CA2193860A1 - T cell epitopes of rye grass pollen allergen - Google Patents

Abstract

The present invention relates to the immunodominant T cell epitope of rye grass, its minimal sequence, its role in hayfever and its use in the manufacture of a medicament for the treatment or prophylaxis of hayfever.
According to the present invention there is provided a polypeptide of nine amino acid residues including the six amino acid residues VXRIDT, and analogues and mimetics thereof. The polypeptide can be administered to provide a method for treatment or prophylaxis of allergies particularly of rye grass allergy or hayfever.

Description

~ ~W096/00238 2 1 938 60 ~ 493 T Cell Ep1topes Of Rye Grass Pollen Allersen The preser~t inVeTltion relaees to the ; ~ n7n~ T cell epitope of rye graC5, its minimal seguence, its role iTl hayfever ard its use i~ the ~-nllf7~tllre of a ';1 t ror the treatment or prophylaxis of hayfever.

Amiao acid residue 8ymbols used hereafter are those ;~ont;f;o~
by the I~PAC-I~8 Ri ~ho~i r~ At~-re C ' cci ~n and they irclude all D or L amino acids or analogues and derivatives thereof. The symbol X L~L~E_~Ls an nn; ~onti c; o~ amino acid or analogue ther_o_ One of the major causes of hayfever in Europe is the pollen of rye grass, ~oliu~ pere~ue, which is a common allergen. Several proteins from the pollen of rye grass have been isolated and the complete amino acid se~ s for three of these proteiTls ~Lol pI, 501 pII and -ol pIII) have beeTl pllhli chor~

The major Cracti- o ~olium pereu~e ~. pereT~ne~ has been found to be 501 pI, a gl~ycoprotein of 240 amiro acid residues, and most hayfever s-~fcrers in Europe sersitive to grass pollen (lbOut 80%~ po5sess specific IgE Anriho~iPc to this proteir..

The complete se.:o ce of Ilol pI ha.s beer. pnhl; cho~ ard it has W096/00238 2 1 9 3 8 6 0 PCT/GB9~01493 been suggested that there is a polypeptide sequence of 14 nn-~rrl-t;ve amino acid residue6 which includes the immunogenic rinAnt of Lol p~. ~Bungy et al. Clin Exp Immunol 1993, ~~, 111-116). When te5ted ~n vivo by ;ntrA~rr_l skin testing such a polypeptide could show typical delayed type hypersensitivity (D~H) reactions after 24-48 hours in atopic rye grass-sensitive patients. Current glliA~l;n~c 6uggest that allergic symptoms ~for example those of hayfever) limited to the nose should be treated by immunotherapy, ~cr~r; Al ly where hayfever symptoms are not rnnrrnllr~ by conventional, ~;rAtinn.
It is also s~gr,~tDd that patierts with pollen asthra should be excluded from such tL~ - because of potential side effects.

Immunotherapy using native allergen to reduce the sensitivity of patients has the disadvantage that all sensitisiug agents have the pot~nt;Al to induce severe allergic r~Art;nnA such as brrnrhnqpA~, anaphylaxis and even death.

Cliuical hyyo5-~itisation for hayfever using an ill~L~ ' Al regime of injection of whole extract of allergens is not curative, although symptoms are ameliorated. However, whole extracts cor,tain an IgE bindir,g site which can result in anaphylactic shock and even death. Although phArr-~nlorJical ~ ~096/00238 21 93860 1 ll~ C~l493 treatment can relieve mild symptoms, there is as yet no ; log;cal "cure" for the severe cases, ~cpP~i~lly those with asthma.

One approach to overcome these problems has been the development of modified allergens for use in vaccines which might first be more effective, and second, much safer.

The availability of a safe and effective peptide vaccine would allow treatment to be given to a very important group of patients.

Studies of the desensitisation effect of an enzyme digest of pollen comprising a T cell epitope suggest that an allergic response to pollen may be reduced in both animal and man (Bungy et al supra). There i8 pot~nt;Al clinical benefit if an im~uno dominant T cell epitope could be used to induce anergy or specific T cell ~ qiveness locally or systemically in patients allergic to a major allergen such as rye or other grasses.

~ayfever sy~nptoms are brought about through specific ly/nphocyte activation leading to the release of vagoactive amine , ~i~tnr~
(e.g. histamine) -om cells into ~Ytr~r~llnl~r space. The role of IgE ~n~;hn~i~c in I A~;ng allergic reactions (for example, those of hayfever) is well known.

The aoility to trigger T cells could ~e used in the production of ~n~ihn~;~c to a B cell epitope by , ~ n~t;nn of this B cell epitope with a T cell epitope. Such ~n~;h~ c could be used to block the IgE'mediated response to allergen (for example blocking of binding of IgE to cells).

IgE comprises two light polypeptide chains and two heavy polypeptide chains linked by ~ic..lphi~ bonds. The heavy chains have one variable domain (V~) and four corstant domains (CN1~
C~2, C~3 and C~4 also known as C~1, CIZ, C~3 and C~4). A region of IgE which ~ c part of the heavy chains is known as the Fc region.

As ~;Rrlos~d by Stanworth et al (WO 90/15778, the content of which is incuL~L~ed herein by reference), inv~cr;g~t;nnR have been carried out in order to define the nature of the "effector"
site within the IgE Fc region which is thought to provide the ; n~ngin~l si~al resulti~g in hista~ine release. S~LUULULe-~ctivity studies have been carried out on model h; c~: ' n~_ releasing polypectides and this has ; n~ that a cluster of basic amino aci~i are ~Rs~n~;~l for the direct triggering of ~ W096l00238 21 93860 r ~ 493 histamine release. Furthermore, the presence of nP;ghho~lr;ng ~ hydrophobic residues and the ~ t;nn of their C-terminal carboxylic acid residues has been found to enhance triggering of histamine release.

Based on the above observations, the Fc region of human IgE, the ~Lu~ULe of which has been ~lllri~t~dt was examined for amino acid residue se~u~.~s which fnlf;llpd such criteria. The amino acid residue sequence spanning residues 496-506 of the CD4 domain~ v~ [SEQ ID:1] seemed the most likely to meet these criteria.

WO 90/15878 ~ r;h~ certain ; ;ve peptides ~ad ~nt;h~ c and their use in allergy treatment by prevention of the triggering of the release of histamine. The Stanworth ~ppl; rAt;nn described an ; , comprising a residue of a histamine-releasing peptide comprisi~g a cationic N-terminal head and a l.~d~ bic C-terminal tail, together with a residue capable of eliciting ~nt;ho~i~c against said peptide whilst ;nh;h;t;ng histamine-release by said peptide is useful in anti-allergy treatment. Preferably the histamine-releasing peptide is of formula: ~A~DDVD tSEQ ID:2], optionally amidated at the C-terminal. ~nt;ho~;~C to the histamine-releasing peptide are used for passive ; ;c~t;nn, W096/00238 ~ ~ 9~865 P~ 'al493 According to the present invention there is provided the minimal T cell epitope of rye grass, which i9 an ; -~ 'n~nt g-mer polypeptide active in ~t; l~t;ng T cells from atopic patients with hayfever. T cells aper;f1r~1ly sensitive to any or some of the individual proteinB Lol pI, Lol pII and Lol FIII may be 8t; late~ by this i -~ n~nt 9-mer.

According to the present invention there is provided a polypeptide having ninc amino acid residues and ;nrln~inrJ the six amino acid sequence VXRIDT [SEQ ID:3]. The sequence VXRIDT
may be referred to as the T cell epitope motif.

Also according to the invention there is provided a polypeptide having nine amino acid residues of the sequence ~v~L~L~a, [SEQ
ID:4].

Also according to the invention there is provided a polypeptide having a se~uence of nine amino acid residues of the sequence AVWRIDTPD [SEQ ID:S~ or ~ J~ [SEQ ID:6] which has 0, 1 or 2 of its amino acid residues s~ostituted for any other amino acid residue.

The polypeptides of the present invention having nune amino acid residues may be ~ _ 1PYPd to a hapten ~e.g. ~hu~hu~ylcholine ~ W096/00238 2 1 ~ 3 8 6 0 PCT/GB9~/01493 or 2,4 dinitrophenyl) or a phArr~ gi~=lly acceptable carrier or both; l~d either directly or linked indirectly by way of a spacer such as a polypeptide or a 11Yd~G,b~11 or co-administered. Accordingly, there is provided a conjugate comprising a phArr-~lo~ically A~r~r~Ahl~ carrier and a polypeptide having nine amino acid residues in~ ;ng the T cell epitope motif VXRIDT, or nine amino acid residues of the se~uence XVX~IDTXX, or the se~uence AVWRIDTPD or ~
having 0, l or 2 of its amino acid residues substituted ~or Any other amino acid re6idue. Furthermore, there is also provided by the present invention a ligand comprising an antibody domain specific for the nine amino acid polypeptide o~ the prese~t inventior.. This antibody domain may be mono- or polyclonal or antigen binding ~ _ -c thereof.

The ; ~ nAnt g-mer polypeptide of the invention may be used to induce T cell activation. l'his, in turn, could lead to T cell arergy, ~;m;m1t;~n or lack of responsiveness to an ~-ntigen In a second aspect, the present invention provide8 an i comprisirg a residue of a h;ct: 'n~ releasing peptide comprising a catioric ~-terminal head and a 1~y1 ~ C-terminal tail, together with a residue capable o~ ;t;ng Ant;hoS;~c against W096/00238 21 9386~ sj ~

this peptide, together with a g-mer polypeptide as previously described A purpose of this second aspect gf the present invention is to induce production of ~ntiho~;~c to the i ~n in individualS
sensitive to rye yrass pollen The present invention can include an i , comprising a residue of a hict=min~-releasing peptide which has a cationic N-terminal head comprising the sequence KT~, and a hydrophobic C-terminal tail ~ _ c; ng the sequence FF The C-terminal may be blocked by '~tinn, The N-terminal head may be separated from the C-terminal tail by from two to six yL~ ' 'nrntly non-polar and non-hydLuyhwbic amino acid residues (for example GSG).

The present invention can provide an i , inr~ ing the peptide sequence h ~ 'V~', together with a polypeptide having g amino acid residues as A~c~r;h~d above, wherein the 9-mer polypeptide is _l~Y~d directly to the i -, or is indirectly linked e.g. by way of a spacer such as a polypeptide or hyd~uu~Lbull or the 9-mer polypeptide is co-r' 'n;ctPr~ with the ; ~g~n, Prefer~bly the present invention provides an ; , which 'W096/00238 2 l ~ 3 8 6 0 PCT/GB95/01493 includes both a residue of a histamine-releasing polypeptide having the se~uence ~L~r~V~, and a T cell epitope polypeptide having the sequence AVWRIDTP~, either ,1~T~ directly or linked indirectly e.g. by way of a spacer such as a polypeptide or I~YdLU~IbUI1 or the 9-mer polypeptide is CQ- ' ~ni~t~red with the i , . Preferably the C-terminal of the ; , is blocked by ~~r;nn.

In a third aspect, the present invention provides a method of using the above ~: _ '- and ; , c of the present invention in the -~n~fAct~re of a ~ for the treatment or prophylaxis of allergies particularly of rye grass allergy or hayfever.

In a fourth aspect, the present invention provides a method for rr~ or prophylaxis of allergies particularly of rye grass allergy or hayfever, by Al' ~n;ctr~t;nn of an effective amount of the above ~ _ ' or i , c, M~ c made Arcnr~ing to the invention may be administered to patients by any of the administration methods already known to those skilled in the art.

In a fifth aspect, the present invention provides an assay for W096/00~8 ~ ~ 9 ~ 8 60 r~

sensitivity to rye grass allergens and hence to ~AtrrminAtirn of hayfever snffrrAnrA In such _n assay p~riphArAl blood lAAr cells (PBMCs) may be cultured in the presence of the ~ nAnt s-mer T cell epitope and lymphocyte activation may be measured using thymidine ;nrrrporAt;rn as detrrm;nAnt.
Cells from subjects allergic to any of the main allergens of rye grass would be expected to react.

In a sixth aspect, the present invention provides an assay for specific T cell prrlllAt;rnc sensitive to rye grass allergen, such as Lol pI, by testing for reaction to the 9-mer ~Qrr;hed above, selecting reactive T cells and forming t_erefrom T cell lines, and cloning therefrom a T cell clone having qper;f;r;ty for the g-mer.

The present invention will be further illustrated with reference to the drawings of which:

Figure 1 shows the peripheral blood , rl rAr cell response cf An atopic pataent (code name A~i3) to Lol pI;

Figure 2 shows PBMC responses to 20 peptide pools;

Figure 3 shows the decoding of the i ~ n~nt Pool 1~;

~ 'W096/00238 2 1 9 3 8 6 ~ 1493 Figure 4 shows dose response curves for peptides of Pool 17;

Figure 5 shows PBMC ~~O~ull~es of atopic patient A83 to 9 mer peptides;

Figure 6 shows T cell line sperif;~ity for Lol pI;

Figure 7 shows a comparison of T cell line and PBMC rocpnnc~c to 9-mer peptidesi Figure ~ shows the results of Lysine substitution when each amino acid residue of the 14 amino acid polypeptide thought to irclude the T cell epitope of Lol pI was individually substituted for Lysine; and Figure 9 shows the results of Glycine substitution when each amino acid residue of the 14 amino acid residue polypeptide thought to include the T cell epitope of Lol pI was individually ~ubstituted for Glycire.

Rye grass (Lolium perenne) pollen extract was supplied by Dome T.~h~r~tnr;~c tStoke Court, U.~.~ And subjected to 33~ ammonium sulphate precipitation. The sllp~rnAtAnt was dialysed agairst phncrh~t~ buffer saline (PBS). The dialysed sample was injected W09~00238 I~1.~.. I493 ~
~ 1 ~386~

on to FPLC (fast protein liquid ~ rgrArhy~(PharmaCia~ LXB, Sweden) for protein separation on a gel f;ltrAt;nn column Superose 12, and 0.3 ml frArt;rnq were collected. Sodium dodecyl sulphate (SDS) polyacrylamide-gel ~ sis (PAGE) was p~-C~ ' on a vertical slab-gel apparatus (Bio-rad, Richmond, CA). 15% gradient acrylamide gels were employed in this study and run at 180 V for 12 hours. The apparent l~r~l1Ar mass of the allergens was ~Ct;~-t~ by using prestained protein standards.

The iso-allergen clone 5A of Lol pI, consists of 240 amino acids. A set of 115 ov~rlArr;nrJ, 12-mer peptides spanning the entire length of the iso-allergen clone 5A of the Lol pI was 5ynth~Q; 7~ on polyethylene rods (pins) using the multi-pin terhn;r~ which enables the rapid mapping of T cell ~r~rm;nAntQ All of the peptides were acetylated at the N
terminus and, as a result of cleavage from the pins, had a C-terminal ~;k~tor;r~rA7;n~ (D~P) group.

A ~-alanine residue was incorporated to space the defined se~uence from the D~P group. The peptides were released from the solid phase according to the method of Maeji et al. using O.lM rh~QrhAte buffer (p~ 7) for 4 hours at room t~ t--re.

Sets of six r~nr;n-lr-lQ peptides were pooled, with the exception ~ ~V096/00238 2 1 93860 ~ s l493 of Pools-1 and 15 (5 peptides) and Pool-20 (3 peptides).
Therefore, 20 peptide pools were used for epitope mapping. The purity of selected peptides inrll~;ng all peptides in Pool-17, was est~hl;ch~ by reverse phase HPLC analysis and the amino acid - t; nn wag checked by amino acid analysis.

100 ml of blood from each subject was rrll~rt~ in ~L~s~Lv~tive free heparin. ~r~rin;7~ venous blood was diluted 1:1 with RPMI 1640 and layered onto L/,,' , el~ (Nycomed, 0810, Norway).
The gradients were centrifuged at 400g for 25 minutes at room t~ , . Mr,nrn--rlr~r cellg were hclY~aLed and washed twice in RPMI 1640 (Gibco, Grand Island, NY, ~SA) and then ~
at a l,,-~ l,~t;nn of 2xlO' cells/ml in RPMI s~rrl~ ~ with 10~ human AB-serum (ICN Flow, Bucks, U.R.), 2mM 1-rl n~ and 20~g/ml g~nt rin (Gibco, Grand Island, NY, USA). Cells were cultured at a ~ t;rn of 2 x 105 per well in 0.2 ml of complete medium in ~-bottomed 96-well microtiter plates.

Purified Lol pI or Lolium pereune extract (LPE) was added at a range of .,." ,I,~li~nc and PPD or Con-A was used as a positive control. Cultures were inrnh~tr~ at 37'C in a hnmi~;~;ed a ~heL~ rrnt~;n;nr 5~ C~2 in air. After 5-6 days, 0.4~Ci/well [methyl-'H] TdR ~ ' , U.~.) was added and 6-18 hours later the cells were harvested onto Titertek glass-fibre W09~00238 2 1 93860 1~1 . l493 paper (TCN Flow, Bucks, U.X.). 3H-thymidine incorporation into DNA was measured by li~uid sr;nt;1lAt;rn (National Diagnostics, Atlanta, USA) counting (Minaxi Tri-Carb 4000 Series, Packard).

Sirce the precursor frequency of T cells specific to any one peptide is very low, 12-24 replicate cultures were used for each of the 20 peptide pools at a final ~ , _ 5 .~t;rn of 1 ~g/ml of each peptide. Twenty pools of peptides were screened, each pool ~ c;nrJ 6 ovrrl~rpinrJ peptides (with the ~r~rt;rnc as noted above) with successive peptides differing by only two amino acids at their starting (N-terminal~ residue. The thymidine in.uL~u~tion of each replicate was measured srr~r~t~ly Purif; r~t; rn of rye grass extract by FPLC yave two peaks (Peak 1 and 2). Flectrophoresis of a L. pere~ne extract under reducing conditions followed by protein staining showed that the molecular weight of peak 1 was approximately ~1 kDa and its location was equivalent to highly purified Lol pI. The other peak was i~nt; f;~ as a mixture of Lol pII and Lol pI~I with a lrrnl~r weight of 11 kDa.

Human p~rirh~rA1 blood rle~r cells (PBMC) proliferative ~nD~u~lseS to the major purified fraction of r, p OEe~e (~.ol pI~

were found (Fig.1). This dose ranging rYr~r; shows the ~ W096/00238 21 ~3860 r~ 493 typical bell-shaped dose response curve of lymphocyte activation seen with allergen and antigen. The peak incorporation of tritiated thymidine is seen at 10~g/ml of purified ~ol pI. This is evidence of lymphocyte reactivity to the major fraction of Rye grass protein Ln atopic subjects. Atopics reayu~ ed to both T~ol pI and LPE as might be expected. Non-atopics did not respond to Lol pI but some did show minor st; lRt;nn with LPE.

The presence of T cell epitopes in the 20 pools of peptides was inV~ct;gRt~ by i~ vitro prnl;f~rRt;nn tests on PBMC of six rye grass-se sitive patients and seven non-atopic control subjects.
Whilst ~ive out of the six patients ~ ded to Pool 17, patient B reacted to Pool 15 and 19. In addition, patient E
responded to Pool 6 and patient F also Lea~v.~ded to Pool 13.
Pool 17 clearly rnntA;n~ the ; -~ nlrt epitcpe. Non-atopic subjects did not respond to any of the 20 peptide pools.
There was no class II MHC (major histc t;h;l;ty complex~ Dr restriction in t_e atopic subjects.

The number of wells nnntR;n;ng p~r;rh~rRl blood _~Rn cells giving positive prnl;f~r~t;ve ~ nses to 20 ~;ff~n~nt peptide pools spanning the entire length of the ~ol pI is shown in Fig. 2. Each pool was composed of 6 (except 1, 15 and 20) ov~nlRrring 12-mer peptides offset by 2 residues, and thus W096l00238 L~ 1493 2~ 93~6~

spanned 22 residues of the protein se~uence, and was tested against 12 identical cultures from each patient. These data identify Pool 17 as rnnt~;ning the major epitope of rye grass.
The cut-off for sirJn;f;r~nrP in terms of number of wells responding is defined by statistical methods based on the Poisson distribution. In this instance, Pool 17 is statistically different from all other pools tested. Further data from other atopic subjects rnnf; 1 the i ~ n~nrP
Of Pool 17. As is ;~nP~ above, Pool 17 is statistically different from all the other pools tested. Further data from other atopic subjects rnnf; ' the ; ~ n~nr~ of Pool 17.
As is r ; nnP~ above, Pool 17 consists Of 6 overlapping peptides covering a span of 22 amino acids.

~aving ;~Pnt;f;P~ Pool 17 as the ; ~ n~nt pool, each of the peptides it rrnt~;nP~ was tested 5pr~r~tply. Experiments showed that peptides 3 and 4 of Pool 17 were the main ~t; l~t~ry peptides in vitro (Fig. 3). The active polypeptides of Lol pI (3) are l"WGAv~ ~ LSEQ ID:9] and (4) 'sAVW~IDTPD~LT'~ [SEQ ID:I0] and are each as active in lymphoproli_er~tive assays as is the whole pool When large numbers of rPrl; r~tPc are tested with any given antigen, it is clear that the positive wells are not normally -~ W096l00238 2 ~ 93~6~ r~ 0.~,3 distributed. This is because of the random ~i Qtr; h -t; nn of the low numbers of antigen specific T cells among the replicate wells. For this reason we have not treated the proliferation data using statistical methods based on normally distributed data but on the Poissor. model. T]1e non-res~.lder wells are normally distributed and we have therefore taken a mean +/- 3SD
as the cut-off to score wells as either positive or negative.
Any well with counts above the means and three standard deviations is considered as positive.

Of Pool 17, only 2 out of the 6 peptides have Sri lAr~r activity. This activity or lack of activity of different Pool 17 peptides could have been dose related and thus dose response curves were s~n~rAt~.

PBMCs were ;n~nhAt~ with a range o~ ~,.,.,. I,_t;~n of a) Pool 17, b) peptide 4 of Pool 17 and c) Pool 8 ~a non-st; lAt~ry peptide pool). Cells were harvested after 6 days in culture.

The dose response of peptide 4 of Pool 17 is parallel to whole Pool 17 whilst the non-st; lAt~ry pool (Pool 8) is still ;n~ffe~t;ve i~ activatirLg lymphocytes at 100 times the ~nn~ntrAt;~n of the peptide.

W096/00238 2 ~ 9 3 8 6 0 PCTIGB95/01493 Thus, finding that Pool 17 is dominant is not due to an effect of final concentration because the non-st; lAtnry pool (Pool 8~ was still ineffective at high .,,,,/~ At;nnc.

Eight overlapping 9-mer peptides offset ~y one residue were screened using PBMCs from the atopic patient (AS3). The presence of minimal T cell epitopes was investigated by in vitro proliferation tests against slngle peptides. The thymidine incorporation of each replicate was measured separately.

Figure 5 shows the percentage of wells cnntA;n;ng peripheral blood l~r cells from a rye grass sensitive~patient (AS3) giving positive proliferative responses to single peptides of the 9-mer peptides spanning the entire length of the ; o~ nAnt epitope of the Lol p~.

Surprisingly, as shown in Fig. 5, the 9 amino acid residue polypeptide of se~uence (5) AVWRIDTPD is found to be the most active of any 9, 10, ll, 12, 13 or 14 amino acid residue polypeptide spanning the length of the two nnnt;nllnnc ; -- nAnt polypeptides. The 9 amino acid residue polypeptide of sequence (6) VWRmTPD~ was also found to be active. More surprisingly, six amino acid residues were ~ound to be the most i L~-L in fl~tormining whether the polypeptide W096/00~8 2 1 9 3 8 6 0 PCT/GB9S/01493 is i ~ n~nt.

To s~rpl~ the data obtaLned from PF3MC ~ e~ SeS to 9-mer peptides, T cell line responses to 9-mer peptides were investigated.

Lol pI-specific T cell lines were obtain as follows: 0.5 x 10' PPMCs per ml from patient AS3 who reactive to the i -' n~nt T cell epitope were sti l~t~2 with Lol pI (lO~g/ml) in PRMI
1640 supplemented with 2mM L-glutamine, ~ x 10-sM Z-merrArto~thAnn1, 20yg/ml g~ntl '~;n, and 105~ human AB serum in 24-well flat-bottomed culture plates for 5 days. 5~h~ y, human r~ ' nAnt IL-2 (hrIL-2~ (25 units/ml) was added and kept in culture for an additional 7 days. Viable T cell blasts were then s~r~rAt~d by a Ficoll-Hypaque density gradient and Lol pI
spen;f;r;ty of T cell line was assessed. The Qre~;f;r;ty of the T cell lines produced by st; l~tinn with IL-2 and Lol pI was checked. 5 x 10' T cell line cells were {n~lhAt~ in dllrl;
200 ~l cultures in the presence of 5 x lO~ ;rr~ t~d autologous antigen presenting cells ~APCs) plus 1, 5 and 10 ~g/ml of Lol pI in 96-well round-bottomed microtiter plates for 3 days at 37~C, in a ' ~;f;~ e of 5~ COI, then pulsed overnight with 0.4 ~Ci/well 3H-thymidine. ~;nnll~ uptake was measured by srintillAt;on counting. As 3hown in Fig. 6, using a dose response of Lol pT ~rom 1 ~g/ml to 10 ~g/ml it is clear that this T celi line is specific for Lol pT, T cell line ~T) plu5 irradiated APC~ (A) gives low background counts in comparison.

5 x 10' T cell line cells were ;nrllhat~d in duplicate 200 ~1 cultures in the presence of 5 x 10' irradiated =l.trlOrJn..c APCs plus 7.5 ~g/well of individual single peptide of the 9-mers in 96-well round-bottomed microtiter plates for 3 days at 37~C, in a 1 '~;~;e~ ~ ,' e of 5~ CO2, then pulsed overnight with 0.4 ~Ci/well 3~-thymidine. RA~; ~n11r1; ~G uptake was measured by Srint;ll~t;~n counting. Two peptides (5) and (6) are clearly more active in sti lAting T cell lines from an atopic donor than other peptides.

A comparison of P3MC and T cell line responses (Fig. 7) shows that the T cell line and P3MC respond similarly.

Fxper; ~ti~n on the previously known 14-mer peptides was carried out to attempt to identify parts of the T sell epitope minimal se~uence which could be substituted with other amino acids. ~esults are shown in Fig. 3 for a series of 14_mer peptides having a single lysine (R) ~ubstitution and in Fig. g ~ W O 96/00238 2 1 9 3 8 b O PC~rlGB95/01493 for a series having a single glycine ~G) substitution.
Sub6titution at a point which causes loss or reduction in rctivity ;nS;r~to~ that the amino ~cid rormally at that position is important or oRs~nt;~. In contrast certain substitutions at certain points have little or no effect on response. From this information it has been deduced that the nine amino acid residue polypeptide ;n~ ;ng the T cell epitope motif VXRIDT, or nine amino acid r~Lsidues having the sequence XVXRIDTXX, or the seriuence AVW~IDTPD or Vw~lJL~K wherein 0, 1 or 2 of the amino acid residues is substituted for any other amino acid residue is the common ; ' nAnt T cell epitope of rye grass. The existence of a common epitope motif in rye grass was not obvious and its ;~ont;f;rAt;nn is of rnnR;~orAhlo clinical cign;f;rAnr~, In other areas of plant allergy (e.g. ragweed allergy) it has not been possible so far to characterise a common epitope to all of the v_rious major allergy inducing proteins.

It will be apparent to those skilled in the art of ; 1 ngy that the surprising charactoni cAti nn of a T cell epitope motif and an ; ~ nAnt T cell epitope for rye grass for the first time allows the possibility of a previously ; ~ hl o detailed analysis of the I 1orlllAr environment of the imm~ne respon6e to rye grass allergen. In particular the motif and epitope can be W096100238 2 ~ 93~0 ~- . ;.1493 modelled to ~t~rnin~ the shape and configuration of the polypeptides, the spatial distrihnt;nn o~ charges, the level of steric constraint on the molecules etc. so as to ~ rm; n~ the ~ lAt i nnq~; n hetween structure and activity. With such information it is foreseen that those skilled in the art could form analogs or mimetics of the motif and epitope, which would exhi_it ~q~nt;Ally the same or similar ~nnrt;nnAl activity whilst differing in chemical formula. For example some or all amino acid residues could be replaced ~y e~uivalent non-amino-acid moieties. It is intended that the present invention should cover such f~nnt;nn~lly active analogue or mimetic T cell epitope or motif e~uivalents.

~ 'W096/00238 21 93 8 6 D r l~ 1493 s~r~uENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLI ~ :
(A) NAME: PEPTIDE T ERAPEUTICS LIMITED
(B) STREET: 321 CAMBRIDGE SCIENCE PARIC
(C) C}TY: CAMBRIDGE
(D) STATE: CAMBRIDGE
(E) COUNTRY: ENGLAND
(F) POSTAL CODE (ZIP): CB4 4WG
(G) TELEP~ONE: 01223 423333 (~) TELEFAX: 01223 423111 (ii) TITLE OF INVENTION: T Cell Epitopes (iii) NUMBER OF SEQUENCES: 20 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC ~ihlr (C) OPERATI~G SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Pa~entT~ Release #1.0, Version ~1.30 (EPO) (~i) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9125024.1 (B) FILING DATE: 25-NOV-1991 (2) s~r~ _.llU~ FOR SEQ ID NO: 1:
(i) SEQUEN OE r~=~rT~qTICS:
(A) LENGT~: 11 amino acids (B) TYPE: ami~o acid (C) 5~ : single (D) TOPOLOGY: lir,ear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE ~U~l~llU~: SEQ ID NO: 1:
Arg Leu Thr Lys Gly Ser Gly Phe Phe Val Phe .

SU~STITUTF SHE~T (RULi' 2b) ~2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQ~ENCE CN~2ACTERISTICS: _ _ _ _ (A) LENGT~: l0 amino acids (B) TYPE: amino acid (C) ST~En~CC single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE ~ ~T~TqTICS:
(A) BENGTX: 6 amino acids (3) TYPE: amino acid (C) ~ l ~h~ N~.:.S single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQ~ENCE ~L~l~lLLN: SEQ ID NO: 3:
Val Xaa Arg Ile Asp Thr (2) lN~ -TnN FOR SEQ ID NO: 4:
(1) SEQUENCE r7~E~T~TICS:
~A) LENGTE: 9 amino acids (B) TYPE: amino acid (C) ST~ nN~c~: single (D) TOPOLQGY: linear (ii) MOLECULE TYPE: peptide SU~3STITUTE SHEET ~r~ULE 26) 21 9386~

xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 4:
Xaa V~l Xaa Arg Ile Aqp Thr Xaa Xaa l S
(2~ INPORMATION FOR SEQ ID NO: S:
(i) SEQUENCE ~ rTEFT.qTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amiro acid (C) STR7''''1RnN~.C.q: single ~D) TOPOLOGY: lirear (ii) MOLECULE m E: peptide (xi) SEQUENOE ~r_~lrLlu~: SEQ ID NO: S:
Ala Val Trp Arg Ile Asp Thr Pro Asp (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUEN OE Fr~ r~T~ T CT ICS:
(A) LENGT~: 9 amino acids (B) TYPE: amino acid (C) STFD~ nN~Cq: single (D~ TOPOLOGY: linear (ii) MOLECULE m E: peptide (xi) SEQUENCE ~r_~ u~ SEQ ID NO: 6:
Val Trp Arg Ile Asp Thr Pro Asp Lys (2) INFO~MATION FOR SEQ ID NO: 7:
(i) SEQ~ENCE ru~
(A) LENGT~: 12 amino ac~ds (B) TYPE: amino acid (C~ ST~ Fn~qq: single (D) TOPOLOGY: l~~ear SUeSTlTUTE CHEET (l~ '.26~

W 096100238 ~ ~ 9 3 8 6 0 PCT/GB95101493 ~ii) MOLECULE TYPE: peptide (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: E:
Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile Asp l 5 l0 (2) INFORMATION FOR SEQ }D NO: 8:
(i) SEQUENCE r~a~T~CTICS:
(A) LENGTU: 12 amino acids (B) TYPE: amino acid (C) ST~a~Rn~qq si~gle (D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE ~r~oKl~1lu~: SEQ ID NO: 8:
Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro l 5 l0 (2) l~ruK~ u~ FOR SEQ ID NO: 9:
(i) SEQ~ENCE ~a~ l~a:
(A) LENGTU: 12 amiro acids (3) TYPE: amino acid (C) ST~a : sirgle (D) TOPOLOGY: li~ear (il) MOLECULE TYPE: pep~ide (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 9:
Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys q 10 (2) INFORMATION FOR SEQ ID NO: l0:

SUBSTITUTE SHEET ~RULE 26~

~ W 096/00238 2 1 938 ~0 .~ 1493 ~i) SEQUENCE r~T~TqTICS:
(A) LENGT~: 12 amino acids (B) TYPE: amino acid ( C ~ 5~ n~nNR.c .C: single (D~ TOP010GY: linear (ii) MOLECULE TYPE: peptide (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO: 10:
Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQVENCE ~ T.~
(A) LENGT~: 12 amino acids (B) TYPE: amino acid (C) ST~ ~Fn~cc single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE ~ 9~ : SEQ ID NO: 11:
Trp Arg Ile ASp Thr Pro A6p Ly3 Leu Thr Gly Pro (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE ~ lC~
(A) LENGT~: 12 ~mi~o acids (B) TYPE: amino acid ~C) ~. : single ~D) TOPOLOGY: liuear ~ii) MOLECULE TYPE: peptide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

SUBSTITUTE SHEET ~RVLE 26~

W 09~00238 2 1 9 3 8 6 0 PCT/GB951014~3 Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Tyr (2) INFORMATION FOR SEQ ID NO: 13~ m (i) SEQUENCE ~D~D~Tr~T~TIcs (A) LENGT~: g amino acids (B) TYPE: amino acid (C) ST~D~n~nN~.C.q: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Glu S ~ Trp Gly Ala Val Trp Arg Ile (2~ INFORM~TION FOR SEQ ID NO: 14:
(i~ SEQIJENCE ~D~a(-TF'l~T.CTICS
(A~ LENGT~: 9 amino acids (B~ TYPE: amino acid (C~ : single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (xi) SEQUENCE ~o~~ u~: SEQ ID NO: 14:
Ser Trp Gly Ala Val Trp Arg Ile Asp (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE ~ ~ D ~ .~ l l Ca:
(A) LENGT~: 9 amino acids (i3) TYPE: amino acid (C) STI~D~''')rn~C:C: siugle (D~ TOPOLOGY: linear (ii~ MOLEC~LE TYPE: peptide SUBSTITUTE SHEET (RULE 26~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: lS:
Trp Gly Ala Val Trp Arg Ile Asp Thr (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE r~ ca:
(A) LENGTE: 9 amino acids (B) TYPE: amino acid ~ c) ST~ nN~C~: sirgle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE ~U~l~lUN: SEQ ID NO: 16:
Gly Ala Val Trp Arg Ile Asp Thr Pro (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQ~ENCE r~o~,, r.~ I ~1~:
(A) LENGTE: 9 amino acids (B) TYPE: a~ino acid (C) 5~Fn~T~zcc: 3 ingle (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: peptide (xi) SEQ~ENCE ~U~l~'lUN: SEQ ID NO: 17:
Trp Arg Ile Asp Thr Pro Asp Lys Leu (2) lNrU~_~IlUN FOR SEQ ID NO: 19:
(i) SEQUENOE r~o~ I h ~
(A) LENGTE: 9 amino acids SUBSTITUTE SHEEl' lRULE 26~

W 096/00238 ~ t 9 3 8 6 0 PCTIGB95/01493 (B) TYPE: amino acid (C) ST~ n~Cq: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEO ID NO: 1~:
Arg Ile Asp Thr Pro Asp Lys Leu Thr (Z~ INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE ~ T~,L~5 (A) LENGTH: 14 amino acids (B) m E: amino acid (C) sT~ n~qq single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1..14 (D) OTHER L~r~ aU~:/note= nEACH RESIDUE IS INDIVIDUALY
su~ u~r~ WITH Lys TO GIVE 14 DIFFERENT SEOUENCES"

(xi) SEQUENCE L~o~IL~llo~: SEQ ID NO: 19:
Trp Asp Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE ~DI I r.~ I ~11~:
(A~ LENGTH: 14 amino acids (B) TYPE: amino acid (C) ST~EnNrCq: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SUBSTITUTE SHEET (RULE 261 ~ W 096/00238 2 ~ 93~ Y~ 1493 (ix) FEATURE:
~A) NAME/~EY: Modified-site (~) LOCATION:1..14 (D) OTHER INFORMATION:/notez ~EACH RESIDUE IS
INDIVIDUALLY ~U~L'.~.~ WITH Gly TO GIVE 14 DIFFERENT
SEQUENCES

(xi) SEQUENCE ~lPll~N: SEQ ID NO: 20:
Trp Gly Ala Val Trp Arg Ile Asp Thr Pro A~p Lys ~eu Thr SUaSTlTUTE SHEET-(RULE 26)

Claims (14)

Claims

1. A polypeptide of nine amino acid residues including the six amino acid residue VXRIDT which is capable of inducing T cell activation, and analogues and mimetics thereof excluding the polypeptides having the sequences Trp-Gly-Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys, Gly-Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys-Leu, and Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys-Leu-Thr corresponding to amino acid residues within the region 193-206 of Lol pI.

2. A polypeptide according to claim 1, having the amino acid residue sequence XVXRIDTXX.

3. A polypeptide capable of inducing T cell activation having the amino acid residue sequence AVWRIDTPD having 0, 1 or 2 of its amino acid residues substituted for any other amino acid residue, and analogues and mimetics thereof excluding the polypeptides having the sequences Trp-Gly-Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys, Gly-Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys-Leu, and Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys-Leu-Thr corresponding to amino acid residues within the region 193-206 32a of Lol pI.

4. A polypeptide capable of inducing T cell activation having the amino acid residue sequence VWRIDTPDK having 0, 1 or 2 of its amino acid residues substituted for any other amino acid residue, and analogues and mimetics thereof excluding the polypeptides having the sequences Trp-Gly-Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys, Gly-Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys-Leu, and Ala-Val-Trp-Arg-Ile-Asp-Thr-Pro-Asp-Lys-Leu-Thr corresponding to amino acid residues within the region 193-206 of Lol pI.

5. A polypeptide having the amino acid residue sequence according to any of claims 1 to 4, together with a polypeptide having the amino acid residue sequence KTKGSGFFVF either complexed directly or linked indirectly by way of a spacer such as a polypeptide or a hydrocarbon, or co-administered.

6. An immunogen comprising a residue of a histamine releasing peptide comprising a cationic N-terminal head and a hydrophobic C-terminal tail, together with a residue capable of eliciting antibodies against this peptide, together with a 9-mer polypeptide according to any of claims 1 to 4.

7. An immunogen comprising a residue of a histamine-releasing peptide which has a cationic N-terminal head comprising the sequence KTK,and a hydrophohic C-terminal tail comprising the sequence FF, the N-terminal head being separated from the C-terminal tail by from two to six predominantly non-polar and non-hydrophobic amino acid residues, together with a 9-mer polypeptide according to any of claims 1 to 4.

8. An immunogen according to claim 7, wherein the C-terminal is blocked by amidation.

9. An immunogen according to claim 7 or 8, wherein the sequence GSG separated the N- from the C-terminal.

10. A polypeptide according to any of claims 1 to 4, complexed to a hapten or a pharmacologically acceptable carrier or both; complexed either directly or linked indirectly by way of a spacer such as a polypeptide or a hydrocarbon or co-administered.

11. Use of polypeptides and immunogens according to any of claims 1 to 10, in the manufacture of a medicament for the treatment or prophylaxis of allergies particularly of rye grass allergy or hayfever.

12. A method for treatment or prophylaxis of allergies particularly of rye grass allergy or hayfever, which comprises administration of an effective amount of a polypeptide or immunogen according to any of claims 1 to 10.

13. An assay for sensitivity to rye grass allergens for determination of hayfever sufferance, in which assay peripheral blood mononuclear cells (PBMCs) are cultured in the presence of a polypeptide according to any of claims 1 to 4, and lymphocyte activation is measured using thymidine incorporation as determinant.

14. An assay for specific T cell populations sensitive to rye grass allergen, such as Lol pI, which comprises testing T cells for reaction to the 9-mer polypeptide of any of claims 1 to 4, selecting reactive T cells end forming therefrom T cell lines, and cloning therefrom a T cell clone or clones having specificity for the 9-mer.