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GB2454687A - MHC binding peptides from GAD65 - Google Patents

MHC binding peptides from GAD65 Download PDF

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GB2454687A
GB2454687A GB0722399A GB0722399A GB2454687A GB 2454687 A GB2454687 A GB 2454687A GB 0722399 A GB0722399 A GB 0722399A GB 0722399 A GB0722399 A GB 0722399A GB 2454687 A GB2454687 A GB 2454687A
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peptides
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Nikolai Franz Gregor Schwabe
Linda Cheng-Choo Tan
Catherine Elizabeth Gouveia
Benjamin Lester Holley
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Proimmune Ltd
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Abstract

MHC binding peptides - in particular MHC class II binding peptides - are derived from the human glutamate decarboxylase, 65kDa isoform (GAD65). The peptides may be used to produce MHC-peptide complexes, or to generate antibodies or CD4+ cell lines specific to these peptides. Pharaceutical and diagnostic compositions to treat or detect insulin dependent diabetes mellitus (IDDM), stiff-man syndrome (SMS) or polyendocrine autoimmune syndrome (PAS).

Description

MHC Binding Peptides And Their Uses
Introduction
The present invention is concerned with MHC binding peptides derived from human glutamate decarboxylase, 65 kDa isoform (C1AD65) and their uses.
Major Histocompatibility Complex (MHC) molecules, which are found on the cell surface in tissues, play an important role in presenting cellular antigens in the form of short linear peptides to T cells by interacting with T cell receptors (TCRs) present on the surface of I cells.
In the above interaction of the MI-IC molecule and the T cell, the T cell recognizes and binds to the peptide bound in the MHC binding groove and the surrounding area of the MHC binding groove itself. In order for a peptide to bind to an MHC molecule and therefore be able to cause an immune response in vivo it needs to fit the properties of the binding groove, which are also referred to as the "binding motif', of the relevant MHC allele in question. This binding motif restricts the type of peptides that can bind to a given MHC allele. Binding peptides matching the :" binding motif of a given MI-IC allele are also referred to as "restricted" to that allele. *....
* It is known that class I MHC molecules typically bind peptide fragments of 8 to 11 amino acid ::* length in their binding groove formed between the MHC class I aiphal and alpha2 domains. For * Class II MHC molecules, binding of the peptides occurs in the groove formed between the alphal and betal domains of the molecule, the length of binding peptide varies more widely, and these peptides are typically 15 to 25 amino acids long or even longer, e.g. up to 30 amino acids length.
Class I MHC molecules are expressed on all nucleated cell and present Cytosolic-denved peptide fragments to cytolytic CD8+ T cells. Class II MHC molecules are expressed on professional antigen presenting cells (APCs) and present peptide fragments derived from exogenous proteins to CD4+ T cells.
Understanding which peptides in a protein sequence can bind to a given MHC molecule and cause a T cell immune response is of considerable interest for understanding cellular immunity and in order to design and monitor the effectiveness of immunotherapeutic products, such as vaccines or compounds for tolerising immunisation. MHC binding peptides that cause T cell immune responses are also referred to as "T cell epitopes".
The relevance of identifying MHC-binding peptides has also been described extensively in the literature, such as in US 5,585,461 or in US 6,733,973, which describe the discovery and uses of MHC class I restricted T cell epitopes for the tumour antigen MAGE-3 and cytomegalovirus (CMV), respectively and EP0665289 which describes the discovery and uses of MHC class II restricted I cell epitopes for the GAD65.
GAD65 is an important autoantigen associated with insulin dependent (type 1) diabetes mellitus (IDDM), stiff-man syndrome (SMS), and polyendocrine autoimniune syndrome (PAS). IDDM is one of the most common autoimmune diseases with several million people affected GAD-65 is expressed at high levels in pancreatic beta cells and cellular autoimmune responses directed against this protein are implicated in the destruction of these cells leading to clinical * -disease onset of diabetes. Suppression of GAD65 expression in the nonobese diabetic (NOD) :" mouse model prevents IDDM, further implicating this protein in the disease. * I....
* By the time symptomatic diabetes has developed, over 90% of pancreatic beta cells have already been destroyed. It is therefore important to develop assays to identify diabetes as early as : possible. Serum antibodies to GAD6S are present in the majority of newly diagnosed diabetics (70%) and can be a useful pre-clinjcal marker for diabetes due to the presence of these antibodies several years prior to disease onset. Autoantibodies are not directly accountable for pancreatic beta cell injuiy, however, they have been implicated in modulating presentation of antigens by professional APCs. GAD65-reactjve antibodies can increase the efficiency of antigen capture by APCs and lower the amount of antigen required to have stimulatory effects on CD4+ T cells through enhanced presentation of certain antigens by MHC Class II molecules on the APC surface.
Serum antibodies to GAD6S, however, are also present in individuals without IDDM and in patients with other autoimmune diseases such as SMS and PAS. New T cell epitopes implicated in the development of JDDM have the potential to be deployed in therapies, such as in tolerising immunisation, or as preclinical markers that could predict the onset of LDDM better.
A strong association is seen between IDDM and certain MHC class II alleles (DRB 1*0401, DRBI*0404, DRBI*0405 and DQBI*0301), indicating the importance of MHC epitope restriction and diabetes susceptibility.
Despite the clear known link between GAD65 and!DDM the relationship between this protein and diabetes is not fully understood. Finding new MHC binding peptides and I cell epitopes of GAD65 is therefore particularly important in order to develop assays to identify diabetes as early as possible, to understand autoimmune disease progression, and for potential therapeutic intervention.
The Invention
S
We have used an MHC-peptide binding assay as described in our co-pending PCT patent application W02007034 151, which is incorporated herein by reference, to identify MHC- :" ; binding peptides from human glutamate decarboxylase, 65 kDa isoform (GAD65), Genbank :.: protein database accession number: AAA58491.
SEQ ID NO: 1: S 1 MASPGSGFWS FGSEDGSGDS ENPGTARAWC QVAQKFTGGI GNKLCALLYG DAEKPAESGG 61 SQPPRAAARK AACACDQKPC SCSKVDVNyA FLHATDLLPA CDGERPTLAF LQDVMNIILQ 121 YVVKSFDRST KVIDFHYPNE LLQEYNWELA DQPQNLEEIL MHCQTTLKYA IKTGHPRYFN 181 QLSTGIJDMVG JJAADWrJTSTA NTNMFTYEIA PVFVLLEYVT LKKMREIIGW PGGSGDGIFS 241 PGGAISNMYA MMIARF1(Mpp EVKEKGMAAL PRLIAFTSEH SHFSLKKGpj ALGIGTDSVI 301 LIKCDERG1 IPSDLERRIL EAKQXGFVPF LVSATAGTTV YGAFDPLLAV ADICKKyKIc.J 361 MHVDAAWGGG LLMSRKHKWK LSGVERANSV TWNPHKMMGV PLQCSALLVR EEGLMQNCNQ 421 MHASYLFQQD KHYDLSYDTG DKALQCGRHV DVFKLWIJMWR AKGTTGFEAH VDKCLELAEy 481 LYNIIKNREG YEMVFDGKPQ HTNVCFWYIP PSLRTLEDNE ERMSRLSKVA PVIKARMMEY 541 GTTMVsYQPL GDKVNFFRMV ISNPAATHQD IDFLIEEIER LGQDL To perform our binding assay we synthesized a library of overlapping I 5-mer peptides, offset from one another by one amino acid for two regions of GAD65 (SEQ ID NO: 1): amino acid region 253-306, SEQ ID NO: 2: I IARFKMFPEV KEKGMAALPR LIAFTSEHSH FSLE(KGAJAL GXGTDSVILI FCCDE and amino acid region 361-414, SEQ IDNO:3: 1 MNVDAAWGGG LLMSRKHKWK LSGVERANSV TfrNPHKMMGV PLQCSALLVR EEGL The following known T cell epitopes were used as controls in our MHC peptide binding assay to assess the binding of peptides to HLA-DRI (DRA*0l01, DRB*OlOl): Table 1: Sequences of control peptides used EQ ID NO: Control Type Peptide Sequence /0 relative to 1EQ ID NO: 4 Positive Control PKYVKQNTLKLAT 687.22 Q ID NO: 5 Pass/Fail Control GPDGRLLRGHDQYAY 100 The following known I cell epitopes were used as controls in our MHC peptide binding assay to assess the binding of peptides to HLA-DR2 (DRAI 0101, DRBI * 1501): S. S. * S.. *S*p
*. Table 2: Sequences of control peptides used L % signal relative1 : EQ ID NO: Control Type Peptide Sequence pass I fail controL :h JEQ ID NO: 6 Positive Control MSIYVYALPLKMLNI 279.06 Q ID NO: 4 Pass/Fail Control PKYVKQNTLKLAT 100 * S *.* I : The following known T cell epitopes were used as controls in our MHC peptide binding assay to assess the binding of peptides to I-ILA-DR3 (DRA 10l01, DRB 1*0301): Table 3: Sequences of control peptides used % signal reJativefl EQ ID NO: Control Type Peptide Sequence.
pass / fail controJ ID NO: 7 Positive Control PSPSMGRDIKVQFQ 395.95 9 ID NO: 4 Pass/Fail Control PKYVKQNTLKLAT 100 The following known T cell epitopes were used as controls in our MI-IC peptide binding assay to assess the binding of peptides to HLA-DR4 (DR.A 1*0101, DRB 1*0401): Table 4: Sequences of control peptides used I. % signal relative to EQ ID NO: Control Type Peptide Sequence pass / fail control EQ ID NO: 4 Positive Control PKYVKQNTLKLAT 386.99 EQIDNO:8 Pass/FajiControl WNRQLYPEWTEAQRLD 100 We have found that surprisingly 28 1 5-mer peptides from the GAD65 protein segments (SEQ ID NO: 2 and SEQ ID NO: 3) bind with an efficiency to HLA-DRI that is similar as or better than the known T cell epitope that has been used as a pass/fail control SEQ ID NO: 5.
These peptides of the invention are listed in Table 5 below and are listed in single letter amino acid nomenclature: TableS: Sequences of peptides of the invention binding to HLA-DR1 T Position of first :. EQ ID NO: GAD65 protein Peptide Sequence % signal relative to * .. (SEQIDNO:2 pass ai Co *.** -SEQIDNO:31 ___________ ___________ EQ ID NO: 9 253 IARFKMFPEVKEKGM 136.88 EQIDNO 10 254 ARFKMFPEVKEKG -13664 : SEQ ID NO: 11 -255 RFKMFPEVKEKGMpJ 140.17 3EQ ID NO: 12 256 FKMFPEVKEKGML 109.16 : EQIDNO: 13 257 KMFPEVKEKGMAALP -100.29 : SEQ IDNO: 14 277 TSEHSHFSLKKGJ 142.07 * JEQ ID NO: 15 278 SEHSHFSLKKGAJL -101.23 -EEQIDNO: 16 279 EHSHFSLKKGAJLG 124.61 EQ ID NO: 17 -280 HSHFSLKKGAALGI 179.82 -EQ ID NO: 18 -281 SHFSLKKGAAALGIG -127.01 SEQ ID NO: 19 -282 HFSLKKGAAALGI -118.64 SEQ ID NO: 20 -283 FSLKKGAAALGIG 101.67 SEQ ID NO: 21 -284 SLKKGAAALGIGTDS 100.23 EQ ID NO: 22 -285 LKKGAAALGIGTDSV 134.83 SEQ ID NO: 23 -373 MSRKHKWKLSGVER -140.54 QIDNO:24 374 SRKHKWKLSGVEPAj 162.71 SEQ ID NO: 25 -375 RKHKWKLSGVERANS 108.08 SEQ ID NO: 26 376 KHKWKLSGVEPANSV 181.42 SEQ ID NO: 27 377 HKt'JKLSGVERANSVT 188.16 EQ ID NO: 28 378 KWKLSGVERANSVTW 231.51 EQ ID NO: 29 379 JWMLSGVERANSVTWN 140.86 EQ ID NO: 30 I 388 NSVTWNPHKMMGVPL 158.63 EQ ID NO: 31 389 -SVTWNPHKMMGVPLQ 306.72 SEQ ID NO: 32 390 VTWNPHKMMGVPLQC 242.12 SEQ ID NO: 33 391 TWNPHKMMGVPLQCS 225.53 SEQ ID NO: 34 -392 WNPHKMMGVPLQCSA 188.41 SEQ ID NO: 35 -393 NPHIQ4MGVPLQCSAL 182.92 EQ ID NO: 36 394 -PHKMMGVPLQCSALL -185.16 Further we have found that surprisingly 37 1 5-mer peptides from the GAD65 protein segments (SEQ ID NO: 2 and SEQ ID NO: 3) bind with an efficiency to HLA-DR2 that is similar as or better than the known T cell epitope that has been used as a pass/fail control SEQ ID NO: 4.
These peptides of the invention are listed in Table 6 below and are listed in single letter amino acid nomenclature: Table 6: Sequences of peptides of the invention binding to HLA-DR2 Position of first * : * EQ ID NO: GAD6Sprotn Peptide Sequence SEQIDNO:3) ______________ SEQ ID NO: 9 253 IARFKMFPEVKEKGM -1666.33 :.: SEQ ID NO: 10 -254 ARFKMFPEVKEKGMA -1631.40 : SEQ ID NO: 11 255 -RFKMFPEVKEKG!,4J 1610.71 EQ ID NO: 12 256 FKMFPEVKEKGMAL 971.05 EQIDNO:37 262 -VKEKGMAALPRLIAF 446.14 EQ ID NO: 38 263 KEKGMAALPRLIAFT 491.67 SEQ ID NO: 39 -264 EKGMAALPRLIAFTS 350.85 SEQ ID NO: 40 265 KGMAALPRLIAFTSE 428.54 SEQ ID NO: 41 266 GMAALPRLIAFTSEH 474.44 SEQ ID NO: 42 267 MAALPRLIAFTSEHS 200.51 SEQ IDNO:43 268 AALPRLIAFTSEHSH -316.59 IEQIDNO:44 269 ALPRLIAFTSEHSHF -254.72 SEQ IDNO:45 -270 -LPRLIAFTSEHSHFS 104.09 3EQ ID NO: 46 -271 PRLIAFTSEHSHF 121.48 SEQIDNO:47 -272 RLIAFTSEHSHFSL 138.55 JEQ ID NO: 48 -273 -LIAFTSEHSHFSLKK 135.66 EQ ID NO: 16 279 EHSHFSLKKGLG 242.92 EQ ID NO: 17 280 IHSHFSL}CIcGAAALGI I 331.96 SEQ ID NO: 18 281 SHFSLKKGAAALGIG 372.64 EQ ID NO: 19 282 HE'SLKKGAAALGIGT 285.75 EQ ID NO: 20 283 FSLKKGAPJALGIGTD 178.74 EQ ID NO: 21 284 SLKKGAAALGIGTDS 124.77 EQIDNO:49 289 AAALGIGTDSVILIK 129.99 SEQ ID NO: 50 361 MHVDAAWGGGLLMSR 244.13 SEQ ID NO: 51 -362 HVDAAWGGGLLMSRK 245.06 EQ ID NO: 52 363 VDAAWGGGLLMSRI<H 332. 11 SEQ ID NO: 53 364 DAAWGGGLLMSRKHK 171.85 SEQ ID NO: 54 372 LMSRKHKWKLSGVER 370.45 SEQ ID NO: 29 379 WKLSGVERANSVTWN 122.67 EQ ID NO: 55 380 KLSGVERANSVTWNP 129.60 SEQ ID NO: 56 385 ERANSVTWNPHKG 220.39 EQ ID NO: 57 386 RANSVTWNPHKMMGV 186.80 EQIDNo:58 -387 ANSVTWNPHKMMGVP 145.19 EQ ID NO: 30 -388 NSVTWNPHKMMGVPL 177.89 EQ ID NO: 31 389 SVTWNPHKMMGVPLQ 172.62 : *.* tSEQ ID NO: 32 390 VTWNPEKMMGVPLQC 121.13 *... r-__________________ ***** EQ ID NO: 59 I 395 IHKMMGVPLQCSALLV 101.91 * * :: Further we have found that surprisingly 14 15-mer peptides from the GAD65 protein segments *.. (SEQ ID NO: 2 and SEQ ID NO: 3) bind with an efficiency to HLA-DR3 that is similar as or b** better than the known I cell epitope that has been used as a pass/fail control SEQ ID NO: 4.
These peptides of the invention are listed in Table 7 below and are listed in single letter amino acid nomenclature: Table 7: Sequences of peptides of the invention binding to HLA-DR3 Position of first I amino acid in.
EQ ID NO: GAD65 protein Peptide Sequence % signal relative to (SEQ ID NO: 2 PS ai control SEQIDNO:3J ___________ ___________ SEQ ID NO: 9 253 IARFKMFE'EVKEKGM 104.77 EQ ID NO: 10 254 ARFKMFPEVKEKG 271.34 EQ ID NO: 11 255 RFKMFPEVKEKGA 289.49 SEQ ID NO: 12 -256 -FKMFPEVKEKGMJ\JL 124.47 !SEQ ID NO: 13 257 KMFPEVKEKGMAALP 106.87 SEQ ID NO: 48 273 -LIAFTSEHSHFSLKK 104.77 SEQ ID NO: 60 -274 IAFTSEHSHFSLKKG 271.34 SEQ ID NO: 61 275 AFTSEHSHFSLKKGA 289.49 EQ ID NO: 62 276 -FTSEHSHFSLKKG -124.47 EQ ID NO: 14 -277 TSEHSHFSLKKGA -106.87 EQ ID NO: 53 364 DAAWGGGLLMSRKHK 176.65 SEQIDNO:63 -371 LLMSRKHKWKLSGVE -210.96 SEQ ID NO: 54 372 LMSRKHKWKLSGVER -100.47 -JEQ ID NO: 28 378 KWKLSGVERANSVTW 109.84 Further we have found that surprisingly 33 1 5-mer peptides from the GAD65 protein segments (SEQ ID NO: 2 and SEQ ID NO: 3) bind with an efficiency to HLA-DR4 that is similar as or better than the known T cell epitope that has been used as a pass/fail control SEQ ID NO: 8.
These peptides of the invention are listed in Table 8 below and are listed in single letter amino acid nomenclature: Table 8: Sequences of peptides binding to HLA-DR4 * * .* .1s * . I Position of first amino acid L in GAD65 protein. % signal relative to pEQ JD NO: (SEQ ID NO: 2 Peptide Sequence pass / fail control L SEQIDNO.3) ___________ SEQ ID NO: 9 -253 IARFKMFPEVKEKj 205.61 SEQ ID NO: 10 254 ARFKMFPEVKEKGMJ -119.89 EQ ID NO: 11 255 -RFKMFPEVKEKG 177.01 EQ ID NO: 13 257 KMFPEVKEJçGMLp 109.62 EQ ID NO: 40 -265 -KGMAALPRLIAFTSE 126.19 SEQIDNO:41 266 GMAALPRLIAFTSEH 211.01 SEQIDNO:42 267 MAALPRLIAFTSEHS -143.52 EQ ID NO: 43 268 AALPRLIAFTSEHSH 200.89 EQIDNO:44 -269 -ALPRLIAFTSEHSHF -242.69 SEQ ID NO: 45 -270 LPRLIAFTSEHSHFS 228.87 -SEQ ID NO: 46 271 PRLIAFTSEHSHFSL 220.88 SEQ IDNO:47 272 RLIAFTSEHSHFSLK 120.61 JEQIDNO:48 273 -LIAFTSEHSHFSLKK 106.13 -JEQ1DNO:60 274 IAFTSEHSHFSLKKG -121.37 EQ ID NO: 17 280 HSHFSLKKGAAAL 120.48 EQIDNO:18 281 ISHFSLKKGAAALGIGjj 1SEQ ID NO: 19 282 HFSLKKGAALGIGT 163.43 SEQ ID NO: 20 283 FSLKKGIALGIGTD 108.72 -EQ ID NO: 21 284 SLKKGAMLGIGTDS 125.25 3EQ ID NO: 22 285 -LFKGAAALGIGTDSV 163.32 SEQ ID NO: 23 373 MSRKHKWKLSGVEpJ 109.41 EQ ID NO: 24 374 SRKHKWKLSGVEN 191.41 EQ ID NO: 25 -375 RKHKWKLSGVER,ANS 173.70 SEQ ID NO: 26 376 KHKWKLSGVERANSV -234.22 SEQ ID NO: 27 -377 HKWKLSGVERANSVT 313.85 SEQ ID NO: 28 378 KWKLSGVERANSVTW 423.26 JEQ ID NO: 29 379 WKLSGVERANSVTWN 286.46 EQIDNO:32 390 VTWNPHKMMGVPLQC 213.01 SEQ ID NO: 33 391 TWNP}IKMMGVPLQCS 273.46 JEQJDNO:34 392 WNPHKMMGVPLQCSA 247.29 JEQ ID NO: 35 393 NPHKMMGVPLQCSAL 278.04 3EQ ID NO: 36 394 PHKMMGVPLQCSALL 256.98 SEQIDNO:59 395 HKMMGVPLQCSALLV 214.79 * .IS In its first aspect the invention therefore concerns one or more of the isolated peptides selected * from the group consisting of: *S *.* * S
S SeS*S
* IARFRMFPEVKEKGM (SEQ ID NO: 9), ARFKMFpEVKEKGnJ (SEQ ID NO: 10), RFKMFPEVKEKGMJ (SEQ ID NO: 11), * FKMFPEVKEKGML (SEQ ID NO: 12), KMFFEVKEKGMJLP (SEQ ID NO: 13), TSEHSHFSLKKG (SEQIDN0: 14), SEHSHFSLKKG1JJL (SEQ ID NO: 15), EHSHFSLKKGLG (SEQ ID NO: 16), HSRFSLKKG1JLGI (SEQIDNO: 17), SHFSLKKGAAJLGIG (SEQ ID NO: 18), HFSLKKGALGIGT (SEQ ID NO: 19), FSLKKGAAALGIGTD (SEQ ID NO: 20), SLKKGkAALGIGTDS (SEQ ID NO: 21), LKKGAAALGIGTDSV (SEQ ID NO: 22), MS RKHKWE<LSG VERA (SEQ ID NO: 23), SRKFIKWKLSGVERAN (SEQ ID NO: 24), RKHKWKLSGVERANS (SEQ ID NO: 25), KHKWKLSGVERANSV (SEQ ID NO: 26), HKWKLSGVERANSVT (SEQ ID NO: 27), KWKLSGVERANSVTW (SEQ ID NO: 28), 1KLSGVEBANSVTWN (SEQ ID NO: 29), NSVTWNPHKMMGVPL (SEQ ID NO: 30), SVTWNPHKMMGVPLQ (SEQ ID NO: 31), VTWNPHKMMGVPLQC (SEQ ID NO: 32), TWNPHKMMGVPLQCS (SEQ ID NO: 33), WNPHKMMGVPLQCSA (SEQ ID NO: 34), NPHKMMGVPLQCSAL (SEQ ID NO: 35), ?HKMMGVPLQCSALL (SEQ ID NO: 36), VKEKGMMLPRLIAF (SEQ ID NO: 37), KEKGMMLPRLIAFT (SEQ ID NO: 38), EKGMAALPRLIAFTS (SEQ ID NO: 39), KGMAALPRLIAFTSE (SEQ ID NO: 40), GMAALPRLIAFTSEH (SEQ ID NO: 41), MALPRLIAFTSEHS (SEQ ID NO: 42), AALPRLIAFTSEHSH (SEQ ID NO: 43), ALFRLIAFTSEHSHF (SEQ ID NO: 44), LPRLIAFTSEISHFS (SEQ1DNO:45), PRLIAFTSEHSHFSL (SEQ ID NO: 46), RLIAFTSEHSHFSLK (SEQIDNO: 47), LIAFTSEHSHFSLKK (SEQ ID NO: 48), AAALGIGTDSVILIK (SEQIDNO:49), MHVDAAWGGGLLMSR (SEQ ID NO: 50), HVDAAWGGGLLMSRK (SEQIDNO:51) :.: . VDAAWGGGLLMSRKH (SEQ ID NO: 52), DAAWGGGLLMSRKHK (SEQ ID NO: 53), LMSRKHKWKLSGVER (SEQ ID NO: 54), KLSGVERNSVT?JNP (SEQ ID NO: 55), ERANSVTWNPHMG (SEQ ID NO: 56), RANSVTWNPHKMMGV (SEQ ID NO: 57), ANSVTWNPHKMMGVP (SEQ ID NO: 58), HKMMGVPLQCSALLV (SEQ ID NO: 59), IAFTSEHSHFSLKKG (SEQ ID NO: 60), AFTSEHSHFSLKKGA (SEQ ID NO: 61), FTSEHSHFSLKKGAJ (SEQ ID NO: 62), LLMSRKI-1KWKLSGVE (SEQ ID NO: 63), In addition the present invention also includes peptides with partial regions of the peptides of the invention SEQ ID NO: 9 to SEQ ID NO: 63 which have a length of at least 6 amino acids, preferably at least 8 amino acids, particularly preferably of at least II amino acids.
The minimum length of a peptide according to the invention is determined by its capability to recognize a MHC molecule, to bind specifically to it and to react with the corresponding T cell receptor.
In addition to peptides having the amino acid sequences SEQ ID NO: 9 to SEQ ID NO: 63 or partial regions thereof, the invention also concerns peptides with amino acid sequences which exhibit an essentially equivalent specificity or/and affinity of binding to MHC molecules as the aforementioned sequences and which are preferably derived by substitution, deletion or insertion of individual amino acid residues or short sections of amino acid residues from these sequences The peptides derived from peptides having the sequences SEQ ID NO: 9 to SEQ ID NO: 63 preferably have a sequence homology of at least 30%, particularly preferably of at least 50% and most preferably of at least 60% to the parent peptides or partial sequences thereof.
:.,. Examples of variants of the specifically stated peptides are the corresponding homologous peptide sections from human GAD 67, Genbank protein database accession number: AAA62368: U..... * .
:: SEQJDN0:64: 1 MASSTPSSSA TSSNAGADPN TTNLRPTTYD TWCGVAHGCT RKLGLKICGF LQRTNSLEEK I.. * 61 SRLVSAFRER QSSKNLLSCE NSDRDARE'RR TETDFSNLFA RDLLPAKNGE EQTVQFLLEv * . 121 VDILLNYVRK TFDRSTKVLD FHHPHQLLEG MEGFNLELSD HPESLEQILV DCRDTLKyGV 181 RTGHPRFFNQ LSTGLDIIGL AGEWLTSTAJ. TNMFTYEIAP VFVLMEQITL KKMREIVGWS 241 SKDGDGIFSP GGAISNMySI P4AARYKYFPE VKTKGMAAVP KLVLFTSEQS HYSIKKAGAA 301 LGFGTDNVIL IKCNERGF<II PADFEAKILE AKQKGYVPFY VNATAGTTVY GAFDPIQEIA 361 DICEKYNLWL HVDAAWGGGL LMSRKHRHKL NGIERANSVT WNPHKMMGVL LQCSAILVKE 421 KGILQGCNQM CAGYLFQPDK QYDVSYDTGD KAIQCGRHVD IFKFWwjçj KGTVGFENQI 481 NKCLELAEyL YAKIKNREEF EMVFNGEPEH TNVCFWYIPQ SLRGVPDSPQ RREKLHKVAP 541 KIKALMMESG TTMVGYQPQG DKANFFRMVI SNPAATQSDI DFLIEEIERL GQDL The term "essentially equivalent specificity or/and affinity of binding to MHC molecules" also includes an improved binding specificity or/and affinity compared to the sequences SEQ ID NO: 9 to SEQ ID NO: 63 which is found particularly in the case of truncated peptides which have a length of6to 14 amino acids.
Moreover the present invention also includes peptide derivatives. This term includes peptides in which one or several amino acids have been derivatized by a chemical reaction. Examples of peptide derivatives according to the invention are in particular those molecules in which the backbone or/and reactive amino acid side groups e.g free amino groups, free carboxyl groups or/and free hydroxyl groups have been derivatized. Specific examples of derivatives of amino groups are sulfonic acid or carboxylic acid axnides, thiourethane derivatives and ammonium salts e.g. hydrochiorides. Examples of carboxyl group derivatives are salts, esters and amides.
Examples for hydroxyl group derivatives are 0-acyl or 0-alkyl derivatives. Furthermore the term peptide derivative according to the present invention also includes those peptides in which one or several amino acids are replaced by naturally occurring or non-naturally occurring amino acid homologues of the 20 "standard" amino acids. Examples of such homologues are 4- * hydroxyproljne, S-hydroxylysjne, 3-methylhistidine, homoserine, ornithine, beta-alanine and 4-S...
*..*. aminobutyric acid.
* : Those peptides are particularly preferred which have an essentially equivalent specificity or/and S....
* affinity of binding to MHC molecules as peptides having the sequences SEQ ID NO: 9 to SEQ : ID NO: 63 but which, in contrast to these peptides, do not cause an activation of T cells but : rather the production of an anergic state in the T cells.
The present invention also covers polypeptides in which the MHC-binding peptide section is a component of a larger polypeptide such as a non-naturally occurring component.
A further subject matter of the present invention is a peptide or peptide derivative which carries a substance that generates a signal or a marker group e.g. a fluorescent marker group (e.g. rhodamjne, phycoerythrjn), digoxin, biotin, a radioactive group or a toxin group (e.g. ricin, cholera toxin etc.). Coupling of the peptide according to the invention to marker groups enables the peptide to be used as a diagnostic agent for in vivo or in vitro (e.g. imaging) applications or as a therapeutic agent. Furthermore the peptide according to the invention can also for example be present in a cyclised form or in an oligomeric form in which the sequences that are important for binding to the MHC molecule are separated from one another by spacer regions.
The binding assay was carried out according the methods described in the applicant's co-pending PCT application W02007034 151, measuring the extent of the peptides at a known concentration to assemble with the HLA alpha and beta chains. The extent of assembly was measured quantitatively for each peptide using a solid phase binding assay (ELISA) with a conformational antibody that only detects fully assembled MI-IC complexes. The ELISA absorption readings were taken for all peptides to be tested and the respective control peptides, which are known T cell epitopes, i.e. they are known to elicit I cell immune responses in humans. SEQ ID NO: 4 is a peptide binding with high efficiency to HLA-DRI, SEQ ID NO: 6 is a peptide binding with high efficiency to HLA-DR2, SEQ ID NO: 7 is a peptide binding with high efficiency to HLA-DR3, SEQ ID NO: 4 is a peptide binding with high efficiency to HLA-DR4, SEQ ID NO: 5 is a peptide binding with intermediate efficiency to HLA-DRI, SEQ ID NO: 4 is a peptide binding with intermediate efficiency to HLA-DR2, SEQ ID NO: 4 is a peptide binding with intermediate efficiency to HLA-DRJ, and SEQ ID NO: 8 is a peptide binding with intermediate efficiency to HLA-DR4. Peptides binding with high efficiency were used as positive controls, peptides * *** *... binding with intermediate efficiency were used as pass / fail controls.
The peptides of the invention were selected based on their ability to bind with similar or better ***..
* : efficiency to the MHC alleles as stated in the tables above than the control peptide is able to * bind to the MHC allele in question. The tables above show the percent signal relative to the pass : / fail control, where the pass / fail control is set to 100 % in each case. * *I*.. * S
The peptides of the invention are useful in a number of ways: Injection of the peptides of the invention could be used to diagnose type 1 diabetes and could be used to inactivate, tolerise or delete T cells to GAD to prevent autoimmune disorders such as IDDM such as described in EP0665289 and also SMS and PAS. They may be used as isolated peptides, e.g. in the form of synthetic peptides in prophylactic or therapeutic vaccines to treat or vaccinate against type I diabetes. They are useful in this way when bound to HLA-DRI, HLA-DR2, HLA-DR3 and HLA-DR4 complexes, as applicable, and on their own.
Alternatively they may be segments in larger protein based vaccine constructs. They may be administered in the form of one or more of the isolated peptides or other methods such as described in EP0665289.
Obtaining the isolated peptides of the invention is well known in the art, e.g. by solid phase peptide synthesis, which is e.g. offered as a routine service to researchers by many companies today, such as Sigma.Genosys, Woodlands, Texas, USA.
The invention further concerns DNA or RNA sequences encoding one or more of the peptides of the invention. These DNA or RNA sequences may be used in DNA or RNA constructs for heterologous expression in a prokaryotic host cell, such as E.coli., or a eukaryotic host cell, such as an insect cell or mammaliaji cell. The constructs may be viral vector based constructs such as vaccinia or pox vector constructs, which may be used as vaccines or other immunotherapies to treat or vaccinate against IDDM, SMS or PAS. The actual sequences of such coding DNA or RNA sequences may be based on the original coding sequences of GA065 studied here and may be adapted based on the host cell in which these sequences are to be expressed. Back-translation from protein to DNA and RNA coding sequences is documented extensively in the s.. literature. Such coding DNA or RNA sequences may offer additional design options, such as S...
* selecting codon-optimised sequences for the expression in the host of interest or other codon * choices, e.g. suited to the restriction sites in the expression system used. S....
* In a third aspect thereof the invention therefore concerns a DNA or RNA sequence encoding one or more of the peptides of the invention and a vector comprising such a DNA or RNA sequence.
S S.... * S
* The construction of vaccines based on the peptides of the invention or RNA, DNA coding therefore is described in some detail in US6,733,973.
In another alternative embodiment the peptides may be incorporated in an oligomeric MHC-peptide complex, such as in fluorescent labelled MHC multimers such as MHC tetramers which may be formed by coupling monomeric MHC-peptide complexes incorporating the peptides of the invention to a multivalent entity at a specific attachment site, as described in US 5,635,363 which is incorporated in herein in its by reference, which are useful for labelling, detecting and isolating antigen-specific T cells. Such MI-IC multimers are consequently useful for immune monitoring of single-antigen specific T cells that are reactive to the peptides of the invention in patients. Immune monitoring by MHC multimers is useful for both understanding autoimmune disease progression, defining preclinical markers of disease and determining appropriate therapeutic treatment.
In a fourth aspect therefore the invention concerns an MHC-peptide complex comprising one or more of the peptides of the invention and in a fifth aspect the invention concerns an oligomeric MHC-peptide complex comprising one or more of the peptides of the invention.
In yet another embodiment the binding peptides of the invention can be used to raise polyclonal or monoclonal antibodies against the peptides of the invention, which may be useful for investigating preclinical markers of autoimnune disease or presentation of the specific peptide on a cell. The detection of such specific peptide presentation can be very important when tracking the distribution of MHC complexes presenting a particular peptide in vivo, e.g. in the context of a vaccination protocol where the vaccination occurs through a particular administration route and/or location and is designed to effect presentation of a specific peptide.
In this case it is often required that the MHC-peptide presentation is then translated to a specific site of action in the vaccinated individual's tissues. Monitoring the effectiveness of such * translocation of antigen presentation is possible by using binding of antibodies that are specific * : to the MHC-peptide combination of interest, whereby the antibodies themselves are labelled to **...
* enable visualisation, e.g. through a fluorescent or radioactive label, which may be traceable in **. vivo. *. * * **.. * *
In a sixth aspect the invention therefore comprises an antibody specific to one or more of the peptides of the invention or specific to an MHC-peptide complex comprising one or more of the peptides of the invention.
In a further embodiment of the invention CD4+ T cell lines or clones that are reactive to the peptides of the invention may be raised. Raising such T cell lines may be effected as described in US5,585,461, US6,733,973, or EP0665289. Such antigen-specific I cell lines can also be generated by incorporating the peptides of the invention in MI-IC tetramers, such as described in USS,635,363. Such MI-IC tetramers can be coupled to paramagnetic beads, such as Dynabeads�, which are useful for isolating peptide-specific T cells. Cells isolated in this manner can be expanded in culture and as described in EP0662289. Such cell lines are further useful as positive controls for monitoring immune responses against the peptides of interest in patients. Alternatively they may be useful in a therapeutic application where it is desirable to administer T cells specific to one or more peptides of the invention to a patient either autologously or allogeneicaliy, e.g. in order to suppress immune responses to the antigen in question, such as described in EP0665289.
In a seventh aspect the invention therefore concerns a CD4+ T cell line or clone specific to one or more of the peptides of the invention.
Alternatively the peptides of the invention may be administered in the form of a cellular vaccine through the administration of autologous or allogeneic antigen presenting cells or dendritic cells that have been treated in vitro so as to present one or more of the peptides of the invention on their surface. The preparation of such antigen presenting cells is described in EP0665289.
In an eighth aspect the invention therefore concerns an isolated dendritic cell presenting one or more of the peptides of the invention. 0***
* In a ninth aspect the invention concerns pharmaceutical composition comprising one or more * : from the group consisting of one or more of the peptides of the invention, a DNA or RNA sequence of the invention, a vector of the invention, an MHC-peptide complex of the invention, an oligomeric MHC-peptjcfe complex the invention, an antibody of the invention, a CD4+ T cell *... line of the invention, a dendritic cell of the invention.
The pharmaceutical composition of the invention can be used for prophylactic treatment of autojmmtme diseases such as JDDM, SMS, and PAS.
Pharmaceutical compositions comprising the peptides are useful for, e.g. parenteral administration, i.e. subcutaneously, intramuscularly or intravenously, but not limited thereto.
The compositions for parenteral administration may for example comprise the active ingredients dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g. buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter and may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active ingredients in these formulations can vary widely, i.e. in the case of soluble matter from less than about I pg/mI, usually at least about 0.1 mg/mI to as much as 10-mg/mI and will be selected primarily based on fluid volumes, viscosities, etc. in accordance with the particular mode of administration selected.
In a tenth aspect the invention concerns a diagnostic composition comprising a label and one or more from the group consisting of one or more of the peptides of the invention, a DNA or RNA sequence of the invention, a vector of the invention, an MHC-peptide complex of the invention, an oligomeric MHC-peptide complex the invention, an antibody of the invention, a CD4+ I cell line of the invention, a dendritic cell of the invention.
The diagnostic compositions of the invention can be used to detect, characterise and monitor ,.. GAD6S specific autoimmune disease progression in individuals, detect cells that are presenting S...
or are reactive to the peptides of the invention in a cell population and generally assess the immune competence of individuals relative to autoimnrnne disease onset and appropriate S....
: therapeutic intervention. For this purpose such reagents may be provided with a label, such as a I....
radioactive or fluorescent label as described in US6,733,973 and US5,635,363. S. * S I. S
It will be understood that the foregoing examples are merely given to illustrate the invention and the invention is not limited thereto. Other uses of the peptide sequences described herein will be evident to the skilled practitioner.

Claims (10)

  1. Claims: 1. An isolated peptide selected from the group Consisting of: IARFKMFPEVKEKGM (SEQ ID NO: 9), ARFKMFPEVKEKG (SEQ ID NO: 10), RFKMFPEVKEKGJ (SEQ ID NO: 11), FRMFPEVKEKGL (SEQ ID NO: 12), KMFPEVKEKGMAALP (SEQ ID NO: 13), TSEHSHFSLKKG (SEQ ID NO: 14), SEHSHFSLKKGL (SEQ ID NO: 15), EHSHFSLKKGAJALG (SEQ ID NO: 16), HSHFSLKKGAALGI (SEQ ID NO: 17), SHFSLRKGAAALGIG (SEQ ID NO: 18), HFSLKKGAA3LGIGT (SEQ ID NO: 19), FSLKKGP,.A.ALGIGTD (SEQ ID NO: 20), SLKKGAAALGIGTDS (SEQ ID NO: 21), S..
    LKKGAAALGIGTDSV (SEQ ID NO: 22), MSRKHKWKLSGVERJ (SEQ ID NO: 23), SRKHKWKLSGVERpN (SEQ ID NO: 24), * RKHKWKLSGVEPNS (SEQ ID NO: 25), KHKWKLSGVEINSV (SEQ ID NO: 26), HKWKLSGVERANSVT (SEQ ID NO: 27), .. KWKLSGVERANSVTW (SEQ ID NO: 28), WKLSGVERAN5Vq (SEQ ID NO: 29), NSVTWNPHKMMGVPL (SEQ ID NO: 30), SVTWNPHKMMGVPLQ (SEQ ID NO: 31), VTWNPHIcMMGVPLQC (SEQ ID NO: 32), TWNPHKMMGVPLQCS (SEQ ID NO: 33), WNPHKM11GVPLQCSA (SEQ ID NO: 34), NPHKMMGVPLQCSAL (SEQ ID NO: 35), PHKMMGVPLQCSALL (SEQ ID NO: 36), VKEKGNALPRLIAF (SEQ ID NO: 37), KEKGMAALPRLIAFT (SEQ ID NO: 38), EKGMAALPRLIAFTS (SEQ ID NO: 39), KGMAALPRLIAFTSE (SEQ ID NO: 40), GMA.ALPRLIAFTSEM (SEQ ID NO: 41), MAALPRLIAFTSEHS (SEQ ID NO: 42), AALPRLIAFTSEHSH (SEQ ID NO: 43), ALPRLIAFTSEHSHF (SEQ ID NO: 44), LPRLIAE'TSEHSHFS (SEQ ID NO: 45), PRLIAFTSEHSHFSL (SEQ ID NO: 46), RLIAFTSEHSHFSLK (SEQ ID NO: 47), LIAFTSEHSHFSLKK (SEQ ID NO: 48), AMLGIGTDSVILIK (SEQ ID NO: 49), MHVDAAWGGGLLMSR (SEQ ID NO: 50), HVDAAWGGGLLMSRK (SEQ ID NO: 51), VDAAWGGGLLMSRKH (SEQ ID NO: 52), DAAWGGGLLMSR1cHK (SEQ ID NO: 53), LMSRKHKWFLSGVER (SEQ ID NO: 54), KLSGVERANSVTWNP (SEQ ID NO: 55), ERANSVTWNPHCJMG (SEQ ID NO: 56), RANSVTWNPHKMMGV (SEQ ID NO: 57), ANSVTWNPHKGVP (SEQ ID NO: 58), HKMMGVPLQCSALLV (SEQ ID NO: 59), IAFTSEHSHFSLKKG (SEQ ID NO: 60), AFTSEHSHFSLKKGA (SEQ ID NO: 61), FTSEHSHFSLKKGIJ (SEQ ID NO: 62), LLMSRKHKWKLSGVE (SEQ IDNO:63), S.. I...
  2. 2. A DNA or RNA sequence encoding one or more of the peptides of claim I. I**SS * . :
  3. 3. A vector comprising a DNA or RNA sequence of claim 2.
  4. 4. An MHC-peptide complex comprising one or more of the peptides of claim 1.
  5. 5. An oligomeric MHC-peptide complex comprising one or more of the peptides of claim I.
  6. 6. An antibody specific to one or more of the peptides of claim I or an MHC-peptide complex comprising one or more of the peptides of claim 1.
  7. 7. A CD4+ T cell line or clone specific to one or more of the peptides of claim 1.
  8. 8. A dendritic cell primed with one or more of the peptide of claims 1.
  9. 9. A pharmaceutical composition comprising one or more from the group Consisting of one or more of the peptides of claims 1, a DNA or RNA sequence of claim 2, a vector of claim 3, an MHC-peptide complex of claim 4, an oligomeric MFIC-peptide complex of claim 5, an antibody of claim 6, a T cell line or clone of claim 7, a dendritic cell of claim 8.
  10. 10. A diagnostic composition comprising a label and one or more from the group consisting of one or more of the peptides of claims I, a DNA or RNA sequence of claim 2, a vector of claim 3, an MHC-peptide complex of claim 4, an oligomeric MHC-peptide complex of claim 5, an antibody of claim 6, a CD4+ I cell line or clone of claim 7, a dendritic cell of claim 8.
    S S.. I,..
    S * . S
    I * I
    I I....
    I I S. * S I. S
    S I....
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US20020114816A1 (en) * 1995-07-14 2002-08-22 Josef Endl Autoreactive peptides from human glutamic acid-decarboxylase (gad)
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Publication number Priority date Publication date Assignee Title
WO2024023521A1 (en) 2022-07-27 2024-02-01 The University Of Birmingham Tolerogenic peptides

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