEELDLK PEQCTIENLG SCDSITVTKE DTVILNGSGP 360 KEAIQERIEQ IKGSIDITTT NSYEKEKLQE RLAKLSGGVA 400 VIRVGGASEV EVGEKKDRYD DALNATRAAV EEGILPGGGT 440 ALVKASRVLD EVVVDNFDQK LGVDIIRKAI TRPAKQIIEN 480 AGEEGSVIIG KLIDEYGDDF AKGYDASKSE YTDMLATGII 520 DPFKVVRSGL VDASGVASLL ATTEVAIVDA PEPPAAAGAG 560 GMPGGMPG MPGMM 600 SEQUENCES OF BACTERIAL ANTIGENS A) Mycobacterium leprae 18 KDa Antigen Nerland A H, Mustapha A S, Sweetser D, Godal T, Young R J Bacteriol 170 5919-5921 (1988) Sequence 148 AA; 16643MW; MLMRTDPFRE LDRFAEQVLG TSARPAVMPM DAWREGEEFV 40 VGFDLPGKA DSLDIDIERD VVTVRAERPG VDPDREMLAA 79 ERPRGVFNRQ LVLGENLDTE RILASYQEGV LKLSIPVAER 119 AKPRKISVDR GNNGHQTINK TPHEIIDA 65 KDa Antigen Mehra V, Sweetser D and Young R A (1986) Proc Natl Acad Sci USA 83 7013 AA 589, MW 61,831 The Underling Amino Acids Correspond To Antigenic Peptides.

VPGRDGETQP ASCGRPSRAL HPASVSNGGC RSPVILASFL 40 IRRNHFAMAK TIAYDEEARR GLERGLNSLA DAVKVTLGPK 80 GRNVVLEKKW GAPTITNDGV SIAKEIELED PYEKIGAELV 120 KEVAKKTDDV AGDGTTTATV LAQALVKEGL RNVAAGANPL 160 GLKRGIEKAV DKVTETLLKD AKEVETKEQI AATAAISAGD 200 QSIGDLIAEA MDKVGNEGVI TVEEESNTFG LQLELTEGMR 240 FDKGYISGYF VIDAERQEAV LEEPYILLVS SKVSTVKDLL 280 PLLEKVIQAG KSLLIIAEDV EGEALSTLW NKIRGTFKSV 320 AVKAPGFGDR RKAMLQDMAI LTGAQVISEE VGLTLENTDL 360 SLLGKARKVV MTKDETTIVE GAGDTDAIAG RVAQIRTEIE 400 NSDSDYDREK LQERLAKLAG GVAVIKAGAA TEVELKERKH 440 REIDAVRNAK AAVEEGIVAG GGVTLLQAAP ALDKLKLTGD 480 EATGANIVKV ALEAPLKQIA FNSGMEPGVV AEKVRNLSVG 520 HGLNAATGEY EDLLKAGVAD PVKVTRSALO NAASIAGLFL 560 TTEAVVADKP EKTAAPASDP TGGMGGMDF 600 70 KDa Antigen Not yet sequenced.Immunological cross-reactivity with the 71 KDa antigen of Mycobacterium tuberculosis (YOUNG ET AL Proc Natl Acad Sci USA 85, 4267-4270 (1988).

B) Mycobacterium tuberculosis 65 KDa Antigen Schinnick, T S (1987). Journal of Bacteriology 169 1080 AA 562, MW 59083 RGCRHPVTPP VSSPIRRNHF AMAKTIAYDE EARRGLERGL 40 NALADAVKVT LGPKGRNVVL EKKWGAPTIT NDGVSIAKEI 80 ELETPYEKIG AELVKEVAKK TDDVAGDGTT TATVLAQALV 120 REGLRNVAAG ANPLGLKRGI EKAVEAKVTET LLKGAKEVET 160 KEQIAATAAI SAGDQSIGDL IAEAMDKVGN EGVITVEESN 200 TFGLQLELTE GMRFDKGYIS GYFVTDPERQ EAVLEDPYIL 240 LVSSKVSTVK DLLPLLEKVI GAGKPLLIIA EDVEGEALST 280 LVVNKIRGTF KSVAVKAPGF GDRRKAMLQD MAILTGGQVI 320 SEEVGLTLEN ADLSLLGKAR KVVVTKDETT IVEGAGDTDA 360 IAGRVAQIRQ EIENSDSDYD REKLQERLAK LAGGVAVIKA 400 GAATEVELKE RKHRIEDAVR NAKAAVEEGI VAGGGVTLL 440 AAPTLDELKL EGDEATGANI VKVALEAPLK QIAFNSGLEP 480 GVVAEKVRNL PAGHGLNAQT GVYEDLLAAG VADPVKVTRS 520 ALQNAASAIG LFLTTEAVVA DKPEKEKASV PGGGDMGGMD 560 F 600 71 KDa Antigen Partial sequence, contains only the homolgy domain with HSP70 Young D, Lathigra R, Hendrix R, Sweetser D, Young R, Proc Acad Sci USA 85, 4265-4270 (1988).

EFQPSVQIQV YQGEREIAAH NKLLGSFELT GIPPAPRGIP 40 (1) QIEVTFDIDA NGIVHVTAKD KGTGKENTIR IQEGSGLSKE 80 DIDRMIKDAE AHAEEDRKRR EEADVRNGAE TLVYNTEKFV 120 3,4 KEQREGGSKV PEDTWRIGYF GHQVGDGEAG PGVAGSGASD 160 (2) LRSSSGCVTG HWRCPPRAAA GRCPPRLGM 200 C) Plasmodium falciparum (MALARIA) 90 KDa Antigen M Jendoubi, S Bonnefoy, Nucl Acids Res 16, 10928 (1988) Partial sequence, contains only the region of homology with HSP90 KDFDGKKLKC CTKEGLDIHH SEEAKKDFET VIKDVLHKKV 40 EKVVVCQRIT DSPCVLVTSE FGWSANMERI MKAQALRDNS 80 MTSYMLSKKI MEINARHPII SALKQKADAD KSDKTVKYLI 120 WLLFDTSLLT SGFFALEEPT TFSKRIHRMI KLGLSIDEEE 160 NNDIDLPPLE ETVDATDSKM EEVD 200 75 KDa Antigen Ardeshir F, Flint J E, Richman S and Reese R T, Embo J.

6, 493-499 (1987).

Partial sequence from the first AA MLKLIERNTT IPAKKSQIFT TYADNQPGVL IQVYEGERAL 40 TKDNNLLGKF HLDGIPPAPR KVPQIEVTFD IDANGILDVT 80 AVEKSTGKQN HITITNDKGR LSQDEIDRMV NDAEKYLAED 120 EENRKRIEAR NSLENYCYGV KSSLEDKIKE KLQPAEIETC 160 MKTITTILEW LEKNQLAGKD EYEAKQKEAE SVCAPIMSKI 200 YQDAAGAAGG MPGGMPGGMP GGMPGGMNFP GGMPGAGMPG 240 NAPAGSGPTV EEVVD 280 APPENDIX 2 DIFFERENTIATION OF HOMOLOGOUS (UNDERLINE) AND NON-HOMOLOUGOS SEQUENCES A) Alignment of Residues 47 to 161 of partial sequence of P.Falciparum 90KD to residues 581 to 699 of human HSP90 --RI-DSPCVLVTSEFGWSANMERIMKAOALRDNSMTSYMLSKKIMEINAR NRLVSSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYIMAKKHLEINPD HPIISALKDADKSDKTVKYLIWLLFDTSLLTSGFFALEEPTTFSKRI HPIVETLRQKAEADKNDKAVKDL W LLFETALLSSG-FSLEDPQTHSNRI HRMIKLGLSIDEEE---NN YRMIKLGLGIDEDEVAAEE B) Alignment of residues 7 to 157 of partial sequence of P. falciparum 70 KDa to residues 411 to 613 of human HSP70.

NTTIPAKKSOIFTTYADNOPGVLIOVYEGERALTKDNNLLGKFHL ALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFEL DGIPPAPRKVPOIEVTFDIDANGILDVTAVEKSTGKQNHITITNDKGRLS SGIPPAP-GVPQIEVTFDIDANGILNVTATDKSTGKMWITITNDKDRLS QDEIDRMVNDAEKYLAEDEENRKRIEARNSLENYCYGVKSSLEDM-IKEKLQ KEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKIS P~ETCMK---TITTILEWLEK~QLAGKDEYEAKQKEAES~CAPIMSKIYQDA EADKKKVLDKCQEVI-SWLDANTLAEKDEFEHKRKELEQVCNPIISGLYQGA C) Alignment of residues 5 to 110 of M tuberculosis 71K to residues 430 to 548 of human HSP70 VQIOVYQGEREIAAHNKLLGSFELTGIPPAPRGIPOIEVTFDI YSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAP-GVPQIEVTFDI DANGIVHVTAKDKGTGKENTIRIQEGSG-LSKEDIDRMIKDAEAHAEEDR DANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDE KRREEADVRNGAE----- VQRERVSAKNALESYAFNM APPENDIX 3 Antigenic peptides of the 65 Kda Antigen of Mvcobacterium leprae MEHRA V, SWEETSER D and YOUNG R A (1986) Proc Natl Acad Sci USA 83 7013 -NSLADAVKVTLGPKGRNVVLEKKWGAPTITNDGVS -RNVAAGANPLGLKRGIEKAV -ALDKLKLTGDEATGA -GEYEDLLKAGVADP -ASDPTGGMGGMDF TABLE 1 One and Three Letter Amino Acid Abbreviations A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glumatic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T The Threonine V Val Valine W Trp Tryptophane Y Tyr Tyrosine B Asx Asp or Asn (not distinguished) Z Glx Glu or Gln (not distinguished) X X Undetermined or atypical amino acid From: IUPAC-IUB Commission on Biochemical Nomenclature, J Biol Chem 243, 3557-3559, 1968.

References 1 Young D B, Lathigra R, Hendrix R, Sweetser D and Young R A 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc Natl Acad Sci USA 85 4267.

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Claims (4)

1. A method of treating an autoimmune disease in a patient comprises introducing a compound, comprising an amino acid sequence of a protein which is not homologous with amino acid sequences synthesised by cells of the patient, into the patient.

2. Use of a compound comprising an amino acid sequence of a protein for the treatment of an autoimmune disease in a patient, wherein the amino acid sequence is not homologous with amino acid sequences synthesised by cells of the patient.

3. A composition for treatment of an autoimmune disease in a patient, comprising a compound which comprises an amino acid sequence of a protein which is not homologous with amino acid sequences synthesised by cells of the patient, in combination with a pharmaceutical carrier.

4. The use of a compound comprising an amino acid sequence of a protein which is not homologous with amino acid sequences synthesised by the cells of a patient for the manufacture of a medicament for the treatment of an auto immune disease in the patient.