CA2157596A1 - T cell epitopes of ryegrass pollen allergen - Google Patents

T cell epitopes of ryegrass pollen allergen

Info

Publication number
CA2157596A1
CA2157596A1 CA002157596A CA2157596A CA2157596A1 CA 2157596 A1 CA2157596 A1 CA 2157596A1 CA 002157596 A CA002157596 A CA 002157596A CA 2157596 A CA2157596 A CA 2157596A CA 2157596 A1 CA2157596 A1 CA 2157596A1
Authority
CA
Canada
Prior art keywords
seq
lpi
peptide
lol
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002157596A
Other languages
French (fr)
Inventor
Irwin J. Griffith
Mei-Chang Kuo
Mohammad Luqman
Stephen Palmer Powers
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immulogic Pharmaceutical Corp
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of CA2157596A1 publication Critical patent/CA2157596A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The present invention provides isolated peptides of Lol p I, a major protein allergen of the species Lolium perenne. Peptides within the scope of the invention comprise at least one T cell epitope, or preferably at least two T cell epitopes of a protein allergen of Lol p I. The invention also provides modified peptides having similar or enhanced therapeutic or diagnostic properties as the corresponding naturally-occurring allergen or portion thereof, but having additional properties, e.g., reduced side effects. The invention further provides nucleic acid sequences coding for peptides of the invention. Methods of treatment and diagnosis of treatment to Lol p I or an allergen immunologically related to Lol p I in an individual (such as Dac g I Poa p I or Phl p I) also are provided. Compositions for therapeutic, diagnostic or reagent uses comprising one or more peptides of the invention are also provided.

Description

WO 94/21675 ~ 1 5 7 5 9 6 PCT/US94/02537 T CELL EPlTOPES OF RYEGRASS POLLEN ~T~T~F~RGEN
k~ d of tlle Invention The most abundant proteins of grass pollen are allergens, which are the major cause of allergic disease in temperate climates (Marsh (1975), "Allergens and the genetics of allergy"; in M. Sela (ed), The Antigens, 3:271-359, Academic Press Inc., London, New York)., Hill et al. (1979) Medical Journal of Australia, 1:426-429).The first descriptions of the allergenic proteins in ryegrass showed that they are immunochemically distinct, and are known as groups I, II, m and IV (Johnson and Marsh (1965), Nature, 206:935-942, and Johnson and Marsh (1966) Immunochemistry, 3:91-100). Using the International Union of Immunological Societies' (IUIS) nomenclature, these allergens are de~.~ign~t~(l Lol p I, Lol p rJ., Lol p m, and Lol p IV. Another important Lolium perenne allergen which has been identified in the liLe,aLu,e is Lol p IX, also known as Lol p V or Lol p Ib, which has been found to be closely related to the Group V protein allergens in grasses.
These proteins have been identified in pollen from ryegrass, Lolium perenne, and act as antigens in triggering immecli~te (Type 1) hypersen~itivity in susceptible hllm~n.~, Lol p I is defined as an allergen because of its ability to bind to specific IgE in sera of ryegrass-sensitive patients, to act as an antigen in IgG responses and to trigger T-cell responses. The allergenic pro~el~ies have been ~e~ecl by direct skin testing of grass pollen-sensitive patients. The results showed that 84~o had a skin sensitivity to Lolp I (Freidhoff, et al., (1986) J. Allergy Clin. Irnmunol., 78:1190-1201) demonstrating the primary importance of this protein as the major allergen.
Furthermore, 95% of patients demonstrated to be grass pollen-sensitive possessedspecific IgE antibody that bound to Lol p I, as demonstrated by immunoblotting (Ford and Baldo (1986) International Archives of Allergy and Applied Immunology, 81: 193-203).
Substantial allergenic cross-reactivity between grass pollens has been demonstrated using an IgE-binding assay, the radioallergo-sorbent test (RAST), for e~rnple, as described by Marsh et al. (1970) J. Allergy, 46:107-121, and Lowenstein (1978) Prog. Allergy, 25:1-62. (Karger, Basel).
The immunochemical relationship of Lol p I with other grass pollen antigens has been demon~tr~t~d using both polyclonal and monoclonal antibodies (e.g., Smart and Knox (1979) Intemational Archives of Allergy and .

WO 94/2l675 2 ~ ~ 7 5 9 ~ PCT/US94/02537 Applied lmmunology, 62: 173-187; Singh and Knox (1985), International Archives of Allergy and Applied Immunology, 78:300-304). Antibodies have been prepared to both purified proteins and IgE-binding components. These data demonstrate that the major allergen present in pollen of closely related grasses is imm~mochemically sirnilar to Lol p I (Singh and Knox, supra).
Grasses that may be considered immunoch~.mic~lly related to Lol p I and that comprise allergens which may be considered immunologically cross-reactive with antibody to Lol p I include:
Pooid (festucoid) grasses of the Poaceae (C~Tramin~ae) family include the following. GROUP 1: Triticanea: Bromus inermis, smooth brome; Agropyron repens, English couch; A. cristatum; Secale cereale rye Triticum aestivum, wheat. GROUP 2: Poanae: Dactylis glomerata, orchard grass of cocksfoot;
Festuca elatior, meadow fescue; Lolium perenne, perennial ryegrass;
Lmultiflorum, Italian ryegrass; Poa pratensis, K~ cky bluegrass;
P.compressa, flattened meadow grass; Avena sativa, oat; Holcus lanatus, velvet grass or Yorkshire fog; Anthoxanthum odoratum; sweet vernal grass;
Arrhenatherum elatius, oat grass; Agrostis alba, red top; Phleum pratense, timothy; Phalaris arundiriacea, reed canary grass. Panicoid grass, Paspalum notatum, Bahia grass, Andropogonoid grasses: Sorghum halepensis, Johnson grass.
In view of the prevalence of ryegrass pollen allergens and related grass allergens all over the world, there is a pressing need for the development of compositions and methods that could be used in detecting sensitivities to Lol p I
or other immlmologically related grass allergens, or in treating sensitivities to such allergens, or in ~ ting in the manufacture of medic~m~.nt.c to treat such sensiliviLies. The present invention provides m~teri~l~ and methods having one or more of those utilities.

Swnmarv of the Inven'don The present invention provides isolated peptides of Lol p I. Peptides within the scope of the invention comprise at least one T cell epitope, preferably at least two T cell epitopes of L,ol p I. The invention further provides peptides comprising at least two regions, each region comprising at least one T cell epitope of Lol p I.
2 ~ 5 ~ S 9 ~ PCT/US94/02537 The invention also provides modified peptides having similar or enh~nced therapeutic ordiagnostic p,opelLies as the corresponding, naturally-occln~ing allergen or portion thereof, but also having advantageous physical or biological propel~ies, such as reduced side effects, reduced IgE binding, imp,vvt;d solubility, increa~.ed in vitro or in vivo T cell stim~ ting ability, increased stability or the like. I~e"ed peptides of the invention are capable ofmodifying, in a Lol p I-sensitive individual to whom they are ~(lmini~tered, theallergic response of the individual to Lol p I or an allergen immunologically cross-reactive with Lol p I, e.g., allergens derived from pollen belonging to the Poaceae (Gr~min~e) family, such as Dactylis glomerata (Dac g I), Poa pretensis (Poa p I) and Phleum pratense (Phl p I), as discussed above.
The present invention also provides non-native (i.e., recombinant or chemi~lly synth~i7~d) Lol p I peptides or their derivatives or homologues and provides non-native allergenic protein or peptides immunologically cross-reactive with antibodies or with T cells of Lol p I or derivatives or homologues thereof.
The present invention also provides Dac g I and Poa p I protein allergens which are immunologically cross-reactive with Lol p I, and fr~gme of Dac g I and Poa p I produced in a host cell transformed with a nucleic acid sequence coding for Dac g I and Poa p I, respectively, and fragmt~.nt~ of Dac g I
and Poa p I pr~p~,d synthetically. The present invention further provides nucleic acid sequences coding for Dac g I, Poa p I and fr~gment.c thereof. Also provided are isolated peptides of Dac g I and Poa p I comprising at least one T
cell epitope which are immunologically cross-reactive with peptides comprising at least one T cell epitope derived from LoE p I.
2s Methods of tre~tment and of diagnosis of sensitivity to ryegrass pollenprotein, Lol p I, or to pollen proteins that are immunologically related to Lol p I
(such as Dac g I, Phl p I and Poa p I), as well as compositions comprising one or more peptides of the invention, are also provided.
Further features of the present invention will be better understood from the following detailed description of the preferred embo~liment.c of the invention in conju~ ion with the appended figures.
r Brief Des~ lion of ~he Fi~ures wo 94/21675 PCT/USg4/02537 Fig. 1 shows the nucleotide sequence of cDNA clone 26j (SEQ ID NO 1) and its predicted amino acid sequence (SEQ ID NO: 2). Clone 26.j is a PCR-generated,full-length clone of Lol p I.
Fig. 2 shows various peptides of desired lengths derived from Lol p I (SEQ ID
NO: 3-30); such peptides include polymorphisms inherent in the Lol p I sequence (i.e., LPI-4.1 (SEQ ID NO: 8) and LPI-16.1 (SEQ ID NO: 23)) or homologues of peptides derived from Lol p I (i.e., LPI- 11 (SEQ ID NO: 15), and LPI- 12 (SEQ ID NO: 17)).
Fig. 3 is a graphic representation depicting responses of T cell lines from thirty-five grass-sensitive patients primed in vitro with purified native Lol p I and 0 analyzed for response to various r ol p I peptides by percent of positive responses (with an S.I. of at least two, shown over each bar), the mean s~im~ n index of positive response for the peptide (shown over each bar in parentheses) and the positivity index (% positive x mean S.I. index, Y axis).
Pig. 4 shows various peptides of desired lengths derived from Lol p I (SEQ ID
NO: 23, 25, 27, 30-50).
Pig. 5 shows the nucleotide sequence of cDNA clone 106.5 (SEQ lD NO: 51) and its predicted arnino acid sequence (SEQ ID NO: 52). Clone 106.5 is a PCR-ge"f,~ed, full-length clone of Dac g I.
Fig. 6 shows the nucleotide sequence of cDNA clone 114 (SEQ ID NO: 53) and its predicted amino acid sequence (SEQ ID NO: 54). Clone 114 is a PCR-gellFI~lecl~ full-length clone of Poa p I.
Pig. 7 shows the nucleotide sequence of cDNA clone 20 (SEQ ID NO: 55) and its predicted amino acid sequence (SEQ ID NO: 56). Clone 20 is a PCR generated, full length clone of Phl p I.
Fig. 8 shows a comparison of the amino acid sequences of the mature protein of Lol p I (SEQ ID NO: 57), Dac g I (SEQ ID NO: 58), Phl p I (SEQ ID NO: 59), and Poa p I (SEQ ID NO: 60), including polymorphisms thereof.
I;ig. 9 shows a comparison of various peptides comprising at least one T cell epitope derived from Lol p I, with homologous peptides derived from the same regions of Dac g I, Phl p I, and Poa p I (SEQ ID NO: 23, 25, 27, 30, 61-70).

Detailed Description of the Invention The present invention provides isolated peptides derived from Lol p I (SEQ
IO NO: 3-50). The present invention also provides Dac g I and Poa p I protein 3s allergerls which are immlmologically cross-reactive with L ol p I. The term "peptide"

WO 94/21675 ~157 5~ ~ PCT/US94/02537 as used herein refers to any protein fragment of Lol p I that induces an immlln response. The terms "fragment" and "antigenic fragment" of a protein as used interch~ng~ably herein refer to an amino acid sequence having fewer amino acid residues than the entire native amino acid sequence of the protein from which the s fragment is derived, and that induces an immune response. The terms "isolated" and "purified" as used herein refer to peptides of the invention which are substantially free of cellular material or culture me-lillm when produced by recombinant DNA
techniques, or substantially free of chemical precursors or other chemicals whensynth~si7Pd chPmic~lly. Preferred peptides of the invention include peptides derived 10 from Lol p I which comprise at least one T cell epitope of the allergen, or a portion of such a peptide which includes at least one T cell epitope.
Peptides comprising at least two regions, each region comprising at least one T cell epitope Lol p I are also within the scope of the invention. Isolated peptides or regions of isolated peptides, each comprising at least two T cell epitopes of the Lol p 15 I protein allergen are particularly desirable for increased thelal~eulic effectiveness.
Peptides that are immunologically related (e.g., by antibody or T cell cross-reactivity) to peptides of the present invention, such as peptides derived from Dac g I and Poa p I, are also within the scope of the invention. Peptides immunologically related by antibody cross-reactivity are recogniæd by antibodies specific for a peptide of Lol p I.
20 Peptides immunologically related to a given peptide by T cell cross-reactivity are capable of also reacting with the same T cells that react with that given peptide.
Isolated protein and peptides of the invention can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid having a sequence encoding such peptide. The isolated peptides of the invention can also be produced by 25 chemical synthesis. When a protein or peptide is produced by recombinant techniques, host cells transformed with a nucleic acid having a sequence encoding a peptide of the invention or the functional equivalent of the nucleic acid sequence are cultured in a me~ lm suitable for the cells. Peptides can be purified from cell culture mPrlillm, host cells, or both, using techniques known in the art for purifying peptides 30 and proteins including ion-exchange chromatography, gel filtration chromatography, ~ filtration, electrophoresis or immunopurification with antibodies specific for the peptide, the protein allergen from which the peptide is derived, or a portion thereof.
The present invention provides expression vectors and host cells transformed to express the nucleic acid sequences of the invention. Nucleic acids coding for Lol p I
3s peptides of the invention, or at least a portion thereof, may be expressed in bacterial WO 94/21675 ~ PCT/US94102537 cells such as ~ , insect cells, yeast, or m~mm~ n cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers, and other expression control elements may be found in Sambrook et al. Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold s Spring Harbor, New York, 1989. Other suitable expression vectors, promoters, enh~nrers, and other expression elements are known to those skilled in the ar~
Expression in m~mm~ n, yeast or insect cells leads to partial or complete glycosylation of the recombinant material and formation of any inter- or intra-chain disulfide bonds. Suitable vectors for expression in yeast include YepSec 1 (Baldari et al. (1987) Embo J., 6: 229-234); pMFa (Kurjan and Herskowitz (1982) Cell, 30: 933-943); JRY88 (Schultz et al. (1987) Gene, 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). These vectors are freely available. Baculovirus andm~mm~ n expression systems are also available. For example, a baculovirus systemis commercially available (PharMingen, San Diego, CA) for expression in insect cells while the pMSG vector is commercially available (Pharmacia, Piscataway, NJ) for ,ssion in m~mm~ n cells.
For expression in E. coli, suitable expression vectors include, among others, pTRC (Amann et al. (1988) Gene, 69: 301-315); pGEX (Amrad Corp., Melbourne, Australia); pMAL (N.E. Biolabs, Beverly, MA); pRIT5 (Pha~macia, Piscataway, NJ);pET-l ld (Novagen, Madison, WI) Ja~meel et al., (1990) J. Virol., 64:3963-3966; and pSEM (Knapp et al. (1990) BioTechniques, 8: 280-281). The use of pTRC, and pET-1 ld, for example, will lead to the expression of unfused protein. The use of pMAL, pRIT5 pSEM and pGEX will lead to the expression of allergen fused to maltose E
binding protein (pMAL), protein A (pRIT5), truncated ~3-galactosidase (PSEM), orglutathione S-tr~n~fer~e (pGEX). When a Lol p I peptide of the invention, is expressed as a fusion protein, it is particularly advantageous to introduce an enzymatic cleavage site at the fusion junction between the carrier protein and the Lol p I peptide.
The Lol p I peptide may then be recovered from the fusion protein through enzymatic cleavage at the enzymatic site and biochemical purification using convçntion~l techniques for purification of proteins and peptides. Suitable enzymatic cleavage sites include those for blood clotting Factor Xa or tnrombin for which the appropliateenzymes and protocols for cleavage are commercially available from, for example,Sigma Chemical Company, St. Louis, MO and N.E. Biolabs, Beverly, MA. The di~rerenl vectors a'lso have different promoter regions allowing con~.lilutive or inducible expression witn, for exarnple, IPTG induction (PRTC, Amann et al., (1988) WO 94/21675 ~ 9 ~ PCT/US94/02537 supra; pET-l ld, Novagen, Madison, WI) or temperature induction (pRIT5, Pharmacia, Piscataway, NJ) . It may also be apprup.iate to express recombin~nt Lol p I peptides in dirrelent E. coli hosts that have an altered capacity to degrade recombinantly expressed proteins (e.g., U.S. Patent 4,758,512). ~ltern~tively, it may 5 be advantageous to alter the nucleic acid sequence to use codons pl~çerelllially utili_ed by E. coli, where such nucleic acid alteration would not affect the amino acid sequence of the expressed protein.
Hûst cells can be transformed to express the nucleic acid sequences of the invention using conventional techniques such as calcium phosphate or calcium chloride 10 co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming the host cells may be found in Sambrook et al. supra, and other laboratory textbooks. The nucleic acid sequences of the invention may also be chemically synth~ci7Pd using standard techniques (i.e., solid phase synthesis). Details of the cloning of Lol p I are given in the Examples.
Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene, 69:301-315) and pET1 ld (Studier et al., Gene Expression Technology:
Methods in Enzymology, Academic Press, San Diego, California (1990), 185:60-89).While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted intopETl ld relies on transcription from the T ,' gnlO-lac 0 fusion promoter m~ tP~l by coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ~ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
One strategy to m~imi7~ recombinant Lol p I peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesm~n, S., Gene Expression Technology:
Methods in Enzymology, Academic Press, San Diego, California (1990), 185:119-128). Another strategy would be to alter the nucleic acid sequence of the desired gene to be inserted into an expression vector so that the individual codons for each amino acid would be those ple~ ially utilized in highly expressed E. coli proteins (Wada et al, ( 1992) Nuc. Acids Resl 20:2111-2118). Such alteration of nucleic acid r sequences of the invention could be carried out by standard DNA synthesis techniques.
The nucleic acids of the invention can also be chernic~lly synthesi7P.d using standard techniques. Various methods of chemically synth~.~i7ing ,2~ 6 polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully ~l~tom~tPd in commercially available DNA synth~ci7Prc (See e.g., Itakura et al. U.S. Patent 4,598,049; Caruthers et al. U.S. Patent 4,458,066; and Itakura U.S. Patents 4,401,796 and 4,373,071, incorporated by reference herein).The present invention also provides fr~gment.c of nucleic acid sequences encoding peptides of the invention. As used herein, the term "fr~gment" of a nucleic acid sequence refers to a nucleotide sequence having fewer bases than the nucleotide sequence coding for the entire amino acid sequence of the protein. Nucleic acid sequences used in any embodiment of this invention can be cDNA obtained as lo described herein, or alternatively, can be any oligodeoxynucleotide sequence having all or a portion of a sequence represented herein, or their functional equivalents. Such oligodeoxynucleotide sequences can be produced ch~.mic~lly or mech~nic~lly, using known techniques. A functional equivalent of an oligonucleotide sequence of Lolp I
is one which is 1) a sequence capable of hybridi_ing to a compl~ment~ry oligonucleotide to which the sequence (or corresponding sequence portions) of Lol p I
as shown in Fig. 1 (SEQ ID NO: 1) or fragments thereof hybridizes, or 2) the sequence (or corresponding sequence portion) complementary to the sequence of L ol p I as shown in Fig. 1 (SEQ ID NO: 1), and/or 3) a sequence which encodes a product (e.g., a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence portion) of Lol p I as shown in Fig. 1 (SEQ ID NO: 1). Whether a functional equivalent must meet one orboth criteria will depend on its use (e.g., if it is to be used only as an oligonucleotide probe, it need meet only the first or second criteria and if it is to be used to produce a Lol p I peptide of the invention, it need only meet the third criterion).
Preferred nucleic acids encode a peptide having at least about 50% homology to a Lol p I peptide of the invention, more preferably at least about 60~o homology and most preferably at least about 70% homology with a Lol p I peptide of the invention.
Nucleic acids that encode peptides having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with Lol p I peptides of the invention are also within the scope of the invention. Homology refers to sequence .cimil~rity between two peptides of Lol p I, or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the comparedsequence is occupied by the same nucleotide or amino acid, then molecules are WO 94/21675 ~ 1 ~i 7 5 !~ 6 PCT/US94/02~37 homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
~ c;re.l~,d nucleic acid fr~gment~ encode peptides of at least 7 amino acid re~i~lues in length, and preferably 13-40 amino acid residues in length, and more 5 preferably at least 16-30 amino acids residues in length, Nucleic acid fragments encoding peptides of at least 30 amino acid residues in length, at least 40 amino acid residues in length, at least about 80 amino acid residues in length, at least about 100 amino acid residues in length or more, are also contemplated.
Also within the scope of the invention are nucleic acid sequences encoding 0 allergens immnnologica'lly cross-reactive with Lol p I, such as full length Dac g I and Poa p I proteins or peptides (Figs 5 (SEQ ID NO: 52), 6 (SEQ ID NO: 54), and 9 (SEQ ID NO: 23, 25, 27, 30, 61-70)). Proteins and peptides of Dac g I and Poa p I
may be produced recombinantly as discussed above, or synthetically. Expression vectors and host cells transformed to express Dac g I and Poa p I proteins or peptides 15 thereof are also within the scope of the invention. Details of the cloning of Dac g I
and Poa p I are given in the examples.
The present invention also provides a method of producing isolated Lol p I
peptides of the invention or a portion thereof, comprising the steps of cult~lring a host cell l,~ sr~ ed with a nucleic acid sequence encoding a Lol p I peptide of the 20 invention in an a~plopliate medium to produce a mixture of cells and me~i~lm c~ ,.;";.,~ said Lol p I peptide; and purifying the mi~ture to produce ~Ubst~nti~lly pure Lol p I peptide. Host cells transformed with an expression vector cont~inin~ DNAcoding for a Lol p I peptide of the invention are cultured in a suitable medium for the host cell. Lol p I peptides of the invention can be purified from cell culture me(~ m, 25 host cells, or both using techniques known in the art for purifying peptides and proteins inc1~l-1ing ion-exchange chromatography, gel f~tration chromatography, llltr~filtr~tion, electrophoresis and immunopurification with antibodies specific for the Lol p I peptides or portions thereof.
Another aspect of the present invention pertains to an antibody specifically 30 reactive with a Lol p I peptide. Such antibodies may be used to standardiæ allergen extracts or to isolate the naturally occurring Lol p I . Also, Lol p I peptides of the invention can be used as "purified" allergens to standardize allergen extracts. For example, an animal such as a mouse or rabbit can be immuniæd with an immunogenicform of an isolated Lol p I peptide of the invention capable of eliciting an antibody 35 response. Techniques for conferring immunogenicity on a peptide include conjugation wo 94/21675 PCT/US94/02537 ~1~75~
to carriers or other techniques well-known in the art. The Lol p I peptide can be ~(lministered in the presence of adjuvant. The progress of immuni7~tit)n can be monitored by detection of antibody titers in plasma or serum standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of 5 antibodies.
Following immllni7~tion~ anti-Lol p I peptide antisera can be obtained and, if desired, polyclonal anti-Lol p I peptide antibodies from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immuniæd animal and fused by standard somatic cell fusion procedures with 10 immortali_ing cells such as myeloma cells to yield hybridoma cells. Hybridoma cells can be screened immunoch~.mic~lly for production of antibodies reactive with the Lol p I peptides of the invention. These sera or monoclonal antibodies can be used to standardi_e allergen extracts.
Through use of the peptides and antibodies of the present invention, lS preparations of consistent, well-defined composition and uniform biological activity can be made. Compositions having therapeutic activity may be ~minictered for therapeutic purposes (e.g., to modify the allergic response of a ryegrass sensitive individual to pollen of such grasses or pollen of an immunologically related grass such as Dac g I, Poa p I and Phl p r). ~lmini~tr~tion of such peptides may, for e~ample, 20 modify B-cell response to Lol p I allergen, T-cell response to Lol p I allergen or both responses. Isolated peptides can also be used to study the mechanism of immllnotherapy of ryegrass pollen allergy and to design modified derivatives or analogues useful in immunotherapy. Compositions according to the invention will have utility in diagnosis of ryegrass sensitivity or sensi~ y to grass allergens cross-2s reactive to ryegrass allergens, because the components include T cell epitopesrecognizing the allergens.
The present invention also pertains to T cell clones which specifically recognize Lol p I peptides of the i lvention. These T cell clones may be suitable for isolation and molecular cloning of the gene for t'ne T ce'll receptor which is specifically reactive with 30 a peptide of the present invention. The T cell clones may be produced as described in Example 4, or as described in Cellular Molecular Immunology, Abdul K. Abbas et al., W.B. S~-ln~lers Co. (1991) pg. 139. The present invention also pertains to soluble T
cell receptors. These receptors may inhibit antigen-dependent activation of the relevant T cell subpopulation within an individual sensitive to Lol p I. Antibodies 35 specifica'lly reactive with such a T cell receptor can also be produced according to the WO 94/21675 ~15 7 5 9 6 PCT/US94/02537 techniques described herein. Such antibodies may also be useful to block T-cell-MHC
interaction in an individual. Methods for producing soluble T cell receptors aredescribed in Immunology: A Synthesis, 2nd Ed., Edward S. Golub et al., Sinaur Assoc., Sun-lerl~nd Massachusetts, (1991) pp. 366-369.
It is also possible to modify the structure of a peptide of the invention to achieve additional advantageous physical or biological properties such as increasing solubility, çnh~ncing therapeulic or preventive efficacy, increasing stability (e.g., shelf life ex vivo or resistance to proteolytic degradation in vivo), decreasing adverse side effects, and the like. A modified peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, in order to modify immunogenicity and/or to reduce allergenicity. Peptides may also be advantageously modified by addition or conjugation with another peptide or othercomponent.
For example, a peptide can be modified so that it m~int~in~ the ability to induce T cell anergy and to bind MHC proteins but reduces the ability to induce a strong prolirt;l~live response, or possibly any proliferative response, when ~Amini~t.ored in immunogenic form. In this in~t~nce~ critical binding residues for the T cell receplor can be determin~d using k~own techniques (e.g., substitution of each residue andde~,rmin~tion of the presence or absence of T cell reactivity). Those residues shown to be e~enti~l to interact with the T cell receptor can be modified by replacing the ec~çnti~l amino acid with another preferably similar amino acid residue (a "conservative substitution") whose presence is shown to enh~nce, ~iminish but not elimin~te, or not affect T cell reactivity. In addition, those amino acid residues that are not ecsenti~l for T cell receptor interaction can be modified by replacement with 2s another amino acid whose incorporation may enhance, flimini~h or not affect T cell reactivity but does not elimin~tlo binding to relevant MHC.
Additionally, peptides of the invention can be modified by replacing an amino acid shown to be ecsenti~l to interact with the MHC protein complex with another.
preferably similar amino acid residue (conse. v~ive substitution) whose presence is shown to enh~nre, rlimini$h but not elimin~tç or not affect T cell reactivity. In addition, amino acid residues that are not essential for interaction with the MHC
protein complex but that still bind the MHC protein complex can be modified by repl~cement with another amino acid whose incorporation may enhance, not affect, or limini~h but not elimin~te T cell reactivity. ~r~fel.Gd amino acid substitutions for non-WO 94/21675 ' 2 15 ~ 5 ~ g PCTIUS94/02537 essential amino acids include, but are not limited to substitutions with alanine, glllt~mi~
acid, or a methyl amino acid.
In order to enhance stability and/or reactivity, peptides of the invention can also be modified to incorporate one or more polymorphisms in the amino acid se(luel-re of the protein allergen resulting from natural allelic variation. Additionally, D-amino acids, non-natural amino acids or non-amino acid analogues can be substituted or added to produce a modified peptide within the scope of this invention.
Furthermore, peptides of the present invention can be modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers (Wie et al., supra) to produce a lo protein or peptide conjugated with PEG. In addition, PEG can be added duringch,~mi~l synthesis of a protein or peptide of the invention. l~o,~lific~tions of peptides or portions thereof can also include reduction/ alyklation (Tarr in: Methods of Protein Microcharacterization, J.E. Silver ed. ~llm~n~ Press, Clifton, NJ, pp 155-194 (1986));
acylation (Tarr, supra); chemical coupling to an appLopliate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA
(1980); U.S. Patent 4,939,239; or mild formalin treatment (Marsh International Archives of Allergy and Applied lmmunology, 41: 199-215 (1971)).
To f~t~ilit~te p~lrific~tion and potentially increase solubility of peptides of the invention, it is possible to add reporter group(s) to the peptide backbone. For e~mple, poly-hi~titline. can be added to a peptide to purify the peptide by immobilized metal ion affinity chromatography (Hochuli, E. et al., Bio~rechnology, 6:1321-1325 (1988)). In addition, specific endoprotease cleavage sites can be introduced, ifdesired, between a reporter group and amino acid sequences of a peptide to facilitate isolation of peptides free of irrelevant sequences. In order to successfully desensitize 2s an individual to a protein antigen, it may be necess~ry to increase the solubility of a peptide by adding functional groups to the peptide or by not including hydrophobic T
cell epitopes or regions cont~ining hydrophobic epitopes in the peptides or hydrophobic regions of the protein or peptide. Functional groups such as chargedamino acid pairs (e.g., KK or RR) are particularly useful for increasing the solubility of a peptide when added to the amino or carboxy terminus of the peptide. Examples of modifications to peptides to increase solubility include modifications to peptide LPI-16.1 (SEQ ID NO: 23) (Fig. 2), such modified peptides include: LPI-16.2 (SEQID NO: 31), LPl-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO 33), LPI-16.5 (SEQ
ID NO: 34), LPI-16.6 (SEQ ID N0: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ ID NO: 38), all as shown in Fig. 4.

WO 94/21675 '2,~ 9 6 PCT/US94/02537 To potentially aid proper antigen proce~cing of T cell epitopes within a peptide, canonical plolease sensitive sites can be recombinantly or synthetically engineered between regions, each comprising at least one T cell epitope. For example, charged amino acid pairs, such as KK or RR, can be introduced between regions within a s peptide during recombinant construction of the peptide or added to the amino or carboxy termimls of a synthetically produced peptide. The resulting peptide can be rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generalc;
portions of the peptide cont~ining one or more T cell epitopes. In addition, as mentioned above, such charged amino acid residues can result in an increase in solubility of a peptide.
Site-directed mut~genç~i~ of DNA encoding a peptide of the invention can be used to modify the structure of the peptide by methods known in the art. Such methods may, among others, include PCR with degenerate oligonucleotides (Ho et al., Gene, 77:51-59 (1989)) or total synthesis of mutated genes (Hostomsky, Z. et al., Biochem. Biophys, Res. Comrn, 161:1056-1063 (1989)). To enhance bacterial expression, the aforementioned methods can be used in conj~nction with other procedures to change the eucaryotic codons in DNA constructs encoding protein orpeptides of the invention to ones pltfel~ tially processed in E. coli, yeast, m~mm~ n cells, or other prokaryotic or eukaryotic host cells.
Peptides of the present invention can also be used for detecting and diagnosing ryegrass pollinosis. For example, this could be done in vitro by combining blood or blood products obtained from an individual to be assessed for sensitivity to ryegrass pollen or another cross-reactive pollen such as Dac g I, Poa p I and Phl p I, with an isolated peptide(s) of Lolp I, under conditions apprupliate for binding of 2s components in the blood (e.g., antibodies, T-cells, B cells) with the peptide(s) and (lele~ ,i"g the extent to which such binding occurs. Other diagnostic methods for allergic diseases in whiçh the protein, peptides or antibodies of the present invention will be useful include radio-allergergosorbent test (RAST), paper radioimmunosorbent test (PRIST), enzyme linked immunosorbent assay (ELISA), r~(lioimmunoassays (RIA), immuno-radiometric assays (IRMA), luminescence immunoassays (LIA), hist~mine release assays and IgE immunoblots.
The presence in individuals of IgE specific for at least one protein allergen and the ability of T cells of the individuals to respond to T cell epitope(s) of the protein allergen can be determined by ~(lmini~tering to the individuals an Tmmerli~te 3s Type Hypersensitivity test and a Delayed Type Hy~ersellsitiity test. The individuals WO 94/21675 2, ~ i 9 ~ PCT/US94/02537 are ~mini~te.red an Immediate Type Hypersensitivity test (see e.g., Immunolog~
(1985) Roitt, I.M., Brostoff, J., Male, D.K. (eds), C.V. Mosby Co., Gower Medical Publishing, London, NY, pp. 19.2-19.18; pp. 22.1-22.10) utili7ing the protein allergen or a portion thereof, or a modified form of the protein allergen or a portion s thereof, each of which binds IgE specific for the allergen. The same individuals are ~rlmini~tered a Delayed Type Hypersensitivity test prior to, cimult~n.o.ously with, or subsequent to ~rimini~tration of the Immediate Type Hypersensitivity test. Of course, if the Tmmetli~te. Type Hypersensitivity test is ~1mini~tered prior to the Delayed Type Hypersensitivity test, the Delayed Type Hypersensitivity test would be given to those 0 individuals exhibiting a specific Immediate Type Hypersensitivity reaction. The Delayed Type Hypersensitivity test utili7es a modified form of the protein allergen or a portion thereof, the protein allergen produced recombinantly, or a peptide derived from the protein allergen, each of which has human T cell stim~ ting activity and each of which does not bind IgE specific for the allergen in a substantial percentage 15 of the population of individuals sensitive to the allergen (e.g., at least about 75%).
Those individuals found to have both a specific Immediate Type Hypersensitivity reaction and a specific Delayed Type Hypersensitivity reaction may be treated with a LLe,dpc;ulic composition comprising the same modified form of the protein or portion thereof, the recombinantly produced protein allergen, or the peptide, each as used in 20 the Delayed Type Hypersensitivity test.
Isolated peptides of the invention, when ~(iminictered in a thela~uLic regimen to a Lol p I-sensitive individual (or an individual allergic to an allergen cross-reactive with ryegrass pollen allergen such as Dac g I, Poa p I and Phl p I) are capable of modifying the allergic response of the individual to Lol p I ryegrass pollen allergen (or 25 such cross-reactive allergen). Preferably peptides of this invention are capable of modifying the B-cell response, T-cell response or both the B-cell and the T-cellresponse of the individu~l to the allergen. As used herein, modification of the allergic response of an individual sensitive to a ryegrass pollen allergen or cross-reactive allergen can be defined as n~n-responsiveness or diminution in symptoms to the 30 allergen, as determined by standard clinical procedures (~ee, e.g., Varney et al, British Medical Journal, 302:265-269 (1990)) including rlimimltion in ryegrass pollen-induced ~thm~tic symptoms. As referred to herein, a ~liminution in symptoms includes any reduction in allergic response of an individual to the allergen after the individual has completed a treatment regimen with a peptide or protein of the 35 invention. This ciiminlltion may be subjective (i.e., the patient feels more comfortable WO 94/21675 ~ ~ r ~ 5 PCT/US94/02537 in the presence of the allergen), or rlimimltiQn in symptoms may be determined clinically, using standard skin tests known in the art and discussed above.
Lol p I peptides of the present invention having T cell stimulating activity, and thus comprising at least one T cell epitope, are particularly pl~rell~d. In referring to 5 an epitope, the epitope will be the basic element or smallest unit of recognition by a receptor, particularly immunoglobulins, histocomp~tiki'lity ~nhgenc and T cell receptors where the epitope comprises amino acids essential to receptor recognition.
Amino acid sequences which mimic those of the epitopes and which are capable of down-regulating or reducing allergic response to Lol p I can also be used. T cell 10 epitopes are believed to be involved in initiation and perpetuation of the immnn~
response to a protein allergen that is responsible for the clinical symptoms of allergy.
Such T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropliate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation. These events lead to T cell 15 proliferation, lymphokine secretion, local infl~mm~tory reactions, recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies. One isotype of these antibodies, IgE, is fi-n~l~mt~nt~lly important to the development of allergic symptoms, and its production is inflllenced early in the c~c~de of events, at the level of the T helper cell, by the nature of the 20 lymphokines secreted.
Exposure of ryegrass pollen-sensitive patients or patients sensitive to an immllnogically cross-reactive protein allergen such as Dac g I, Poa p I and Phl p I, to isolated Lol p I peptides of the present invention which comprise at least one T cell epitope and are derived from Lol p I protein allergen, may toleriæ or anergize 2s appl-~pliate T cell subpopulations such that they become unresponsive to the protein allergen and do not participate in stim~ ting an immune response upon such exposure.
In addition, ~qrlminictration of a peptide of the invention or portion thereof which comprises at least one T cell epitope may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring L ol p I protein allergen or portion 30 thereof (e.g., may result in a decrease of IL-4 and/or an increase in IL-2).
Furthermore, exposure to such peptide of the invention may influence T cell subpopulations which normally participate in the response to the naturally occurring allergen such that these T cells are drawn away from the site(s) of normal exposure to the allergen (e.g., nasal mucosa, skin, and lung) towards tne site(s) of thel~eulic 35 ~ mini~tration of the fragment or protein allergen. This redistribution of T cell WO 94/21675 21 ~i 7 ~ 9~ PCT/US94/02537 subpopulations can have the effect of ameliorating or reducing the ability of anindividual's immlme system to stim~ ts the usual immune response at the site of normal exposure to the allergen, resulting in a dimunution in allergic symptoms.The isolated Lol p I peptides of the invention can be used in methods of S diagnosing, treating or preventing allergic reactions to Lol p I allergen or an imm~mQgically related protein allergen such as Dac g I, Poa p I and Phl p I. Thus, the present invention provides compositions useful in allergery diagnosis and/or useful in allergy therapy comprising isolated Lol p I peptides or portions thereof. Such compositions will typically also comprise a ph~rm~ceutically acceptable carrier or 10 diluent when inten~led for in vivo a-lminictration. Therapeutic compositions of the invention may include synthPtic~lly prepared Lol p I peptides.
Administration of the therapeutic compositions of the present invention to an individual to be desen~iti7~d can be carried out using known techniques. Lol p Ipeptides or portions thereof may be ~lministered to an individual in combination with, 15 for exarnple, an app.opliate diluent, a carrier and/or an adjuvant. Ph~rm~ceutically acceptable diluents include saline and aqueous buffer solutions. Ph~rm~,ceutic~lly acceptable carriers include polyethylene glycol (Wie et al. (1981) Int. Arch. Allergy Appl. Immunol., 64:84-99) and liposomes (Strejan et al. (1984) J. Neuroimmunol.,7:27). For purposes of inducing T cell anergy, the therapeutic composition is 20 preferably ~rlminictered in nonimmunogenic form, i.e., it does not contain adjuvant.
The therapeutic compositions of the invention are a lmini~t~ored to ryegrass pollen-sensitive individuals or individuals sensitive to an allergen which is immllnolQgically cross-reactive with ryegrass pollen allergen (i.e., Dactylis glomerata, or Sorghum halepensis, etc.). Therapeutic compositions of the invention may also be used in the 25 m~mlf~cture of meAic~mentc for treating sensitivity to ryegrass pollen allergen or an immunologically related pollen allergen.
Administration of the therapeutic compositions of the present invention to an individual to be desen~iti7~cl can be car-ried out using known procedures at clos~gt~.s and for periods of time effective to reduce sensitivity (i.e., to reduce the allergic 30 response) of the individual to the allergen. Effective amounts of the theLdpeu~ic compositions will vary according to factors such as the degree of sensitivity of the individual to ryegrass pollen, the age, sex, and weight of the individual, and the ability of the protein or fragment thereof to elicit an antigenic response in the individual.
The active compound ~i.e., protein or fragment thereof) may be ~(lminictered 35 in any convenient manner such as by injection (subcutaneous, intravenous, etc.), oral ~ ~ 3~5~
WO 94/21675 ~ PCT/US94/02537 ~ lminictration, inhalation, tr~ncderm~l application, or rectal ~clminictr~tion.Depen-1ing on the route of ~lmini~stration~ the active compound may be coated within a m~t~ri~l to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
s For example, preferably about 1 ,ug- 3 mg and more preferably from about 20-750 llg of active compound (i.e., protein or fragment thereof) per dosage unit may be ~lministered by injection. Dosage regimen may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may be ~(lminist~red daily or the dose may be proportionally reduced as in~ ted by the exigencies of the therapeutic situation.
To ~(lminicter a peptide by other than parenteral ~(lminictration, it may be n-ocecc~ry to coat the protein with, or co-~clminicter the protein with, a material to prevent its inactivation. For example, the peptide or portion thereof may be co-~lminictered with enzyme inhibitors or in liposomes. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
Liposomes include water-in-oil-in-water CGF emulsions as well as convention~l liposomes (Strejan et al., (1984), J. Neuroimununol., 7:27).
The active compound may also be ~clminict~red p~cn~l~lly or al)eliLulleally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these p.cp~dtions may contain a presel vaLivt; to prevent the growth of mlcroorg~nicmc.
Ph~rm~eutical compositions suitable for injection include sterile aqueous solutions (where the peptides are water soluble) or dispersions and sterile powders for 2s the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition intended for in vivo use must be sterile and must be fluid to the extent n~cess~ry to provide easy syringability. It should preferably be stable under the conditions of manufacture and storage and be preserved against the cont~min~tingaction of microorg~nicm~ such as bacteria and fungi. The carrier can be a solvent or dispersion medium cont~ining, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be m~int~in~ for example, by the use of a coating such as lecithin, by the m~int~.n~nce of the required particle size in ~e case of dispersion, and by ~e use of surfactants. Prevention of the action of microorg~nicmc can be achieved by various antibacterial and antifungal ,~ ~,l57S9~ --agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thirmerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol and sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought 5 about, including in the composition, an agent which delays absorption, for example, al~ l..., monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (i.e., protein or peptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by 10 filtered sterili~tion. Generally, dispersions are p-Gpared by incorporating the active compound into a sterile vehicle which contains a basic dispersion m~ m and the required other ingredients from those enumerated above. In the case of st~,rile powders for the preparation of sterile indectable solutions, the plcreL.Gd methods of plGpardLLon are vacuum drying and freeze-drying which yields a powder of the active 15 ingredient (i.e., protein or peptide) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When a peptide of the invention is suitably protected, a~s described above, the peptide may be orally ~timini~tered, for example, with an inert diluent or an ~imil~hle edible carrier. The peptide and other ingredients may also be enclosed in a hard or soft 20 gelatin capsule, compressed into tablets, or incorporated directly into the individual's food. For oral therapeutic ~llministration, the active compound may be form~ tedwith conventional excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspension~, syrups, wafers, and the like. Such compositions and p-cpaL~LLLons should contain at least 1% by weight of active compound. The 25 pelcenldge of the composition and p,cpaldLLons may, of course, be varied and may conveniently be between about 5 to 80% by weight of the dosage unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or plcparaLions according to thepresent invention are prepared so that an oral dosage unit contains from about 10 ~Lg 30 to about 200 mg of active compound.
The tablet~s, troches, pills, capsules and the like may also contain the following:
a binder such as gum gr~g~c~nth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as m~gn~.sium stearate; and a sweetening agent such 35 as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of WO 94/21675 21~ 6 PCT/US94/02537 w~lLe~lc;cn, or cherry flavoring. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar 5 or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and p~pylpalabens as presw ~a~ivc, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit formshould be ph~rm~ceutically pure and substantially non-toxic in the amounts employed.
In addition, the active compound may be incorporated into s~lst~ined-release 10 plcpala~ions and formulations.
As used herein "ph~rrn~ceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for ph~rm~ceutically active substances is well known in the art. Except insofar as any 15 conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.
Various isolated peptides of the invention derived from ryegrass pollen protein Lol p I are shown in Figs. 2 and 4 (SEQ ID NO: 3-50) . Peptides comprising at least 20 two regions, each region comprising at least one T cell epitope of Lol p I are also within the scope of the invention. As used herein a region may include the amino acid sequence of a peptide of the invention as shown in Figs. 2 and 4 (SEQ ID NO: 3-50) or the amino acid sequence of a portion of such peptide.
To obtain isolated peptides of the present invention, L ol p I is divided into non-25 overlapping peptides of desired length or overlapping peptides of desired lengths asdiccuccecl in Example 4 which can be produced recombinantly, or synthetically.
Peptides comprising at least one T cell epitope are capable of eliciting a T cell response, such as T cell proliferation or lymphokine secretion, and/or are capable of inducing T cell anergy (i.e., toleri7~tit n). To determine peptides comprising at least 30 one T cell epitope, isolated peptides are tested by, for example, T cell biology techniques, to determine whether the peptides elicit a T cell response or induce T cell anergy. Those peptides found to elicit a T cell response or to induce T cell anergy are defined as having T cell stim~ ting activity.
As &scussed in Example 4, human T cell stimulating activity can be tested by 35 c~ rin~ T cells obtained from an individual sensitive to Lolp I allergen, (i.e., an WO 94/21675 ,~ 'ig~' PCT/US94/02537 individual who has an IgE-mt~ t.o.d immune response to Lol p I allergen) with a peptide derived from the allergen, then determining whether proliferation of T cells occurs in response to the peptide. T cell proliferation may be measured in several ways, e.g., by cellular uptake of tritiated thymidine. Stimulation indices for responses s by T cells to peptides can be calculated as the m~imllm counts-per-minute (CPM) in response to a peptide divided by the control CPM. A stimulation index (S.I.) equal to or greater than two times the background level is considered "positive". Positive results are used to calculate the mean stim~ tion index for each peptide for the group of patients tested. Plc;fellt;d peptides of this invention comprise at least one T cell 0 epitope and have a mean T cell stim~ tion index of greater than or equal to 2Ø A
peptide having a mean T cell stimulation index of greater than or equal to 2.0 in a significant number of ryegrass pollen sensitive patients tested (i.e., at least 10% of patients tested) is considered useful as a thelap~ulic agent. Ple~e.l~,d peptides have a mean T cell stimulation index of at least 2.5, more preferably at least 3.0, more preferably at least 3.5, more preferably at least 4.0, more preferably at least 5, and most preferably at least about 6. For example, peptides of the invention having a mean T cell stim~ tion index of at least 5, as shown in Fig. 3, include LPI-2 (SEQ ID NO:
5), LPI-3 (SEQ ID NO: 6), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-17 (SEQ ID NO: 24), LPI-l9 (SEQ ID NO: 26), LPI-20 (SEQID NO: 27), LPI-22 (SEQID NO: 29) and LPI-23 (SEQ ID NO: 30).
For example, peptides of the invention having a mean T cell stimulation index of at least 6, as shown in Fig. 3, include LPI-2 (SEQ ID NO: 5), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: 30).
In addition, plGfell~,d peptides have a positivity index (P.I.) of at least about 100, more preferably at least about 200 and most preferably at least about 300. The positivity index for a peptide is detPrrnined by multiplying the mean T cell stimulation index by the percent of individuals, in a population of individuals sensitive to ryegrass pollen (e.g., preferably at least 15 individuals, more preferably at least 30 individuals or more), who have a T cell stimulation index to such peptide of at least 2Ø Thus, the positivity index represents both the strength of a T cell response to a peptide (S.I.) and the frequency of a T cell response to a peptide in a population of individuals sensitive to ryegrass pollen. For example, as shown in Fig. 3, Lol p I peptide LPI- 15 (SEQ ID
NO: 21) has a mean S.I. of 12.2 and 11% of positive responses in the group of individuals tested resulting in a positivity index of 134.2. Lol p I peptides having a WO 94/21675 21~ ~59~ PCT/US94/02537 positivity index of at least about 100 and a mean T cell stimulation index of at least about 4 include: LPI-2 (SEQ ID NO: 5), LPI-ll (SEQ ID NO: 15), LPI-13 (SEQ ID
NO: 19), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID
NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID
NO: 29), and LPI-23 (SEQ ID NO: 30).
In order to clet~.rrnine. precise T cell epitopes by, for example, fine mapping techniques, a peptide having T cell stimulating activity and thus comprising at least one T cell epitope as dele. . .~ ~ by T cell biology techniques is modified by addition or ~le.l~.tion of amino acid residues at either the amino or carboxy terminus of the peptide lo and ~ested to determine a change in T cell reactivity to the modified peptide. If two or more peptides which share an area of overlap in the native protein sequence are found to have human T cell stimulating activity, as detP.rrnint cl by T cell biology techniques, additional peptides can be produced comprising all or a portion of such peptides and dhese additional peptides can be tested by a similar procedure. Following this technique, peptides are selected and produced recombinandy or synthetically.
Examples of fine map peptides are as follows: modified versions of peptide LPI-18 (SEQ ID NO: 25) (Fig. 2) include peptides: LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42) all as shown in Fig. 4; modifled versions of peptide LPI-20 (SEQ ID NO: 27) (Fig. 2) include peptides: LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), and LPI-20.6 (SEQ ID NO: 47) all as shown in Fig. 4; modified versions of peptide LPI-23 (SEQ ID NO: 30) (Fig. 2) include peptides: LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49) and LPI-23.4 (SEQ ID NO: ~0) all as shown in Fig. 4.
Peptides are selected for diagnostic or ~,eldp(,ulic uses based on various factors, including the strength of the T cell response to the peptide (e.g., stimulation index), the frequency of the T cell response to the peptide in a population of individuals sensitive to ryegrass pollen, and the potential cross-reactivity of the peptide with other allergens from other species of grasses as discussed earlier. The physical and chemical properties of these selected peptides (e.g., solubility, stability) are e~minP.d to determine whether the peptides are suitable for use in therapeutic compositions or whether the peptides require modification as described herein. The ability of the selecte-l peptides or selected modified peptides to stimulate human T cells (e.g., induce proliferation, lymphokine secretion) is determined.

WO 94121675 21~}75~6 PCT/US94/02537 The most prere,l~d T cell epitope-cont~ining peptides of the invention do not bind immunoglobulin E (IgE) of an allergic individual or bind IgE to a subst~nti~lly lesser extent (e.g., at least 100 fold less and more preferably, at least lO00 fold less) than the protein allergen from which the peptide is derived. The major complications s of standard immllnotherapy are IgE-me~ t~-l responses such as anaphylaxis.
Immunoglobulin E is a me(li~tor of anaphylactic reactions which result from the binding and cross-linking of antigen to IgE on mast cells or basophils and the consequent release of m~Ai~tors (e.g., hist~mine7 seiolo~ , eosinophil chemotacic factors). Anaphylaxis in a substantial percentage of a population of individualssensitive to Lol p I could be avoided by the use in immunotherapy of a peptide which do not bind IgE in a substantial percentage (e.g., at least about 75%) of a population of individuals sensitive to Lol p I allergen, or, if the peptides do bind IgE, such binding does not result in the release of m~ tors from mast cells or basophils. The risk of anaphylaxis could be reduced by the use in immunotherapy of a peptide or peptides lS which have reduced IgE binding. Moreover, peptides having minim~1 IgE stimul~ting activity are desirable for therapeutic effectiveness. Minimal IgE stim~ tin~ activity refers to IgE production that is less than the amount of IgE production stimulated by the native Lol p I protein allergen. Similarly, IL-4 production can be compared, with reduces IL-4 production inrlic~ting 1t~ssen~cl IgE stimlll~ting activity.
F~ere.l~,d T cell epitope-co"~i.,;"g peptides of the invention, when d to a ryegrass pollen-sensitive individual or an individual sensitive to an allergen which is immlmo1Ogically related to ryegrass pollen allergen (such as Dac g I, Poa p I, and Phl p I) in a therapeutic treatment regimen, are capable of modifying the allergic response of the individual to the allergen. Particularly, such pleft;ll~,d Lol p I
2s peptides of the invention comprising at least one T cell epitope of Lol p I or at least two regions derived from Lol p I, each comprising at least one T cell epitope, when ~tlmini~tered to an individual sensitive to ryegrass pollen are capable of modifying T
cell response of the individual to the allergen, and they will thus be useful astherapeutics in addressing sensitivity to grasses.
A pl~;fell~;d isolated Lol p I peptide of the invention or portion thereof comprises at least one T cell epitope of Lol p I and accordingly, the peptide comprises at least appro~im~t~ly seven amino acid re~itlues For purposes of therapeutic effectiveness, pl~;fell~d therapeutic compositions of the invention preferably comprise at least two T cell epitopes of L ol p I, and accordingly, the peptide comprises at least approxim~tely eight amino acid residues and preferably at least fifteen amino acid WO94/21675 ~7596 PCT/US94/02537 re~i~lues. Additionally, therapeutic compositions comprising prerGll~d isolated peptides of the invention most preferably comprise a sufficient p~ ;ent~ge of the T cell epitopes of the entire protein allergen so that a therapeutic regimen of aclmini~tration of the composition to an individual sensitive to ryegrass pollen results in T cells of the individual being toleri7P.rl to the protein allergen. Synthetically produced peptides of the invention comr~ri~in~ up to approxim~t~ly forty-five amino acid residues in length, and most preferably up to app-o~ ately thirty amino acid residues in length are particularly desirable, as increases in length may result in difficulty in peptide synthesis.
Peptides of the invention may also be produced recombinantly as described above, and peptides exceerling 45 amino acids will be more easily produced recombinantly.
Peptides derived from the Lol p I protein allergen which exhibit T cell stim~ tQry pl~pe-Lies and thus are believed to be useful therapeutics and/or intsrmeAi~te?~ in developing tolerizing peptides comprise all or a portion of the following peptides: LPI-1 (SEQ ID NO: 3), LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID
NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID NO: 8), LPI-5 (SEQ ID NO:9), LPI-6 (SEQ ID NO:10), LPI-7 (SEQ ID NO:ll), LPI-8 (SEQ ID NO: 12), LPI-9 (SEQ ID NO: 13), LPI-10 (SEQ ID NO: 14), LPI-l l (SEQ
ID NO: 15), LPI-12 (SEQ ID NO: 17), LPI-13 (SEQ ID NO:l9), LPI-14 (SEQ ID
NO: 20), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID
NO: 23), LPI-17 (SEQ ID NO: 24), LPI-18 (SEQ ID NO: 25), LPI-l9 (SEQ ID
NO: 26), LPI-20 (SEQ ID NO: 27), LPI-21 (SEQ ID NO: 28), LPI-22 (SEQ ID
NO: 29), and LPI-23 (SEQ ID NO: 30) (Fig. 2) wherein the portion of the peptide preferably has a mean T cell stim~ tion index equivalent to, or greater than the mean T cell stimulation index of the corresponding peptide from which it is derived, as shown in Fig. 3. Even more preferably peptides derived from the Lol p I protein allergen comprise all or a portion of the following peptides: LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID NO: 8), LPI-8 (SEQ ID NO: 12), LPI- 10 (SEQ ID NO: 14), LPI- 11 (SEQ
ID NO: 15), LPI-13 (SEQ ID NO:l9), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ rD NO: 25), LPI-l9 (SEQ ID
NO: 26), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29)and LPI-23 (SEQ ID
- NO: 30), as shown in Fig. 2. Additionally, even morG pl~GîellGd peptides derived from the Lol p I protein comprise the following peptides: LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-l l (SEQ ID NO: 15), LPI-15 (SEQ
ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID

W094/21675 ~Sl~ PCT~S94/02537 NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: 30), all as shown in Fig. 2. Additional ~-Grel-~,d pep~des believed to T cell stim~ l~fing activity comprise the following pep~des: LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQID NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ ID NO: 38), LPI-18.5 (SEQIDNO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQIDNO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID
NO: 50).
One embodiment of the present invention features a peptide or portion thereof of L ol p I which comprises at least one T cell epitope of ~e protein allergen and has a formula Xn-Y-Zm. According to the formula, Y is an amino acid sequence selected from ~e group consis~ng of LPI-l (SEQ ID NO: 3), LPI-l.l (SEQ ID NO: 4), LPI-2 (SEQ ID NO:5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID
NO: 8), LPI-S (SEQ ID NO: 9), LPI-6 (SEQ ID NO:10), LPI-7 (SEQ ID NO: 11), LPI-8 (SEQ ID NO: 12), LPI-9 (SEQ ID NO: 13), LPI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-12 (SEQ ID NO:17), LPI-13 (SEQ ID NO:l9), LPI-14 (SEQ
ID NO: 20), LPI-lS (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID
NO: 23), LPI-17 (SEQ ID NO: 24), LPI-18 (SEQ ID NO: 25), LPI-l9 (SEQ ID
NO: 26), LPI-20 (SEQ ID NO: 27), LPI-21 (SEQ ID NO: 28), LPI-22 (SEQID
NO: 29), LPI-23 (SEQ ID NO: 30), LPI-16.2 (SEQID NO: 31), LPI-16.3 (SEQ ID
NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID
NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ
ID NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ
ID NO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ
ID NO: 44), LPI-20.4 (SEQID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ
ID NO: 47), LPI-23.1 (SEQ ID ~0: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO: S0) and prefe`rably selected from the group consisting of LPI-l.l (SEQ
ID NO:4), LPI-2 (SEQIDNO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQID NO:7), LPI-4.1 (SEQ ID NO: 8), LPI-8 (SEQ ID NO: 12), LPI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-13 (SEQ ID NO: 19), LPI-lS (SEQ ID NO: 21), LPI-16 (SEQ
ID NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-l9 (SEQ ID
NO: 26), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), LPI-23 (SEQ ID
NO: 30), LPI-16.2 (SEQID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID

21~75~
NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID NO- 35), LPI-16.7 (SEQ ID
NO: 36), LPI-16.9 (SEQIDNO: 37), LPI-16.10 (SEQ ID NO: 38), LPI-18.5 (SEQ
ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ
ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ
ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ
ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQIDNO:50) and more preferably selected from the group consisting of LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-15 (SEQ
ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQID
lû NO: 27), LPI-22 (SEQ ID NO: 29), LPI-23 (SEQ ID NO: 30), LPI-16.2 (SEQ ID
NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID
NO: 34), LPI-16.6 (SEQIDNO: 35), LPI-16.7 (SEQIDNO: 36), LPI-16.9 (SEQID
NO: 37), LPI-16.10 (SEQ ID NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ
ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQID NO: 42), LPI-20.2 (SEQ
ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQIDNO: 45), LPI-20.5 (SEQ
ID NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ
ID NO: 49), and LPI-23.4 (SEQIDNO:50), and most preferably selected from the group cnnsi~ting of LPI-16.1 (SEQIDNO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO: 30), LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ ID NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQIDNO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO:50). In addition, Xn are amino acid residues contiguous to the amino tenninll~ of Y in the amino acid sequence of the protein allergen and Zm are amino acid residues contiguous to the carboxy terminus of Y in the arnino acid sequence of the protein allergen. In the formula, n is 0-30 and m is 0-30. Preferably, 3û the peptide or portion thereof has a mean T cell stim~ tion index equivalent to greater than the mean T cell stim~ ti~n index of Y as shown in Fig. 3. Preferably, aminoacids compricing the amino t~ mls of X and the carboxy terrninus of Z are selected from charged amino acids, i.e., arginine (R), lysine (K), histidine (H), pl~lt~mic acid (E) or aspartic acid (D); amino acids with reactive side chains, e.g., cysteine (C),asparagine (N) or glut~minP.(Q); or amino acids with sterically small side chains, e.g., WO9421675 ai~ g6 PCT/IJ594/02537 alanine (A) or glycine (G). Preferably n and m are 0-5; most preferably n + m is less than 10.
Another embodiment of the present invention provides peptides comprising at least two regions, each region comprising at least one T cell epitope of Lol p I and s accordingly each region comprises at least approximately seven amino acid resi(llles These peptides comprising at least two regions can comprise up to 100 or more amino acid residues but preferably comprise at least about 14, even more preferably at least about 20, and most preferably at least about 30 amino acid residues of the Lol p I
allergen. If desired, the amino acid sequences of the regions can be produced and 0 joined by a linker to increase sensitivity to processing by antigen-pr~senting cells.
Such linker can be any non-epitope amino acid sequence or other approp~iate linking or joining agent. To obtain preferred peptides comprising at least two regions, each comprising at least one T cell epitope, the regions are arranged in the same or a different configuration from a naturally-occurring configuration of the regions in the allergen. For example, the regions cont~ining T cell epitope(s) can be arranged in a noncontiguous configuration and can preferably be derived from the same protein allergen. Noncontiguous is defined as an arrangement of regions cont~ining T cell epitope(s) which is different than that of the native amino acid sequence of the protein allergen from which the regions are derived. Furthermore, the noncontiguous regions co"~ g T cell epitopes can be arranged in a nonsequential order (e.g., in an order dirrGlGn~ from the order of the amino acids of the native protein allergen from which the region co~ i"i,-~ T cell epitope(s) are derived in which amino acids are arranged from an amino terminus to a carboxy terminus). A peptide of the invention can comprise at least 15%, at least 30%, at least 50% or up to 100% of the T cell epitopes of L,ol p I.
The individual peptide regions can be produced and tested to determine which regions bind immlln~globulin E specific for Lol p I and which of such regions would cause the release of m~ tors (e.g., hi.~t~mine) from mast cells or basophils. Those peptide regions found to bind immunoglobulin E and to cause the release of me~ tors from mast cells or basophils in greater than approximately 10-15% of the allergic sera tested are preferably not included in the peptide regions arranged to form ~,GfellGd peptides of the invention.
Examples of preferred peptide regions which do not bind to IgE (data not shown) include: LPI-1 (SEQ ID N0: 3), LPI-l.l (SEQ ID NO: 4), LPI-2 (SEQ ID
NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4. l (SEQ ID NO: 8), WO 94/21675 ~ ~ 5 7 5 g 6 PCT/US94/02537 LPI-5 (SEQ ID NO:9), LPI-6 (SEQ ID NO: 10), LPI-7 (SEQ ID NO: 11), LPI-8 (SEQ ID NO: 12), LPI-9 (SEQ ID NO: 13), LPI-10 (SEQ IDNO:14), LPI-ll (SEQ
ID NO:15), LPI-12 (SEQ ID NO: 17), LPI-13 (SEQ ID NO:l9), LPI-14 (SEQ ID
NO: 20), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID
NO: 23), LPI-17 (SEQ ID NO: 24), LPI-18 (SEQ ID NO: 25), LPI-l9 (SEQ ID
NO: 26), LPI-20 (SEQ ID NO: 27), LPI-21 (SEQ ID NO: 28), LPI-22 (SEQ ID
NO: 29), LPI-23 (SEQ ID NO: 30), LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID
NO: 32), LPI-16.4 (SEQ IDNO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID
NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ
ID NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ
ID NO: 41), LPI-18.8 (SEQ IDNO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ
ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ
ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO: 50), ~e amino acid sequences of such regions being shown in Figs. 2 or 4, or portions of said regions comprising at least one T cell epitope.
Preferred peptides comprise various combinations of two or more of the above-discussed p,e~e..Gd regions, or a portion thereof. Preferred peptides compri.~ing a combination of two or more regions (each region having an amino acid sequence as shown in Fig. 2 or Fig. 4), include the following:
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ
ID NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), and LPI-ll (SEQ ID NO: 15);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), PLI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16 (SEQ ID
NO: 22);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16.1 (SEQ ID
NO: 23);
- LPI-10 (SEQ ID NO:14), LPI-ll (SEQ ID NO:15), LPI-15 (SEQ ID
NO: 21), and LPI-16.1 (SEQ ID NO: 23);

wo 94/21675 21~ ~ S ~ 6 PCT/US94/02537 LPI-10 (SEQ ID No:i4)l LPI-ll (SEQ ID NO:15), LPI-15 (SEQ ID
NO:21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), and LPI-20 (SEQ ID NO: 27);
LPI-10 (SEQ ID NO: 14), LPI-ll (SEQ ID NO: 15), LPI-15 (SEQ ID
NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ
ID NO: 27), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30);
LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID
NO: 25), and LPI-20 (SEQ ID NO: 27);
LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID
NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO:30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID
NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-18 (SEQ ID NO: 25) and LPI-20 (SEQ ID NO: 27);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-23 (SEQ ID
NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-16.1 (SEQ ID
NO: 23);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30) and LPI-16.1 (SEQ ID NO: 23);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23) and LPI-ll (SEQ ID NO:15);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23) and LPI-4.1 (SEQ ID NO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23), LPI-4.1 (SEQ ID NO: 8) and LPI-22 (SEQ ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23), LPI-ll (SEQ ID NO: 15) and LPI-4.1 (SEQ ID NO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23), LPI-ll (SEQ ID NO:15), LPI-4.1 (SEQ ID NO: 8) and LPI-22 (SEQ ID NO: 29);
LPI-18(SEQ ~ NO:25),LPI-20(SEQ ~ NO:27),LPI-22(SEQ
NO: 29), and LPI-23 (SEQ ID NO: 30);

wo 94/2l675 2 l ~ ~ ~ 9 6 PCT/US94102537 LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID
NO: 23), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQID NO: 30); and LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID
NO: 23) and LPI-22 (SEQID NO: 29).
Additional p~ -ed peptides comprising various combin~tion.c of two or more of the above discussed preferred regions include:
LPI-16.2 (SEQ ID NO: 31), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQID NO: 30);
LPI-16.3 (SEQ ID NO: 32), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.4 (SEQ ID NO: 33), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQID NO: 30);
LPI-16.5 (SEQ ID NO:34), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.6 (SEQ ID NO: 35), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.7 (SEQ ID NO: 36), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.9 (SEQ ID NO: 37), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30); and LPI-16.10 (SEQ ID NO: 38), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQID NO: 30).

2s In yet another aspect of the present invention, a composition is provided comprising at least two peptides (e.g., a physical mixture of at least two peptides), each comprising at least one T cell epitope of Lol p I. Such compositions can be in the form of a composition ~d~lition~lly with a ph~rm~ceutically acceptable carrier of diluent for therapeutic uses, or with conventional non-ph~rrn~ceutical excipients for reagent use. When used therapeutically, an effective amount of one or more of such compositions can be ~tlmini.ctered .sim~lt~neously or sequentially to an individual sensitive to ryegrass pollen.
In another aspect of the invention, combinations of L,ol p I peptides are provided which can be ~rlmini.~tered simultaneously or sequentially. Such combin~tion.c may comprise therapeutic compositions comprising only one peptide, or WO 94/2167~ 7 ~ 9 ~ PCT/US94/02~37 more pep~des if desired. Such composi~ons may be used simultaneously or sequen~ally in ~lGr~lr~d combinations.
Preferred compositions and pi~r~lr~d combina~ons of Lol p I pep~des which can be ~mini~tered or o~erwise used cim~ n~ously or sequen~ally (compri~.ing S pep~des having am~no acid sequences shown ~n Fig. 2) include the following combinations:
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO:15), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ
ID NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), and LPI-11 (SEQ ID NO: 15);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), PLI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO:15), LPI-15 (SEQ ID NO: 21), and LPI-16 (SEQ ID
NO: 22);
LPI-3 (SEQ ID NO:6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO:lS), LPI-15 (SEQ ID NO: 21), and LPI-16.1 (SEQ ID
NO: 23);
LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID
NO: 21), and LPI-16.1 (SEQ ID NO: 23);
LPI-10 (SEQ ID NO:14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID
NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), and LPI-20 (SEQ ID NO: 27);
LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID
NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ
ID NO: 27), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30);
LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID
NO: 25), and LPI-20 (SEQ ID NO: 27);
LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID
NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ rD NO: 29), and LPI-23 (SEQ ID NO:30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID
NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-18 (SEQ ID NO: 25) and LPI-20 (SEQ ID NO: 27);

LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-23 (SEQ ID
NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-16.1 (SEQ ID
NO: 23);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30) and LPI-16.1 (SEQ ID NO: 23);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23) and LPI-ll (SEQ ID NO:15);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23) and LPI-4.1 (SEQ ID NO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23), LPI-4.1 (SEQ ID NO: 8) and LPI-22 (SEQ ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23), LPI-ll (SEQ ID NO: 15) and LPI-4.1 (SEQ IDNO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID
NO: 30), LPI-16.1 (SEQ ID NO: 23), LPI-ll (SEQ ID NO:15), LPI-4.1 (SEQ ID NO: 8) and LPI-22 (SEQ ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID
NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID
NO: 23), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30); and LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID
NO: 23) and LPI-22 (SEQ ID NO: 29).

Additional preferred compositions and p~ ed combinations of Lol p I peptides which can be ~lminist~red or used .~imlllt~n~ously or sequentially (comprising peptides having amino acid sequences shown in Figs. 2 or 4) include the following 30 combinations:
LPI-16.2 (SEQ ID NO: 31), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.3 (SEQ ID NO: 32), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO:30);

WO 94/21675 ~,157551 6 PCTIUS94/02537 LPI-16.4 (SEQ ID NO: 33), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.5 (SEQ ID NO: 34), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.6 (SEQ ID NO: 35), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.7 (SEQ ID NO: 36), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30);
LPI-16.9 (SEQ ID NO: 37), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30); and LPI-16.10 (SEQ ID NO: 38), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), and LPI-23 (SEQ ID NO: 30).

In each of the above plc;rellGd compositions, peptides LPI-16.1 (SEQ ID
NO: 23), LPI-18 (SEQ IDNO: 23), LPI-20 (SEQ ID NO: 27), and LPI-23 (SEQ ID
NO: 30) may be substituted as follows: peptide LPI-16.1 (SEQ ID NO: 23) (Fig. 2)may be substituted with LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), and LPI-16.10 (SEQ ID
NO: 38), all as shown in Fig. 4; peptide LPI-18 (SEQ ID NO: 25) (Fig. 2) may be substituted wit'n peptides LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42) all as shown in Fig. 4;
peptide LPI-20 (SEQ ID NO: 27) may be substituted with peptides LPI-20.2 (SEQ IDNO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID
NO: 46), and LPI-20.6 (SEQ ID NO: 47) all as shown in Fig. 4; peptide LPI-23 (SEQ
ID NO: 30) may be substituted with peptides LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49) and LPI-23.4 (SEQ ID NO:SO), all as shown in Fig. 4.
The present invention is further illustrated by the following non-limiting Figures and Examples.
F.~MPLE~S

Example 1 - l~ol~tion and Cloning of Nucleic Acid Sequence Coding for Lol p I
Total mRNA was extracted from mature ryegrass pollen by the phenol method 35 of Herrin and ~i~h~ , supra. Double-stranded cDNA was synthPsi7~cl from 1,ug of WO 94/21675 215 7 5 9 ~ y PCT/US94/02537 total mRNA using a commercially available kit (cDNA SYNTHESES SYSTEM PLUS
Krr, BRL, Gaithersburg, MD). After a phenol extraction and ethanol precipitation, the cDNA was blunted with T4 DNA polymerase (Promega, Madison, WI) and ligated to ethanol-precipitated, self-annealed AT and AL oligonucleotides for use in a modified Anchored PCR reaction, according to the method in Rafnar et al. (1991), J.
Biol. Chem., 266: 1229-1236; Frohman etal. (1990), Proc. Natl. Acad. Sci. USA, 85:8998-9002; and Roux et al. (1990), BioTech., 8: 48-57. Oligonucleotide AT hasthe sequence 5'-GGGTCTAGAGGTACCGTCCGATCGATCATT-3' (SEQ ID
NO: 71) (Rafnar et al. supra). Oligonucleotide AL has the sequence AATGATCGATGCT (SEQ ID NO: 72) (Rafnar et al. supra.).
Polymerase chain reactions (PCR) were carried out using a commercially available kit (GeneAmpt~) DNA Amplification kit, Perkin Elmer Cetus, Norwalk, CT) whereby 10 ~11 lOx buffer cont~ining dNTPs were mixed with l ,ug each of primer AP, which has the sequence 5'-GGGTCTAGAGGTACCGTCCG-3' (SEQ ID NO: 73) (Rafner et al. supra.) and LpA-5, which has the sequence 5'-CCCTGCAGATTATTTGAGATCTTGAG-3' (SEQ ID NO: 74), cDNA (3-5 111 of a 20 ',11 linkered cDNA reaction mix), 0.5 ,ul Amplitaq DNA polymerase, and distilled water to 100 ,ul.
Nucleotides 1 through 8 (5'-CCCTGCAG) of LpA-5 correspond to a Pst I site added for cloning purposes; the rem~ining nucleotides correspond to the non-coding strand sequence complerne.~t~ry to nucleotides 483 through 500 as shown in Fig. 6.
The samples were amplified with a programmable thermal controller (MJ
Research, Inc., Cambridge, MA). The first 5 rounds of amplification consisted ofdenaturation at 94C for 1 minute, ~nn~ling of primer to the template at 45C for 1.5 mimlt~s, and chain elongation at 70C for 2 min-ltes The final 20 rounds of amplifi~tic n consisted of denaturation as above, ~nnto.~ling at 55C for 1.5 min~l~s~
and elongation as above. Five percent (5 ~1) of this initial ~mplific~tion was then used in a secondary amplific~tion whereby lO 111 lOx buffer co~ .;r-g dNTPs was mixedwith 1 ,ug each of primer AP and primer LpA-3, which has the sequence 5'-CCCTGCAGTCATGCTCACTTGGCCGAGTA-3' (SEQ ID NO: 75), 0.5 1ll Amplitaq DNA polymerase, and distilled water to lOO ,ul. The secondary PCR reaction was pe,~lllled as described herein. Nucleotides 1 through 8 (5'-CCCTGCAG-3') of LpA-3 correspond to a Pst I site added for cloning purposes; nucleotides 9 through 12 (5'-TCA-3') correspond to the complementary sequence for a new stop codon, and the rem~ining nucleotides correspond to the non-coding strand sequence complementary WO 94/21675 ~ PCT/US94/02537 2~7~
to nucleotides 793 through 810 of the full length clone of L ol p I as shown in Fig. 1, including tr~n~l~t~d sequence of Lol p I (Fig. 1), the native stop codon and 3' untr~n~l~t~d sequence.
Amplified DNA was recovered by sequential chloroform, phenol, and s chloroform extractions, followed by precipitation at -20C with 0.5 volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. After precipitation and washingwitn 70% ethanol, the DNA was ~im~llt~3nP.ously fligtosted with Xba I and Pst I in a 15 ~Ll reaction and electrophoresed through a preparative 3% GTG NuSieve low melt gel (FMC, Rockport, ME). The appropriate sized DNA band was visualiæd by EtBr 0 st~ining, exci~ecl, and ligated into appro~liately digested M13mpl8 for sequencing by the dideoxy chain te~Tnin~tiOn method (Sanger et al. (1977), Proc. Natl Acad Sci USA, 74: 5463-5476) using a commercially available sequencing kit (Sequenase kit, U.S.
Biochemicals, Cleveland, OH).
Both strands were sequenced using M13 forward and reverse primers (N.E.
BioLabs, Beverly, MA) and internal sequencing primers LpA-13, LpA-12, LpA-9, LpA-2, LpA-7, LpA-10, and LpA-IA. LpA-13 has the sequence 5'-GAGTACGGCGACAAGTGGC-3' (SEQ ID NO: 76), which corresponds to nucleotides 121 through 139 as shown in Fig. 1. LpA-12 has the sequence 5'-TTCGAGATCAAGTGCACC-3' (SEQ ID NO: 77), which corresponds to nucleotides 310 through 318 as shown in Fig. 1. LpA-9 has the sequence 5'-GTGACAGCCTCGCCGG-3' (SEQ ID NO: 78), which corresponds to the non-coding strand sequence complementa~y to nucleotides 335 through 350 as shown in Fig. 1. LpA-2 has the sequence 5'-GGGAATTCCATGGCGAAGAAGGGC-3' (SEQ
ID NO: 79). Nucleotides 1 through 7 (5-GGGATT-3') of LpA 2 correspond to part of an Eco-RI restriction site added for cloning purposes; the rem~ining sequence of LpA-2 corresponds to nucleotides 425 through 441 of Fig. 1. LpA-7 has the sequence 5'-GTGCCGTCCGGGTACT-3' (SEQ ID NO: 80), and corresponds to non-coding strand sequence complementaly to nucleotides 503 throllgh 518 of Fig. 1. LpA-10 has the sequence 5'-CCGTCGACGTACTTCA-3' (SEQ ID NO: 81), which corresponds to non-coding strand sequence compl~.ment~ to nucleotides 575 through 590 of Fig. 1.
LpA-IA has the sequence 5'-GGAGTCGTGGGGAGCAGTC-3' (SEQ ID NO: 82), which corresponds to nucleotides 654 through 672 of Fig. 1.
Multiple clones from several independent PCR ;eactions were sequenced. The nucleotide (SEQ ID NO: 1) and deduced amino acid sequences (SEQ ID NO: 2) of a represen~~ e clone of Lol p I, clone 26 j are shown in Fig. 1. As shown in Fig. l, the nucleic acid sequence coding for Lol p I has an open reading frame beginning with an ATG initiation codon at nucleotides 16-18 ending with a TGA stop codon at nucleotides 805-807. The translated protein has a deduced amino acid sequence of263 amino acids with a predicted molecular weight of 28.4 kD and a pI of 5.55. The 5 initi~tinp~ methionine is numbered amino acid -23, with amino acid numbered +1corresponding to the NH2-terminus of the mature protein, as defined by amino acid sequencing (Cottam et al. (1986), Biochem. J., 234: 305-310). Amino acids -23 through - 1 (Fig. l), correspond to a leader sequence that is cleaved to yield the mature protein; the mature protein is therefore composed of 240 amino acids and has a predicted molecular weight of 26.1 kD and a pI of 5.38. There is a single potential N-linked glycosylation site at amino acid 9.
Amino acids 1 through 30 of clone 26j (Fig. 1) correspond exactly to the published sequence of the NH2 terminus of Lol p I (Cottam et al., supra). Amino acids 213 through 240 of clone 26j (Fig. 1) correspond exactly to the published intPrn~l amino acid sequence of Lol p I (Esch and Klapper (1989), Mol. Immunol., 26: 557-561).

Example 2 ~ ntification of Polymo pl~ . in Lolp I
A number of polymorphisms in the nucleotide sequence coding for Lol p I were 20 discovered during the ~mplifiç~tic-n and sequen~ing of dir~elGll~ Lol p I clones. Some of the polymorphisms cause an amino acid change relative to that of clone 26j, while others are silent polymorphisms that do not cause an amino acid change. The polymorphisms found in the sequence coding for Lol p I are sllmm~ri7t~d in Table 1.
The nucleotide base numbers are those of the sequence of clone 26 j shown in Fig 1.

Wo 94/2l675 PCT/US94/02537 2~51 ~9~

Table 1 Poll,"~o ~hi~ Detected in Lol p I

N~.clE~lide Polymorphism An~ino Acid Polym~
GGC21~j~GGA/GGT None 2 G2~4AC23~s~GAT D4~N
3 GTT?~g~GTC None
4 CGT~1~CGC None GGC~5fi~GGT None 6 AAC~g~AAT None 7 CCC39~;~CCT None 8 CAT4, ~CAC None 9 GCC4~4~GCA None GAC~n~GAT None 11 GG5~2C~GAC Gl44~D
12 CCG~42~CCA None 13 ACA~4~ACG None 14 GC~s?T~GGT Al ~4~G
1~ CTC~ ~CTG None 16 GCGfi?fj~GCC None 17 ATC7~ATT None 18 CCT7~CCC None All cnnfirmed nucleotide polymorphisms (polymorphisms observed in the sequence analysis of clones from two independent PCR reactions) are shown relative to the sequence of clone 26j (Fig.l) (SEQ ID NO: 1). The polymorphic residues in10 their respective codon triplets are numbered. Productive amino acid changes are also shown; most nucleotide polymorphisms are silent and do not result in an amino acid change. Twenty-eight potential polymorphisms have only been observed in clones from single PCR reactions. Seventeen of these 28 potential polymorphisms are silent mutations and do not result in an amino acid polymorphism; the rem~ining 11 potential 15 polymorphic sites would result in the following amino acid changes, specifically: Tl 1
5 ~ 15 75 ~ PCT/US94102537 ~M, A4g~V, R67~S, K7g~R, Vgo~L Q133~R, I162~T~ V173~E~ I187 V223~F and K232~R. The potential polymorphism at amino acid 223 (V223~F) has been previously reported. (Perez et al., supra) Exa~ 3 - ~nm~n IgE reactivity to Purified R~comh;snt and Native Lol p I
Cloned DNA encoding L ol p I and Lol p IX was expressed in E. coli and purified on a Ni-chelating affinity column. Monodonal antibodies were also used to affinity purify and distinguish isoforrns of these and native grass proteins. The recombinant Lol p I was compared to biochemically purified native L ol p I and Lol p ~ in mAb and human IgE reactivity studies (data not shown). The reactivity of human IgE to the recombinant and native forms was equivalent when measured by direct binding ELISA. In conlpeliLion assays, the native Lol p I and Lol p IX proteins could completely inhibit IgE binding to Lol p soluble pollen extract (SPE), whereas the recombinant form of l,ol p I and Lol p lX could only partially inhibit IgE binding to the extract. However, the recombinant Lol p I and Lol p ~ was still active in these colllpe~ilion assays. These asays were then ext~n-le~l to western blot inhibition studies;
both methods confirm the previous finding that Group I and Group ~ constitute one of the major allergenic proteins of Lolium perenne grass pollen. Furthermore, the Lol p I and Lol p ~ native and recombinant allergens showed ihibition of grass allergic patient IgE binding to soluble pollen extracts of other grass species (Dac g, Phl p and Poa p). The degree to which Lol p I and Lol p IX proteins succescfully compete for IgE binding to these other grasses implies a hierarchy of homology between the species. These studies confirm and extend the fin-lings of shared IgE epitopes between temperate grass allergens.
2s Ihe procedures used for the foregoing examples were as follows:

WO 94/21675 ~3 5~9~ PCT/US94/02537 , F.~;traction and Depi~mentation of Aller~ens Defatted Lol p I pollen was extracted twice, overnight at 4C in 50mM
phosphate buffer, 15mM NaCl, pH 7.2 and protease inhibitors (PMSF, Luepeptin, SPTI and pepstatin). The extract was then depigmente~l by batch absorption with DE-52 (Whatman) in 50mM phosphate buffer, 0.3M NaCl, pH 7.2.

Biochemiç~l Purification of Lol p I Allergen Depi~me.ntecl Lol p I extract was dialyzed into H20, pH 8.0 by addition of NH40H. This mateial was loaded onto a DE-52 column and eluted stepwise with lo lmM, 4.5mM and 7.5mM NaH2PO4. The majority of the Group I allergens was eluted with 4.5mM NaH2P04. A further separation of Group I was accomplished by running this DE-52 enriched fraction over A (26/60) superdex 75 column (Pharmacia).

Tmmuno~ffinity Purifir~ti~n of Lol p ~ Aller~en lB9 ascites was precipitated by 50% (NH4)2S04, followed by p~lrifi~ation over Q-sepharose (Pharmacia). Purified lB9, an anti-Lol p IX antibody, was then coupled to Affigel-10 (Biorad), according to the m~nllf~rt~lrer's instructions. Either depigmented pollen extract or DE-52 enriched material was circulated over the lB9 affigen column overnight at 4C. The column was washed with PBS, PBS ~ 0.5M
MaCl and then eluted with O.lM Glycine, pH2.7. Eluted Lol p IX fractions were neutralized with lM tris-base, pH 11.

Ex~ression and pllrific~tion of Recombinant Lol p I
Lol p I cDNA's encoding from the first amino acid of the mature protein to the stop codon were ligated into pETl ld~HR cont~ining a leader which encoded 6 hi.~ti-lin.os. The HIS6 was used for pllrific~tion over a nickel-NTA agarose column (Qiagen). rLol p I was expressed in E. coli.

SDS-PAGE. Electroblottin~ and Immunoblottin~
Electrophoresis was pc,Çolllled using 12.5~o polyaclylarnide gels. The samples were run under reducing conditions (4 hours at 40mA constant current). After electrophoresis the protein was transferred to nitrocellulose membrane (1.5 hours at 1.5A). The blots were stained with 1% India ink, and then blocked with 1 % defatted milk, 1% FCS in Tween solution (2mM Tris-HCI pH 7.5, 0.71M NaCl, and 0.05%
Tween 20) for 1 hour. The human plasma samples were pre-absorbed with blank Wo 94/21675 2 ~ 5 ~ ~ 9 6 PCT/USg4/02537 nitrocellulose for 1.5 hours prior to incubation. Blot sections were incubated with 1st antibodies diluted in 1% miL~/Tween solution overnight at room tempelaLult; (RT).
The blot sections were washed three times and inucbated in the appr~pliate biotinylated 2nd AB (1:2500) for 2 hours at RT. The blot sections were washed three 5 times and finally inrubated with 125I-streptavidin 1 hour at RT. The sections were washed extensively to remove unbound label and exposed to film. Autoradiography was carried out at -80C.

D;rect. Competition and Depletion ELISA
Microtiter plates were coated with 2.5-lO.O,ug/mL of coating antigen (grall soluble pollen extract (SPE), L ol p I, Lol p IX, L,ol p IX, recombinant Lol p I, and/or recombinant Lol p IX) in PBS at lOO,uL/well and incubated overnight at 4C. The plates were washed three times between each step with PBS-T (Phosphate buffered saline + 0.05~o Tween 20). The unbound antigen was removed and the plate blockedwith 300~LL/well of lMG/ML PVP in 0.5% gelatin/PBS for one hour at room tempe. ~t~ ~ e (RT). All subsequent reagents were added at lOO~LL/well for direct ELISA, serially diluted human plasma was added to duplicate wells and incubated overnight at 4C. This was followed by biotinylated goat anti-human IgE (1:1,000) for 1 hour at RT, then streptavidin-HRPO (1:10,000) for 1 hour at RT. TMB
20 substrate and H22 were freshly mixed and added; the color was allowed to develop for 2-5 minllt.oc The reaction was stopped by the ~ itiQn of lM phosphoric acid.The plates were read on a dynatech plate reader at 450NM and the absorbances of duplicate wells were averaged.
For the competition ELISA, the human plasma samples were mixed with an 2s equal volume of serially diluted antigen or with PBS-T (as a control). These samples were incubated overnight at 4C before ~ ition to the microtiter plate and performing the rem~ining steps of the ELISA as stated above.
For the depletion ELISA, the human plasma was pre-incubated on antigen or PBS coated wells, collected and re-incubated on freshly coated wells. The ELISA was 30 then performed as outlined above.

Wo 94/21675 C;~ 5'C? Ç~ PCT/US94/02537 FX~MP~ 4- Human~ Cell Studies with r~71p I

Synthesis of Overlappin~ Pe~tides Ryegrass Lol p I overlapping peptides were synthe~i7~d using standard Fmoc/tBoc synthetic chemistry and purified by Reverse Phase HPLC. Fig. 2 shows Lol p I peptides used in these studies (SEQ ID NO: 3-30). The peptide names are consistent throughout.

~F B;ntiin~ Studies with overlappin~ peptides None of the peptides shown in Fig. 2 bound a detectable amount of IgE from pooled human plasma when analyæd in a solid phase ELISA assay (data not shown).
The procedure for the ELISA assay with the overlapping peptides was substantially the same as that described in Example 3.

T CP11 Responses to ~ye~r~cc Anti~en Pep~ s Peripheral blood mononuclear cells (PBMC) were purified by lymphocyte separation m~ m (LSM) centrifugation of 60 ml of hep~rini7Pcl blood from grass-allergic patients who exhibited clinical symptoms of seasonal rhinitis and were MAST
and/or skin test positive for grass. Long-term T cell lines were established by stim~ tion of 2X106 PBL/ml in buLk cultures of complete me~ lm IRPMI-1640, 2 mM
L-gl~ ., 100 U/ml penicillin/sL~ep~ll.ycin, 5x10-5M 2-mercaptoethanol, and 10 mM HEPES, supplemented with 5% heat-inactivated human AB serum) with 25 mg/ml of purified native Lol p I (95~o pure with a single band on protein gel) for 6 days at 37C in a hllmitlifiP11 5~O C02 incubator to select for L ol p I reactive T Cells.
This amount of priming antigen was determin~cl to be optimal for the activation of T
cells from most grass-allergic patients. Viable cells were purified by LSM
centrifugation and cultured in complete me~ m, supplemented with 5 units recombinant human IL-2/ml and 5 units recombinant human IL-4/ml for up to 3 weeks until the cells no longer responded to lymphokines and were considered "rested." The ability of the T cells to proliferate to selected peptides, recombinant Lol p I (rLol p 1), purified native Lolp I, recombinant Lol p JX (rLolp IX)~ or Derp I (rDerp I) wasthen assessed. For assay, 2x104 rested cells were restimulated in the presence of 2x104 autologous Epstein-Barr virus (EBV)-transformed B cells (prepared as described below) with 2-50 mg/ml of rLol p I, purified native Lol p I, rDer p I, or rLol 3s p IX, in a volume of 200 ml complete m~tlillm in duplicate wells in 96-well round-Wo 94/21675 PCT/US94/02537 ~7596 bottom plates for three days. Each well then received 1 mCi triti~ted thymidine for 16-20 hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. The varying antigen dose in assays with rLol p I, purified native Lol p I, and recombinant Lol p IX and several ~ntigenic peptides (i.e., peptides that induce an immune response, or, specifically, a positive T
cell response in these assays) syntheci7~-l as described above were determin~o-l The titrations were used to optimi_e the dose of peptides in T cell assays. The maximum response in a titra*on of each peptide is expressed as the stimula*on index (S.I.). The S.I. is the counts per minute (CPM) incorporated by cells in response to peptide, divided by the CPM incorporated by cells in medium only. An S.I. value equal to or greater than 2 *mes the background level is considered "positive" and in~licates that the peptide contains a T cell epitope. The positive results were used in calculating mean stimnl~*on indices for each peptide for the group of pa*ents tested. The results (not shown) demonstrate that one patient responds well to rL ol p I and purified native Lol p I, as well as to L ol p I peptides but not to recombinant Der p I. This incliç~tecl that Lol p I T cell epitopes are recogni_ed by T cells from this particular allergic patient and that rLol p I contains such T cell epitopes. T cells from the majority of patients also reacted to rLol p ~, suggesting a presence of Lol p IX antigen in the purified native L ol p I prep that was used to prime T cells.
The above procedure was followed with a number of other p~*ent.~. Individual patient results were used in calculating the mean S.I. for each peptide if the patient responded to the Lol p I protein at an S.I. of 2.0 or greater and the padent responded to at least one peptide derived from Lol p I at an S.I. of 2.0 or greater. A s--mm~ry of positive e~cl.el;.,.lont.~ from 35 patients is shown in Fig. 3. All 35 T cell lines responded 2s to purified native Lol p I and rL olp I. The numbers enclosed in the parentheses denote pelcell~ge of patients responding to that particular peptide. The bar represents the positivity index for each peptide (% of patients responding multiplied by mean S-I-)-P`lG~a,dlion of EBV-transf~rmed B Cells for Use as ~nt~en-presentin~ Cells Autologous EBV-transformed cell lines were derived by incubating 5x106 PBL
with 1 ml of B-59/8 Marmoset cell line (ATCC CRL1612, ~meric~n Type Culture Collection, Rockville, MD) conditioned mP-lillm in the presence of 1 mg/ml phorbol 12-myristate 13-acetate (PMA) at 37~C for 60 min~l~s in 12x75 mm polypropylene 3s round-bottom Falcon snap cap tubes (Becton Dickinson Labware, Lincoln Park, NJ).

æl~7ss6 These cells were then diluted to 1.25x106 cells/ml in the RPMI-1640 me~ m that was supplemented with 10% head-inactivated fetal bovine serum in place of the 5% human AB serum and cultured in 200 ml aliquots in flat-bottom culture plates until visible colonies were detected. They were then transfered to larger wells until the cell lines s were established.
Those skilled in the art will appreciate that the invention described is susceptible to variations and modification other than those specifically described. It is understood that the invention includes all such variations and mo-lific2tic)ns. The invention also includes all steps, features, compositions and compounds referred to or 0 in~lic~tt-d in this specific~tion, individually or collectively, and any and all combinations of any two or more of said steps or features.

Example 5 - Cloning and Expression of Dac g I, Poa p I and Phl p I

A. Clonin~ of Dac g 1.
RNA was obtained from the pollen of Dactylis glomerata using a standard acid phenol extraction procedure (Sambrook et al. (1989), Moleculor Cloning: A
laboratory manual. 2nd Edition., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). This and other pollens described below were purchased from Greer Laboratories (Lenoir, NC). Single and double stranded cDNA was plGpar~d from total D. glomerata RNA using the BRL cDNA Synthesis System (Gaithersberg, MD), blunted using standard procedures (Sambrook et al. (1989) supra), and ligated to self-~nne~l~(l oligonucleotides AT (5'-GGGTCTAGAGGTACCGTCCGATCGATCATT-3') (SEQ ID NO: 71) and AL (5'-AATGATCGATGCT-3') (SEQ ID NO: 72) (Rafnar et al. (1991), J. Biol. Che~, 266: 1229-1236).
The amino portion of the gene encoding Dac g 1, including 5' untr~n~l~tP.d sequence, nucleotide sequence encoding the predicted leader sequence and nucleotide sequence encoding the first portion of the mature protein, was cloned using the polymerase chain reaction (PCR). Oligonucleotide primers AP-2 (5'-GGGTCTAGAGGTACCGTCC-3') (SEQ ID NO: 83) and LpA-7 (5'-GTGCCGTCCGGGTACT-3') (SEQ ID NO: 80) were used in a primary amplification. Oligonucleotide primers AP-2 and LpA-9 (5'-GTGACAGCCTCGCCGG-3') (SEQ ID NO: 78) were used in a secondary ~mplific~tion using 10% of the p ima~,~ amplification as template cDNA. PCRs were carried out using ~e GeneAmp DNA Amplification kit (Perkin Elmer, Norwalk, CT) WO 94/21675 ~ l ~i 7 ~i 9 6 PCT/US94/02537 using a programmable thermal controller from MJ Research, Inc. (Cambridge, MA).
S~mples were amplified for 24 cycles by heating to 94C for 1 min, 54C for 1.5 min and 70C for 1 min.
The resulting PCR product was blunted with T4 DNA polymerase (Sarnbrook et al. (1989) supra) and digested with the restriction endonuclease XbaI. Unlessotherwise stated, all endonucleases and polymerases were obtained from New Fn~l~n~l BioLabs (Beverly, MA). A band of app~ ately 400 base pairs was isolated from a low melting temperature agarose gel (FMC, Rockland, ME) and ligated into appropriately digested pUCl9. The clones 22.2 and 22.5 were subsequently i(lentifie~l by dideoxysequencing (Sanger et al. (1977), Proc. Natl. Acad. Sci. USA, 74:5460-5463) to contain nucleotide sequence of the gene encoding Dac g 1.
A 600 base pair cDNA cont~ining intern~l nucleotide sequence of the gene encoding Dac g 1 was amplified using the primers DGI-3 (5'-TTGGATCCTACGGCAAGCCGACCGGC-3') (SEQ ID NO: 84) and LpA-10 (5-CCGTCGACGTACTTCA-3') (SEQ ID NO: 81). A 300 base pair cDNA cont~ining internal Dac g 1 sequence was amplified using the primers DGI-4 (5'-TTGGATCCATCCCGAAGGTGCCCCCGGG-3' (SEQ ID NO: 85), wherein G at position 14 can also be A) and LpA-9 (5'-GTGACAGCCTCGCCGG-3') (SEQ ID
NO: 78). The cDNAs were amplified for 34 cycles by heating to 94(~ for 45 sec, 60C for 45 sec and 72C for 1 min. These PCR products were blunted with T4 DNA polymerase as above, digested with BamHI and ligated into apploplialely gested pUCl9. Clones 86.1 (600 base pairs) and 88.6 (300 base pairs) were sequenced and found to contain sequence of the gene encoding Dac g 1.
The carboxy portion of the gene encoding Dac g 1, including tne 3' untranslated region, was cloned using oligonucleotide primers AP (5'-GGGTCTAGAGGTACCGTCCG-3') (SEQ ID NO: 73) and DGI-8 (~'-AGGTGACCTTCCACGTCG-3') (SEQ ID NO: 86) in a primary PCR and oligonucleotide primers AP and DGI-9 (5'-TTGGATCCTGGCGCTGCTGGTGAAGTA-3') (SEQ ID NO: 87) in a secondary PCR. Material was amplified for 25 cycles of heating to 94C for 1 min, 60C for 40 sec and 74C for 1 min. The 700 base pair PCR product was digested with BamHI
and Asp718 (Boehringer M~nnheim, Tntli~n~polis, IN), isolated and digested into appropliatelyrlige.steclpUC19asdescribedabove. Theclones 119.2, 119.4, 119.6, 119.9 and 119.12 were isolated, sequenced and found to contain sequence of tne gene encoding Dac g 1.

WO 94/2167~i i PCT/US94/02537 ~
~5~

cDNA clones encoding the mature Dac g 1 protein were obtained by PCR with the oligonucleotide primers DGI-7Eco (5'-TTGAATTCATCCCGAAGGTGCCCCCG-3' (SEQ ID NO: 88), wherein G at position 14 can also be A) and PhA- 1.2 (5'-TTGGTACCTCACTTGGACTCGTAGCT-3') (SEQ ID NO: 89). The cDNAs were amplified for 24 cycles of heating to 94C for 1 min, 54C for 1.5 min and 70C for 1 min. The ~mplifitod cDNA was digested with EcoRI and Asp718, isolated, and ligated into the ayplopliately digested pUCl9. The cDNA clones 106.5, 106.6, 106.9 and 106.12 were identified as Cont~inin~ Dac g 1 sequence by dideoxysequencing. The 0 nucleotide (SEQ ID NO: 51) and deduced amino acid (SEQ ID NO: 52) sequences of clone 106.5 are shown in Fig. 5. Nucleotides 509-515 (encoding amino acids 171 and 172) are from the sequence of clone 106.12. The sequence of clone 106.5 was not resolved in this region.
The insert from clone 1065 was isolated and ligated into a~ropliately digested expression vector pET-l ld (Novagen, Madison, WI: Jameel et al. (1990), J.
Virol., 64:3963-3966). The pET-lld vector had been modified to contain a sequence encoding 6 hi~ti-lines (His 6) imme~ t~.ly 3' of the ATG initiation codon followed by a unique EcoRI endonuclease restriction site. A second EcoRI endonuclease restriction site in the vector, along with neighboring ClaI and Hindm endonuclease restriction sites, had previously been removed by digestion with EcoRI and Hindm~ blunted and reli~te-l A recombinant clone was used to transform Escherichea coli strain BL21-DE3. A culture was grown to A600 of 1.0, IPTG was added to 1 mM final concentration and grown for an additional 2 hours. Bacteria was recovered by 2s centrif~ ti-~n (7,930 G, 10 min) and lysed in 90 ml of 6 M G~l~ni~line-HCl, 0.1 M
Na2HPO4, pH 8.0 for 1 hour with vigorous .~h~ki~g The recombinant Dac g 1 was purified from the extract on a Ni+2 chelating column (Hochuli et al. (1987) J.
Chromatog., 411:177-184; Hochuli et al. (1988), Bio~Tech., 6:1321-1325).

R. Clonin~ of Poa p I.
RNA was isolated from the pollen of Poa pratensis, double stranded cDNA
was prepared and self-annealed oligonucleotides AT and AL were added as described in section A, above. PCR product was ~mplifiP.rl using oligonucleotide primers Phl-7 (5'-CCGAATTCGTGGAGAAGGGGTCCAA-3') (SEQ ID NO: 90) and Poa-l (5'-3s TTAGGATCCTCACTTATCATAIGACGTATC-3' (SEQ ID NO: 91), wherein C at WO 94/21675 2I ~ 7~ ~ 6 PCT/US94/02537 position 13 can also be T, A at position 16 can also be G, A at position 19 can also be G, G at position 23 can also be C, A at position 24 can also be T, C at position 25 can also be T or A or G and A at position 28 can be G). All Poa p 1 clones were ~mpli~l~.d by 20 cycles of heating to 94C for 1 min, 55C for 1 min and 72C for 1 min. The ~mplifit-d m~ttori~l was finally heated to 72C for 5 min. Three clones, 11, 15 and 17, were isolated that contained part of the nucleotide sequence for the gene that encodes Poa p 1. The Dac g 1 sequence encoded by clones 11, 15 and 17 corresponds to amino acids 151 - 240 of Fig. 6.
Clones cont~ining partial nucleotide sequences of the gene encoding Poa p 1 were derived from PCRs that used oligonucleotide primers AP and Poa-3 (5'-TTGAATTCCTTGTCATTGCCCTTCTG-3') (SEQ ID NO: 92) in the primary PCR
and AP and Poa-4 (5'-AAGAATTCCTTCTGCTTGATGTCCAC-3') (SEQ ID
NO: 93) in the secondary PCR. Other clones were derived from PCRs that used oligonucleotide primers AP and Poa-6 (5'-ATGAATTCGAGTCGTGGGGAGCCGTC-3') (SEQ ID NO: 94) in the primary PCR
and AP and Poa-7 (5'-ATGAATTCGTCTGGAGGATCGACACC-3') (SEQ ID
NO: 95) in the secondary PCR. Clones 58, 59 and 63 were derived from the PCR
using primers AP and Poa-4. Clones 91 and 97 were derived from the PCR using primers AP and Poa-7.
Additional clones were derived from a PCR that used oligonucleotide primers Poa-l and Poa-S (S'-ATGAATTCATCGCAAAGGTTCCCCCC-3' (SEQ ID NO: 96), wherein A at position 14 can also be G or C or T). These clones, 113, 114 and 115, corresponded to the portion of the gene that encoded amino acids 1 - 240 of Poa p 1 (see Fig. 6). The nucleotide (SEQ ID NO: 53) and deducecl amino acid (SEQ ID
NO: 54) sequences of clone 114 are shown in Fig. 6. Nucleotide 93 in Fig. 6 was not resolved and could be a G or a C or a T or an A and is represented by the letter "N".
Nucleotide 94 in Fig. 6 was not conclusively resolved and could be a G or a C or a T
but not an A and is represented by the letter "B". The codon cont~ining nucleotide 93 (GGN) encodes a Glycine at residue 31. The codon Cont~ining nucleotide 94 (BCC) encodes an Alanine (GCC), a Proline (CCC), or a Serine (TCC) at amino acid 32. The amino acid at residue 32 in Fig. 6 is represented by an "X".
Inserts from clones 11 and 114 were isolated and ligated into appropliately digested expression vector pET-1 ld (Novagen, Madison, WI: Jameel et al. (1990) J.
Virol. 64:3963-3966). Recombinant proteins were expressed as descibed in section A, above.

WO 94/21675 ~ 96 PCT/US94/02537 C. Clonin~ of Phl p 1.
RNA was isolated from the pollen of Phleum pratense, double stranded cDNA
was prepared and self-annealed oligonucleotides AT and AL were added as described in section A, above. Clones were derived from a PCR that used oligonucleotide primers PhAl.l (5'-TTTGGATCCTCACTTGGACTCGTAGCT-3') (SEQ ID NO: 97) and Phl-2 (5'-TTGAATTCTCGCGAAGGTGCCCCCG-3' (SEQ ID NO: 98), wherein G at position 13 can also be A). These clones, 20 and 22, corresponded to the portion of the gene that encoded amino acids 1 - 240 of Phl p 1 (see Fig. 7). The nucleotide (SEQ ID NO: 55) and deduced amino acid (SEQ ID NO: 56) sequences of clone 20 are shown in Fig. 7.
Clones cont~ining partial nucleotide sequence of the gene encoding Phl p 1 were derived from a PCR using oligonucleotide primers Phl-7 (5'-CCGAATTCGTGGAGAAGGGGTCCAA-3') (SEQ ID NO: 90) and PhAl.l. Clones 47-52 were derived from this PCR. These clones encoded amino acids 151 through 240 of Fig. 7.
Inserts from clones 22 and 51 were isolated and ligated into appropriately digested expression vector pET-1 ld (Novagen, Madison, WI: Jameel et al. (1990) J.
Virol. 64:3963-3966). Recombinant proteins were expressed as descibed in section A, above.

Example 6 - CO~ r;S~n of Dac g 1, Phlp 1 and Poa p 1 With Lolp 1.

The sequences for Dac g 1 (Fig. 5) (SEQ ID NO: 58), Phlp 1 (Fig. 7) (SEQ
ID NO: 59) and Poa p 1 (Fig. 6) (SEQ ID NO: 60) were compared with Lol p 1 (SEQ
ID NO: 57). The amino acid sequenccs of these Group 1 allergens had 95% (Dac g 1), 91% (Phl p 1) and 91 % (Poa p 1) identity, respectively, with Lol p 1. This comparison is shown schem~ti~lly in Fig. 8. The complete sequence of Lol p 1 is shown in standard one letter code. Only differences from the Lol p 1 sequence are shown for the other Group 1 allergens; identity is inclir~t~d by a dash (-). Potential amino acid polymorphisms were predicted by detected nucleotide polymorphisms in each sequence. Such potential polymorphisms are shown by superscript and subscript letters at the site of the polymorphism.
T cell epitope Co~ g peptides of L,ol p 1, peptides 16.1 (SEQ ID NO: 23), 18 (SEQ ID NO: 25), 20 (SEQ ID NO: 27) and 23 (SEQ ID NO: 30), were defined in WO 94/21675 ~15 7 5 ~ 6 PCT/US94/02537 Example 4 (Fig. 3). The sequences of the other Group 1 allergens are very conserved in these regions. Since the Group 1 allergens are homologous, the major T cell epitope cont~ining peptides of Lol p 1 are likely to be the major T cell epitopeco"l~;"illg regions in the related grasses. Comparison of the sequences of the Lolp 1 5 peptides with the homologous peptides cont~ining Dac g 1, Phlp 1 and Poap 1 polymorphisms are shown in Fig. 9 (SEQ ID NO: 23, 25, 27, 30, 61-70).

WO 94/21675 2 ~1 S ~ ~ f PCTrUS94/02537 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: IMMULOGIC PHARMACEUTICAL CORPORATION
(B) STREET: 610 LINCOLN STREET
(C) CITY: WALTHAM
(D) STATE: MASSACHUSETTS
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02154 (G) TELEPHONE: (617) 466-6000 (H) TELEFAX: (617) 466-6010 (ii) TITLE OF INVENTION: T CELL EPITOPES OF RYEGRASS POLLEN
ALLERGENS
(iii) NUMBER OF SEQUENCES: 98 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD
(B) STREET: 60 State Street, suite # 510 (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: US
(F) ZIP: 02109-1875 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII text (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/106,016 (B) FILING DATE: 31-AUG-1993 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/031,001 (B) FILING DATE: 12-MAR-1993 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Amy E. Mandragouras (B) REGISTRATION NUMBER: 36,207 (C) REFERENCE/DOCKET NUMBER: (IMI-040PC) (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400 (B) TELEFAX: (617) 227-5941 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1124 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 94/21675 2 1 5 7 5 9 ~ PCT/US94/02537 (ii) MOLECULE TYPE: cDNA

( iX) FEATURE:
- (A) NAME/KEY: CDS
(B) LOCATION: 16..804 (ix) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 85..804 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CAAATTCAAG ACAAG ATG GCG TCC TCC TCG TCG GTG CTC CTG GTG GTG GCG

Met Ala Ser Ser Ser Ser Val Leu Leu Val Val Ala CTG TTC GCC GTG TTC CTG GGC AGC GCG CAT GGC ATC GCG AAG GTA CCA

Leu Phe Ala Val Phe Leu Gly Ser Ala His Gly Ile Ala Lys Val Pro CCG GGC CCC AAC ATC ACG GCC GAG TAC GGC GAC AAG TGG CTG GAC GCG

Pro Gly Pro Asn Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp Ala AAG AGC ACC TGG TAT GGC AAG CCG ACC GGC GCC GGT CCC AAG GAC AAC

Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn GGC GGC GCG TGC GGG TAC AAG GAC GTT GAC AAG GCG CCG TTC AAC GGC

Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro Phe Asn Gly ATG ACC GGC TGC GGC AAC ACC CCC ATC TTC AAG GAC GGC CGT GGC TGC

Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly Arg Gly Cys GGC TCC TGC TTC GAG ATC AAG TGC ACC AAG CCC GAG TCC TGC TCC GGC

Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly GAG GCT GTC ACC GTC ACA ATC ACC GAC GAC AAC GAG GAG CCC ATC GCA

Glu Ala Val Thr Val Thr Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala CCC TAC CAT TTC GAC CTC TCG GGC CAC GCG TTC GGG TCC ATG GCG AAG

Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys AAG GGC GAG GAG CAG AAG CTC CGC AGC GCC GGC GAG CTG GAG CTC CAG

Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln WO 94/21675 PCT~uS94/02537 ~51~

TTC AGG CGG GTC AAG TGC AAG TAC CCG GAC GGC ACC AAG CCG ACA TTC

Phe Arg Arg Val Lys Cys Lys Tyr Pro Asp Gly Thr Lys Pro Thr Phe CAC GTC GAG AAG GCT TCC AAC CCC AAC TAC CTC GCT ATT CTG GTG AAG

His Val Glu Lys Ala Ser,Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys TAC GTC GAC GGC GAC GGT GAC GTG GTG GCG GTG GAC ATC AAG GAG AAG

Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys GGC AAG GAT AAG TGG ATC GAG CTC AAG GAG TCG TGG GGA GCA GTC TGG

Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp AGG ATC GAC ACC CCC GAT AAG CTG ACG GGC CCA TTC ACC GTC CGC TAC

Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr ACC ACC GAG GGC GGC ACC A~A TCC GAA GTC GAG GAT GTC~ATC CCT GAG

Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu Asp Val Ile Pro Glu GGC TGG AAG GCC GAC ACC TCC TAC TCG GCC AAG TGAGCAAGAA GTGGAGTGAT

Gly Trp Lyæ Ala Asp Thr Ser Tyr Ser Ala Lys CCAA TCAGCTTAAT TTTGACTCAA GATCTCA~AT AATCCAGCCG CACATATATA

CGAGGCGGTG AGACATACAA GCTCCTCCAT GAGTATATTC ATTCATGCCG TATAGAGAGG

AGA~AGATGC CTGAATAAGA GTTTGAGGTC GACACCTTGT GAGAAGTGTA TATAGGAGGA

ACCCAATCTG GCTCCATCTT TCTTTGCTCG CACGGTGTAC TGCTAAGGTT A~ AAC

AGGCCAGATT AACCTACTAT CTAATATATG CAACGTATGG TCATTTTCCC TA~A~PAA~

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 263 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ser Ser Ser Ser Val Leu Leu Val Val Ala Leu Phe Ala Val 2~ 37~
WO 94l21675 PCT/US94/02537 Phe Leu Gly Ser Ala His Gly Ile Ala Lys Val Pro Pro Gly Pro Asn - Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro Phe Asn Gly Met Thr Gly Cys 15 Gly Asn Thr Pro Ile Phe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Ala Val Thr Val Thr Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Asp Gly Thr Lys Pro Thr Phe His Val Glu Lys Ala Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly 45 Gly Thr Lys Ser Glu Val Glu Asp Val I le Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser.Ala Lys .

WO 94/21675 ~1~7 5~ 6 PCT~US94/02~37 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acld (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp 20 .
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ile Ala Lys Val Xaa Pro Gly Xaa Asn Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr (2) INFORMATION FOR SEQ ID NO:6:

~ 2~7~96 , (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asn Val Asp Lys Ala Pro (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:

WO 94121675 ~ ~ ~ S ~ ~ PCTrUS94/02537 (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro Phe Asn Gly Met Thr Gly l 5 l0 15 Cys Gly Asn Thr (2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:
Phe Asn Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly l 5 l0 15 ~ 2~7~9~

Arg Gly Cys Gly (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Pro Ile Phe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
40 Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Ala Val Thr Val (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Glu Ser Cys Ser Gly Glu Ala Val Thr Val Thr Ile Thr Asp Asp Asn Glu Glu Pro Ile WO 94121675 PCTrUs94/02537 ~
~,~5~9~

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Thr Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Asp Asp Gly Glu 2 ~ g 6 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys Lys Gly Glu (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 a~ino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Phe Gly Ser Met Ala Asp Asp Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu (2) INFORMATION FOR SEQ ID NO:19:

WO 94/21675 . PCT/US94/02537 2ls~s~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Glu Leu Gln Phe Arg Arg Val Lys Cys Lys ~yr Pro Asp Asp Thr Lys Pro Thr Phe His (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Tyr Pro Asp Asp Thr Lys Pro Thr Phe His Val Glu Lys Ala Ser Asn Pro Asn Tyr Leu (2) INFORMATION FOR SEQ ID NO:22:

W O 94/21675 215 7 ~ ~ 6 PCT~US94/02537 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Val Glu Lys Ala Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly Asp - 20 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly Asp (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ala Ile Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

WO 94/21675 ~$1 S96 PCTrUS94/02537 (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile Asp (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr (2) INFORMATION EOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids W O 94121675 215 7 5 9 6 PCTrUS94/02537 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu l 5 l0 15 Gly Gly Thr Lys W O 94/21675 2~ ~7 ~ 9 ~ PCTrUS94/02537 (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu Asp Val Ile Pro Glu Gly (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v~ FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser Ala Lys (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Asp Glu Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly Asp (2) INFORMATION FOR SEQ ID NO:32:

W O 94/21675 PCTrus94/02537 ~15759~

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Asp Glu Ala Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly Asp (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Lys Lys Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Lys (2) INFORMATION FOR SEQ ID No:34:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Asp Glu (2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid W O 94/21675 2 ~ ~ 5 ~ 6 PCTrUS94/02537 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Ala Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Asp Glu (2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Asp Glu Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Asp Glu (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Lys Lys Ala Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val 1 5 10 . 15 Lys Lys (2) INFORMATION FOR SEQ ID NO.38 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal W 0 94/21675 215 7 ~ ~ ~ PCTrUS94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Asp Glu Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Glu (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys W O 94l21675 21~ 75 9 6 PCTnJS94/02537 (2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:~0:
Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys WO 94/21675 PCT~US94/02537 ~1~7~

(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr (2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr WO 94/21675 2 1 5 7 $ 9 6 PCTrUS94/02537 ~2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys (2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Ala Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr W094/21675 21575~ PCT/US94/02537 (2) INFORMATION FOR SEQ ID No:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser Ala Lys (2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 723 base pairs (B) TYPE: nucleic.acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.... 720 WO 94/21675 PCTrUS94/02537 ~575~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
ATC CCG AAG GTG CCC CCG GGC CCG.AAC ATC ACG GCG ACC TAC GGT GAC

5 Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp AAG TGG CTG GAC GCG AAG AGC ACA TGG TAC GGC AAG CCG ACG GGC GCC
960 Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala GGC CCC AAG GAC AAC GGC GGC GCG TGC GGG TAC AAG GAC GTG GAC AAG
1445 Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys GCG CCG TTC AAC GGC ATG ACC GGG TGC GGC AAC ACC CCC ATC TTC AAG
1920 Ala Pro Phe Asn Gly ~et Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys GAC GGG CGC GGG TGC GGT TCC TGC TTC GAG ATC AAG TGC ACG AAG CCC

25 Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro GAG TCG TGC TCC GGC GAG GCC GTC ACC GTC CAC ATC ACC GAC GAC AAC
2880 Glu Ser Cys Ser Gly Glu Ala Val Thr Val His Ile Thr Asp Asp Asn GAG GAG CCC ATC GCG CCC TAC CAC TTC GAC CTT TCC GGC CAC GCG TTC
3365 Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe GGT TCC ATG GCG AAG AAG GGC GAG GAG CAG AAG CTG CGC AGC GCG GGC
3840 Gly Ser Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly 115 120 ~ 125 GAG CTG GAG CTG CAG TTT AGG CGG GTG AAG TGC AAG TAC CCC GAG GGC

45 Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly ACC AAG GTG ACC TTC CAC GTC GAG AAG GGT TCC AAC CCC AAC TAC CTG

50 Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu 145 15p 155 160 GCG CTG CTG GTG AAG ~AC GTC GAC GGC GAC GGC GAC GTG GTG GCG GTG
5285 Ala Leu Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val GAT ATC AAG GAG AAG GGC AAG GAC AAG TGG ATC GCG CTC AAG GAG TCA
5760 Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser TGG GGA GCC ATC TGG AGG GTG GAC ACC CCC GAC AAG CTG ACG GGC CCA

WO 94/21675 2 ~ ~ 7 ~ g ~

WO 94/21675 PCT/us94/02537 ~ 2~5~59~
TTC ACC GTT CGC TAC ACC ACC GAG GGA GGC ACC AAG TCC GAA GTT GAG
Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu GAC GTC ATC CCC GAG GGC TGG AAG GCC GAC GCC AGC TAC GAG TCC AAG

Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Ala Ser Tyr Glu Ser Lys TGA

(2) INFORMATION FOR SEQ ID NO:52:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp 30 Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro Phe Asn Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Ala Val Thr Val His Ile Thr Asp Asp Asn 45 Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly 115 120 . 125 Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val 0 Asp Ile Lys GlU Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Val Asp Thr Pro Asp Lys Leu Thr Gly Pro WO 94/2167~ ~ . . PCT/US94/02~37 r~ ' Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Ala Ser Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID No:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 723 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..720 (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 32 (D) OTHER INFORMATION: /note= "Xaa is Ser, Pro or Ala"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
ATC GCG AAG GTT CCC CCC GGC CCG AAC ATC ACG GCG ACC TAC GGC GAC

Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp AAG TGG CTT GAC GCG AAG AGC ACC TGG TAC GGC AAG CCG ACC GGN BCC

Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Xaa GGT CCC AAG GAC AAC GGC GGC GCG TGC GGA TAC AAG GAC GTG GAC AAG

Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys CCC CCG TTC AGC GGC ATG ACC GGC TGC GGC AAC ACC CCC ATC TTC AAG

Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys TCC GGC CGC GGC TGC GGC TCC TGC TTC GAG ATC AAG TGC ACC AAG CCC

Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro GAG TCC TGC TCC GGG GAG CCC GTC CTG GTC CAC ATC ACC GAC GAC AAC

Glu Ser Cys Ser Gly Glu Pro Val Leu Val His Ile Thr Asp Asp Asn GAG GAG CCC ATC GCC GCC TAC CAC TTC GAC CTC TCC GGC AAG GCG TTC

Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Lys Ala Phe WO 94/21675 ~ ~ S ~ ~ PCTrUS94/02537 GGG GCC ATG GCC AAG AAG GGT GAG GAG CAG AAG CTG CGC AGC GCC GGC

Gly Ala Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly GAG CTG GAG CTC AAG TTC CGC CGC GTC AAG TGC GAG TAC CCG AAG GGC

Glu Leu Glu Leu Lys Phe Arg Arg Val Lys Cys Glu Tyr Pro Lys Gly ACC AAG GTT ACC TTC CAC GTC GAG AAG GGG TCC AAC CCC AAC TAC CTT

Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu GCG CTG CTG GTG AAG TAC GTC GAC GGC GAC GGG GAC GTG GTG GCG GTG

Ala Leu Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val GAC ATC AAG CAG AAG GGC AAG GAC AAG TGG ATC GAG CTC AAG GAG TCG

Asp Ile Lys Gln Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser TGG GGA GCC GTC TGG AGG ATC GAC ACC CCC GAC AAG CTC ACC GGC CCC

Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro TTC ACC GTC CGC TAC ACC ACC GAG GGC GGC ACC AAG GCC GAA GCC GAG

Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ala Glu Ala Glu GAC GTC ATC CCC GAG GGC TGG AAG GCC GAC ACC GCC TAC GAG GCC AAG

Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ala Lys TGA

(2) INFORMATION FOR SEQ ID NO:54:
(i~ SEQUENCE CHARACTÉRISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 94/21675 ~15 75~ 6 PCT/US94/02537 ( ix ) FEATURE:
(A) NAME/KEY: Modified-site ( B ) LOCATION: 32 (D) OTHER INFORMATION: /note= "Xaa is Ser, Pro or Ala"

(xi) SEQUENCE DESCRIPTION: SEO ID NO:54:
Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Xaa Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys 20 Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Pro Val Leu Val His Ile Thr Asp Asp Asn Glu G1U Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Lys Ala Phe Gly Ala Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Lys Phe Arg Arg Val Lys Cys Glu Tyr Pro Lys Gly 35 Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Le~ Val Lys Tyr Val Asp Gly ASp Gly Asp Val Val Ala Val Asp Ile Lys Gln Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr GlU Gly Gly Thr Lys Ala GlU Ala Glu 50 Asp Val Ile Pro G1U Gly Trp Lys Ala Asp Thr Ala Tyr G1U Ala Lys W O 94/2l675 a~57~ PCTrUS94/02537 (2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 723 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..720 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
ATC GCG AAG GTG CCC CCG GGT CCG AAC ATC ACG GCG ACC TAC GGC GAC

Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp AAG TGG CTC GAC GCG AAG AGC ACA TGG TAC GGC AAG CCG ACG GGG GCC

Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala GGT CCC AAG GAC AAC GGC GGC GCT TGC GGG TAC AAG GAC GTG GAC AAG

Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys CCC CCG TTC AGC GGC ATG ACC GGC TGC GGC AAC ACC CCC ATC TTC AAG

Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys TCC GGC CGT GGC TGC GGC TCC TGC TTT GAG ATC AAG TGC ACG AAG CCC

Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro GAG GCC TGC TCC GGC GAG CCC GTG GTA GTC CAC ATC ACC GAC GAC AAC

Glu Ala Cys Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn GAG GAG CCC ATC GCC CCC TAC CAC TTC GAC CTC TCC GGC CAC GCG TTC

Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe GGG GCG ATG GCC AAG AAG GGC GAT GAG CAG AAG CTG CGC ACG GCC GGC

Gly Ala Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Thr Ala Gly GAG CTG GAG CTC CAG TTC CGG CGC GTC AAG TGC AAG TAC CCG GAG GGG

Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly WO 94/21675 21~ 7 ~ g R ~i PCT/US94/02537 ACC AAG GTG ACC TTC CAC GTG GAG AAG GGG TCC AAC CCC AAC TAC CTG

Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu GCG CTG CTT GTG AAG TAC GTT AAC GGC GAC GGA GAC GTG GTG GCG GTG

Ala Leu Leu Val Lys Tyr Val Asn Gly Asp Gly Asp Val Val Ala Val GAC ATC AAG GAG AAG GGC AAG GAC AAG TGG ATC GAG CTC AAG GAG TCG

Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser TGG GGA GCC ATC TGG AGG ATC GAC ACT CCC GAC AAG CTC ACG GGC CCC

Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro TTC ACC GTC CGC TAC ACC ACC GAG GGC GGC ACC AAG ACC GAA GCC GAG

Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Thr Glu Ala Glu GAC GTC ATC CCT GAG GGC TGG AAG GCC GAC ACC AGC TAC G~ AG TCC AAG

Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Glu Ser Lys TGA

35 (2) INFORMATION FOR SEQ ID NO: 56:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys 5 Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys
6~
Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ala Cys Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Se~ Gly His Ala Phe W O 94121675 - 2 ~ S ~ S 9 6 PCT~US94/02537 Gly Ala Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Thr Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asn Gly Asp Gly Asp Val Val Ala Val 5 Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Thr Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID No:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 45 (D) OTHER INFORMATION: /note= "Xaa is Asn or Asp"
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 144 (D) OTHER INFORMATION: /note= "Xaa is Asp or Gly"
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 154 (D) OTHER INFORMATION: /note= "Xaa is Gly or Ala"
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 187 (D) OTHER INFORMATION: /note= "Xaa is Ile or Thr"
-(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 223 (D) OTHER INFORMATION: /note= "Xaa is Val or Phe"

WO 94/21675 21 5 7 ~ PCT/US94/02~37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Xaa Val Asp Lys Ala Pro Phe Asn Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Ala Val Thr Val Thr Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys Lys Gly Glu GlU Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Asp Xaa Thr Lys Pro Thr Phe His Val Glu Lys Xaa Ser Asn Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Gly ASp Gly Asp Val Val Ala Val Asp Ile Lys GlU Lys Gly Lys Asp Lys Trp Xaa Glu Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu Xaa Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser Ala Lys (2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME~KEY:

WO 94/21675. 2~ 5~ ~ PCTrUS94/02537 (B) LOCATION: 199 (D) OTHER INFORMATION: /note= "Xaa is Val or Ile"
(ix) FEATURE:
( A) NAME/KEY:
(B) LOCATION: 235 (D) OTHER INFORMATION: /note= "Xaa is Ala or Thr (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Ala Pro Phe Asn Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Ala Val Thr Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly WO 94/21675 21 5 7 5 ~ 6 PCTtUS94tO2~37 Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Xaa Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Xaa Ser Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ala Cys Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ala Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Thr Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly WO 94/21675 PCTrUS94/02537 35~
Thr Lys Val Thr Phe His val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asn Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys G1U Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Thr Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME~KEY:
(B) LOCATION: 88 (D) OTHER INFORMATION: /note= "Xaa is Val or Ile"
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 90 (D) OTHER INFORMATION: /note= "Xaa is Val or Ile"
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION: 180 (D) OTHER INFORMATION: /note= "Xaa is Gln or GlU"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Gly Xaa Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu Pro Xaa Leu Xaa His Ile Thr Asp Asp Asn WO 94/21675 ,~ j 9 ~ PCT/US94/02537 Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Lys Ala Phe Gly Ala Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Lys Phe Arg Arg Val Lys Cys Glu Tyr Pro Lys Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Xaa Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ala Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ala Lys (2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asp Gly Asp (2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID No:62 W O 94/21675 , PCTrUs94/02537 ~515~
Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Val Asn Gly Asp _ (2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys (2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Gly Asp Val Val Ala Val Asp Ile Lys Gln Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys (2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

WO 94121675 ~59~ PCTIUS94/02~37 Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr (2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
Glu Ser Trp Gly Ala Ile Trp Arg Val Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr (2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: .20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Thr Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
Ala Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala W O 94/21675 2 ~ ~ 7 ~ ~ ~ PCTrUS94/02537 Tyr Glu Ala Lys (2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Ala Ser l 5 10 15 Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Glu Ser Lys (2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
GGGTCTAGAG GTACCGTCCG ATCGATCATT

9~

(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
AATGATCGAT GCT

(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73 GGGTCTAGAG GTACCGTCCG

(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
CCCTGCAGAT TATTTGAGAT CTTGAG

(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 60 (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

WO 94/21675 2 ~ 57 ~ 9 6 PCT/US94/02537 CCCTGCAGTC ATGCTCACTT GGCCGAGTA

(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
GAGTACGGCG ACAAGTGGC

(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
TTCGAGATCA AGTGCACC

(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

~7~9~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
GTGACAGCCT CGCCGG

(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
GGGAATTCCA TGGCGAAGAA GGGC

(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
GTGCCGTCCG GGTACT

(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
CCGTCGACGT ACTTCA

.

W O 94/21675 ~ ~ ~57 5~ PCTrUS94/02537 (2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
GGAGTCGTGG GGAGCAGTC

(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
GGGTCTAGAG GTACCGTCC

(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
TTGGATCCTA CGGCAAGCCG ACCGGC

(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:

WO 94/21675 . ` , . ~ PCT/US94/02537 2 1 5 ~

TTGGATCCAT CCCGAAGGTG CCCCCGGG

(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
AGGTGACCTT CCACGTCG

(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
TTGGATCCTG GCGCTGCTGG TGAAGTA

(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

WO 94/21675 2~!~15~ PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
TTGAATTCAT CCCGAAGGTG CCCCCG

(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
TTGGTACCTC ACTTGGACTC GTAGCT

(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
CCGAATTCGT GGAGAAGGGG TCCAA

(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 22 (D) OTHER INFORMATION: /note= "Xaa is Iosine"
7~ 57 5 ~ 6 PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
TTAGGATCCT CACTTATCAT ANGACGTATC

(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
TTGAATTCCT TGTCATTGCC CTTCTG

(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:93:
AAGAATTCCT TCTGCTTGAT GTCCAC

40 (2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
ATGAATTCGA GTCGTGGGGA GCCGTC

WO 94/21675 157 $36 PCT/US94/02537 (2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
ATGAATTCGT CTGGAGGATC GACACC

(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
ATGAATTCAT CGCAAAGGTT CCCCCC

(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
TTTGGATCCT CACTTGGACT CGTAGCT

(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:

WO 94/21675 215 7 ~ 9 6 PCT/US94/02537 TTGAATTCTC GCGAAGGTGC CCCCG

Claims (71)

Claims
1. An isolated peptide of Lol p I or an isolated portion thereof, said peptide or portion thereof comprising at least one T cell epitope of Lol p I, said peptide comprising an amino acid sequence selected from the group consisting of: LPI- 1 (SEQ ID NO: 4).1, LPI-2 (SEQ ID NO: S), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ
ID NO: 7), LPI-4.1 (SEQ ID NO: 8), LPI-8 (SEQ ID NO: 12), LPI-10 (SEQ ID
NO: 14), LPI-11 (SEQ ID NO: 15), LPI-13 (SEQ ID NO: 19), LPI-15 (SEQ ID
NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ
ID NO: 25), LPI-19 (SEQ ID NO: 26), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30), all as shown in fig. 2.
2. An isolated peptide of Lol p I or an isolated portion thereof, said peptide or portion thereof comprising at least one T cell epitope of Lol p I, said peptide having an amino acid sequence selected from the group consisting of: LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 373, LPI-16.10 (SEQ ID NO: 38), LPI-18.5 (SEQ ID
NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID
NO: 50), all as shown in Fig. 4.
3. An isolated peptide or portion thereof according to claim 1, wherein said portion of a peptide has a mean T cell stimulation index approximately equivalent to or greater than the mean T cell stimulation index of the corresponding peptide shown in Fig. 3.
4. An isolated peptide or portion thereof of claim 1 or 2 which comprises at least two T cell epitopes.
5. An isolated peptide or portion thereof of claim 1 or 2 which induces T cell nonresponsiveness or modifies the lymphokine secretion profile of appropriate T
cell subpopulations.
6. An isolated peptide or portion thereof of claim 1 or 2 which, when administered to an individual sensitive to an allergen of the family, Poacea induces T cell anergy or modifies the lymphokine secretion profile of approprate T cell populations.
7. A portion of an isolated peptide of claim 1 or 2 which has a mean T cell stimulation index of at least 3.5.
8. An isolated peptide or a portion thereof of claim 1 or 2 which does not bind immunoglobulin E specific for Lol p I in a substantial percentage of individuals sensitive to Lol p I, or if binding of the peptide or portion thereof to said immunoglobulin E occurs, such binding does not result in release of mediators from mast cells or basophils in a substantial percentage of individuals sensitive to Lol p I.
9. An isolated peptide of claim 1 or 2 which binds immunoglobulin E to a substantially lesser extent than purified native Lol p I binds immunoglobulin E.
10. An isolated peptide or portion thereof of claim 1 or 2 which, when administered to an individual sensitive to Lol p I allergen, modifies the allergic response of the individual to ryegrass pollen allergen.
11. An isolated peptide or portion thereof of claim 1 or 2 which, when administered to an individual sensitive to an allergen of the family Poacea, modifies the allergic response of the individual to said allergen.
12. A portion of an isolated peptide of claim 1 or 2 wherein said portion comprises at least 15 amino acid residues.
13. An isolated nucleic acid having a sequence encoding all or a portion of a peptide of claim 1 or 2.
14. A functional equivalent of a nucleic acid sequence encoding all or a portion of a peptide of claim 1 or 2.
15. An isolated peptide that is immunologically cross-reactive with T cells reactive with a peptide of claim 1 or 2.
16. An isolated peptide or portion thereof of Lol p I, said peptide or portion thereof comprising at least one T cell epitope of Lol p I, said peptide having apositivity index of at least about 100 and mean T cell stimulation index of at least about 3.0 determined in a population of individuals sensitive to said protein allergen.
17. An isolated peptide or portion thereof of claim 16 wherein said population of individuals is at least thirty individuals.
18. An isolated peptide or portion thereof of claim 17 wherein said population of individuals is at least thirty-five individuals.
19. An isolated peptide or portion thereof of claim 17 wherein said mean T
cell stimulation index is at least about 4Ø
20. An isolated peptide or portion thereof of claim 17 wherein said mean T
cell stimulation index is at least about 6Ø
21. A peptide or portion thereof of claim 17 wherein said peptide is selected from the group consisting of: LPI-2 (SEQ ID NO: 5), LPI-11 (SEQ ID NO:15), LPI-13 (SEQ ID NO: 19), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30).
22. An isolated peptide of Lol p I, or a portion thereof wherein said peptide isselected from the group consisting of: LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID
NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID
NO:8), LPI-8 (SEQ ID NO: 12), LPI- 10 (SEQ ID NO: 14), LPI-11 (SEQ ID
NO: 15), LPI-13 (SEQ ID NO: 19), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-19 (SEQ
ID NO: 26), LPI-22 (SEQ ID NO: 29), LPI-23 (SEQ ID NO: 30), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ ID
NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO: 50) or portion thereof.
23. A modified peptide or a modified portion of a peptide of claim 22.
24. A modified peptide of claim 23 wherein said peptide is selected from the group consisting of: LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID
NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), and LPI-16.10 (SEQ ID NO: 38), all as shown in Fig. 4.
25. A modified peptide or a modified portion of a peptide of claim 23 or 24 which does not bind immunoglobulin E specific for Lol p I in a substantial percentage of individuals sensitive to Lol p I, or if binding of the peptide or portion thereof to said immunoglobulin E occurs, such binding does not result inrelease of mediators from mast cells or basophils in a substantial percentage ofindividuals sensitive to Lol p I.
26. A modified peptide or a modified portion of a peptide of claim 23 or 24 which modifies, in an individual sensitive to Lol p I or an immunologically related allergen, the allergic response of the individual to Lol p I allergen or said related allergen.
27. An isolated peptide comprising at least two regions, each region comprising at least one T cell epitope of Lol p I, said regions each comprising all or a portion of an amino acid sequence selected from the group consisting of:
LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID NO: 8), LPI-8 (SEQ ID NO: 12), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-13 (SEQ ID NO: 19), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), LPI-23 (SEQ ID NO: 30), LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID

NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ ID NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID
NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO: 50).
28. An isolated peptide of claim 27 wherein said regions comprise an amino acid sequence selected from the group consisting of: LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID
NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), LPI-23 (SEQ ID
NO: 30), LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ ID
NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO: 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20.5 (SEQ ID NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID
NO: 49), and LPI-23.4 (SEQ ID NO: 50), or a portion thereof containing at least two Lol p I epitopes.
29. An isolated peptide of Lol p I, wherein said peptide comprises a combination of regions selected from the group consisting of:
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ
ID NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), and LPI-11 (SEQ ID NO: 15);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16 (SEQ ID
NO: 22);

LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16.1 (SEQ ID
NO: 23);
LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO:
21), and LPI-16.1 (SEQ ID NO: 23);
LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO:
21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), and LPI-20 (SEQ
ID NO: 27);
LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO:
21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID
NO: 27), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30);
LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO:
25), and LPI-20 (SEQ ID NO: 27);
LPI-15 (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO:
25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ
ID NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO:
29), and LPI-23 (SEQ ID NO: 30);
LPI-18 (SEQ ID NO: 25) and LPI-20 (SEQ ID NO: 27);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-23 (SEQ ID
NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-16.1 (SEQ ID
NO: 23);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30) and LPI-16.1 (SEQ ID NO: 23);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23) and LPI-11 (SEQ ID NO: 15);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23) and LPI-4.1 (SEQ ID NO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16 (SEQ ID NO: 23).1, LPI-4.1 (SEQ ID NO: 8) and LPI-22 (SEQ
ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23), LPI-11 (SEQ ID NO: 15) and LPI-4.1 (SEQ
ID NO: 8);

LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23), LPI-11 (SEQ ID NO: 15), LPI-4.1 (SEQ ID
NO: 8) and LPI-22 (SEQ ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO:
29), and LPI-23 (SEQ ID NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID NO:
23), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30); and LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID NO:
23) and LPI-22 (SEQ ID NO: 29).
30. An isolated peptide of Lol p I, wherein said peptide comprises a combination of regions selected from the group consisting of:
LPI-16.2 (SEQ ID NO: 31), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.3 (SEQ ID NO: 32), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.4 (SEQ ID NO: 33), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.5 (SEQ ID NO: 34), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.6 (SEQ ID NO: 35), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.7 (SEQ ID NO: 36), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.9 (SEQ ID NO: 37), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30); and LPI-16.10 (SEQ ID NO: 38), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30).
31. A monoclonal antibody, polyclonal antibody, or immunoreactive fragment thereof specifically reactive with a peptide of claim 1 or 2.
32. An isolated peptide produced in a host cell transformed with the nucleic acid of claim 13.
33. An isolated peptide produced in a host cell transformed with the nucleic acid of claim 14.
34. An isolated nucleic acid having a sequence encoding a peptide of claim 27 or 29.
35. The functional equivalent of an isolated nucleic acid sequence encoding a peptide of claim 27 or 29.
36. An isolated peptide produced in a host cell transformed with the nucleic acid of claim 34.
37. An expression vector comprising a nucleic acid sequence coding for a peptide of claim 1 or 2.
38. An expression vector comprising the functional equivalent of a sequence coding for a peptide of claim 1 or 2.
39. An expression vector comprising a nucleic acid sequence coding for a peptide of claim 27 or 29.
40. An expression vector comprising the functional equivalent of a nucleic acid sequence coding for a peptide of claim 27 or 29.
41. All or a portion of an isolated peptide of Lol p 1, said peptide or portion thereof comprising at least one T cell epitope of said protein allergen, said peptide having the formula Xn-Y-Zm, wherein Y is an amino acid sequence selected from the group consisting of: LPI-1 (SEQ ID NO: 3), LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ
ID NO: 8), LPI-5 (SEQ ID NO: 9), LPI-6 (SEQ ID NO: 10), LPI-7 (SEQ ID
NO: 11), LPI-8 (SEQ ID NO: 12), LPI-9 (SEQ ID NO: 13), LPI-10 (SEQ ID
NO: 14), LPI-11 (SEQ ID NO: 15), LPI-12 (SEQ ID NO: 17), LPI-13 (SEQ ID
NO: 19), LPI-14 (SEQ ID NO: 20), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-16.1 (SEQ ID NO: 23), LPI-17 (SEQ ID NO: 24), LPI-18 (SEQ
ID NO: 25), LPI-19 (SEQ ID NO: 26), LPI-21 (SEQ ID NO: 28), LPI-22 (SEQ

LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO: 50) wherein Xn are amino acid residues contiguous to the amino terminus of Y in the amino acid sequence of said protein allergen, wherein Zm are amino acid residues continuous to the carboxy terminus of Y in the amino acid sequence of said protein allergen, wherein n is 0-30 and wherein m is 0-30.
42. A portion of an isolated peptide of claim 40 wherein the portion comprises at least fifteen amino acid residues.
43. A composition comprising at least one isolated peptide or a portion thereof of claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.
44. A composition comprising at least one isolated peptide or portion thereof of claim 23 or 24 and a pharmaceutically acceptable carrier or diluent.
45. A therapeutic composition comprising an isolated peptide or portion thereof of claim 27 or 29 and a pharmaceutically acceptable carrier or diluent.
46. Use of a composition of claim 43 in the manufacture of a medicament for treating sensitivity to Lol p I protein allergen or an allergen which is immunologically cross reactive with Lol p I protein allergen.
47. Use of a composition of claim 44 in the manufacture of a medicament for treating sensitivity to Lol p J protein allergen or an allergen which is immunologically cross reactive with Lol p I protein allergen.
48. Use of at least two compositions of claim 43 in the manufacture of medicament for treating sensitivity to Lol p I protein allergen or an allergen which is immunologically cross reactive with Lol p I protein allergen.
49. The use of the composition of claim 46 in the manufacture of a medicament for treating sensitivity to Lol p I protein allergen, wherein said immunologically cross reactive allergen is Dac g I, Poa p I or Phl p I.
50. A method of detecting sensitivity to Lolp I protein allergen or an immunologically cross reactive allergen in an individual, comprising combining a blood sample obtained from the individual with at least one peptide of claim 1 or 2, in vitro, under conditions appropriate for binding of blood components with the peptide, and determining extent to which such binding occurs as indicative of sensitivity in the individual to ryegrass pollenallergen or said immunologically cross-reactive allergen.
51. A method of claim 50 wherein the extent to which binding occurs is determined by assessing T cell function, T cell proliferation or a combination thereof.
52. A composition comprising a pharmaceutically acceptable carrier or diluent and at least two peptides, selected from the group consisting of: LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID NO: 5), LPI-3 (SEQ ID NO: 6), LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID NO: 8), LPI-8 (SEQ ID NO: 12), LPI-11 (SEQ ID NO: 15), LPI-13 (SEQ ID NO: 19), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID NO: 22), LPI-16.1 (SEQ ID NO. 23), LPI-18 (SEQ ID NO; 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), LPI-23 (SEQ ID
NO: 30), LPI-16.2 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.
(SEQ ID NO: 33), LPI-16.5 (SEQ ID NO: 34), LPI-16.6 (SEQ ID NO: 35), LPI-16.7 (SEQ ID NO: 36), LPI-16.9 (SEQ ID NO: 37), LPI-16.10 (SEQ ID
NO: 38), LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 40), LPI-18.7 (SEQ ID NO 41), LPI-18.8 (SEQ ID NO: 42), LPI-20.2 (SEQ ID NO: 43), LPI-20.3 (SEQ ID NO: 44), LPI-20.4 (SEQ ID NO: 45), LPI-20. (SEQ ID
NO: 46), LPI-20.6 (SEQ ID NO: 47), LPI-23.1 (SEQ ID NO: 48), LPI-23.2 (SEQ ID NO: 49), and LPI-23.4 (SEQ ID NO: 50) and wherein said composition comprises a sufficient percentage of the T cell epitopes of said protein allergen such the T cells of an individual sensitive to Lol p I protein pollen or an immunologically cross reactive allergen, are tolerized to said an least one protein allergen.
53. A composition of clam 45 comprising a combination of peptides selected from the group consisting of:
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID NO:
14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), LPI-16 (SEQ ID
NO: 22), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: 30);
LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: 8), LPI-10 (SEQ ID
NO: 14), and LPI-11 (SEQ ID NO: 15);

LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23) and LPI-11 (SEQ ID NO: 15);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23) and LPI-4.1 (SEQ ID NO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23), LPI-4.1 (SEQ ID NO: 8) and LPI-22 (SEQ
ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23), LPI-11 (SEQ ID NO: 15) and LPI-4.1 (SEQ
ID NO: 8);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO:
30), LPI-16.1 (SEQ ID NO: 23), LPI-11 (SEQ ID NO: 15), LPI-4.1 (SEQ ID
NO: 8) and LPI-22 (SEQ ID NO: 29);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO:
29), and LPI-23 (SEQ ID NO: 30);
LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID NO:
23), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 30); and LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-16.1 (SEQ ID NO:
23) and LPI-22 (SEQ ID NO: 29).
54. A composition of claim 43 comprising a combination of peptides selected from the group consisting of:
LPI-16.2 (SEQ ID NO: 31), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.3 (SEQ ID NO: 32), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.4 (SEQ ID NO: 33), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.5 (SEQ ID NO: 34), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.6 (SEQ ID NO: 35), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);
LPI-16.7 (SEQ ID NO: 36). LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30);

LPI-16.9 (SEQ ID NO: 37), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30); and LPI-16.10 (SEQ ID NO: 38), LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO:
27), and LPI-23 (SEQ ID NO: 30).
55. Use of composition of claim 52, 53 or 54 in the manufacture of a medicament for use in treating sensitivity to Lol p I allergen or an immunologically cross-reactive allergen.
56. A method of designing antigenic fragments of Lol p I, which when administered to ryegrass pollen sensitive individuals in sufficient quantity will modify the individual's allergic reaction to ryegrass pollen comprising the steps of:
(a) recombinantly or synthetically producing peptides of Lol p I;
(b) examining said peptides for their ability to influence B cell and/or T cell responses in ryegrass pollen sensitive individuals;
(c) selecting appropriate peptides which contain epitopes recognized by the cells, and (d) combining epitope-containing regions to include multiple epitopes in one peptide.
57. A method of designing antigenic fragments of Lol p I, which when administered to ryegrass pollen sensitive individuals in sufficient quantity will modify the individual's allergic reaction to ryegrass pollen comprising the steps of:
(a) recombinantly or synthetically producing peptides of Lol p I;
(b) examining said peptides for their ability to influence B cell and/or T cell responses in ryegrass pollen sensitive individuals; and (c) selecting appropriate peptides which contain epitopes recognized by the cells.
58. A T cell capable of recognizing a peptide of claim 1 or 2.
59. A receptor of a T cell capable of recognizing a peptide of claim 1 or 2.
60. An isolated nucleic acid having a nucleotide sequence coding for Dac g I, orthe functional equivalent of said nucleotide sequence.
61. An isolated nucleic acid sequence of claim 60 wherein said nucleotide sequence comprises the nucleotide sequence of Fig. 5.
62. An expression vector comprising a nucleotide sequence coding for Dac g I, orthe functional equivalent of said nucleotide sequence.
63. A host cell transformed to express a protein encoded by the nucleic acid of claim 60.
64. Isolated Dac g I protein produced in a host cell transformed with the nucleic acid of claim 60.
65. An isolated nucleic acid having a nucleotide sequence coding for Poa p I, orthe functional equivalent of said nucleotide sequence.
66. An isolated nucleic acid sequence of claim 65 wherein said nucleotide sequence comprises the nucleotide sequence of Fig. 6.
67. An expression vector comprising a nucleotide sequence coding for Poa p I, orthe functional equivalent of said nucleotide sequence.
68. A host cell transformed to express a protein encoded by the nucleic acid of claim 65.
69. Isolated Poa p I protein produced in a host cell transformed with the nucleic acid of claim 60.
70. An isolated protein allergen that is immunologically related to Lol p I.
71. An isolated protein allergen of claim 70 wherein said protein allergen is Dac g I
or Poa p I.
CA002157596A 1993-03-12 1994-03-09 T cell epitopes of ryegrass pollen allergen Abandoned CA2157596A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3100193A 1993-03-12 1993-03-12
US08/031,001 1993-03-12

Publications (1)

Publication Number Publication Date
CA2157596A1 true CA2157596A1 (en) 1994-09-29

Family

ID=21857138

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002157596A Abandoned CA2157596A1 (en) 1993-03-12 1994-03-09 T cell epitopes of ryegrass pollen allergen

Country Status (10)

Country Link
EP (1) EP0688338A1 (en)
JP (1) JPH08509966A (en)
AU (1) AU684501B2 (en)
CA (1) CA2157596A1 (en)
FI (1) FI954269A (en)
IL (1) IL108940A0 (en)
NO (1) NO953571L (en)
NZ (1) NZ263913A (en)
WO (1) WO1994021675A2 (en)
ZA (1) ZA941708B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7485708B2 (en) 1996-09-23 2009-02-03 University Of Arkansas Nucleic acids encoding ara h 3 polypeptides
US7879977B2 (en) 1998-01-31 2011-02-01 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy
US8153414B2 (en) 2000-04-06 2012-04-10 Allertein Therapeutics, Llc Microbial delivery system
US8246945B2 (en) 2000-04-06 2012-08-21 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858689A (en) * 1993-07-22 1999-01-12 Ludwig Institute For Cancer Research Isolated peptides derived from the gage tumor rejection antigen precursor and uses thereof
SE9402089D0 (en) * 1994-06-14 1994-06-14 Rudolf Valenta Recombinant allergen, fragments thereof, corresponding recombinant DNA molecules, vectors and hosts containing the DNA molecules, diagnostic and therapeutic uses of said allergens and fragments
US6759234B1 (en) 1994-09-02 2004-07-06 Immulogic Pharmaceutical Corporation Compositions and methods for administering to humans, peptides capable of down regulating an antigen specific immune response
EP0783322A1 (en) * 1994-09-02 1997-07-16 Immunologic Pharmaceutical Corporation Peptide compositions capable of down regulating an antigen specific immune response
DK1612555T3 (en) 1996-03-21 2009-02-09 Circassia Ltd Cryptic peptides for use in inducing immunological tolerance
US5817463A (en) * 1996-06-28 1998-10-06 Abbott Laboratories Nucleic acid primers and probes for detecting Mycoplasma pneumoniae
FR2809415B1 (en) * 2000-05-29 2004-10-08 Tabacs & Allumettes Ind CLONING AND SEQUENCING OF DAC G1 ALLERGEN FROM DACTILYS GLOMERATA POLLEN, ITS PREPARATION AND ITS USE
AUPR779201A0 (en) * 2001-09-20 2001-10-11 University Of Melbourne, The Immunotherapeutic and immunoprophylactic reagents
AU2003213861B2 (en) * 2002-04-02 2010-04-29 Circassia Limited Immunotherapeutic and immunoprophylactic reagents
AUPS148202A0 (en) * 2002-04-02 2002-05-09 Monash University Immunotherapeutic and immunoprophylactic reagents
GB0710529D0 (en) 2007-06-01 2007-07-11 Circassia Ltd Vaccine
US8551493B2 (en) 2007-08-15 2013-10-08 Circassia Limited Peptide with reduced dimer formation
EP2891662A1 (en) * 2009-02-05 2015-07-08 Circassia Limited Grass peptides for vaccine
EP2249162A1 (en) * 2009-04-30 2010-11-10 Stallergenes Sa Method for grass species identification
US11013781B2 (en) 2015-07-01 2021-05-25 Alk-Abelló As Peptide combinations and uses thereof for treating grass allergy
EP3672600A4 (en) 2017-08-21 2021-05-19 Glycom A/S Synthetic composition for reducing allergy symptoms

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1340729C (en) * 1988-03-23 1999-09-07 Mohan Bir Singh Ryegrass pollen allergen
WO1992003550A1 (en) * 1990-08-17 1992-03-05 The University Of Melbourne Ryegrass pollen allergen
CA2115579C (en) * 1991-08-16 2008-03-25 Mohan Bir Singh Ryegrass pollen allergen

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7485708B2 (en) 1996-09-23 2009-02-03 University Of Arkansas Nucleic acids encoding ara h 3 polypeptides
US7879977B2 (en) 1998-01-31 2011-02-01 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy
US8153414B2 (en) 2000-04-06 2012-04-10 Allertein Therapeutics, Llc Microbial delivery system
US8246945B2 (en) 2000-04-06 2012-08-21 University Of Arkansas Methods and reagents for decreasing clinical reaction to allergy
US8815251B2 (en) 2000-04-06 2014-08-26 Allertein Therapeutics, Llc Microbial delivery system

Also Published As

Publication number Publication date
NZ263913A (en) 1997-10-24
FI954269A0 (en) 1995-09-12
WO1994021675A2 (en) 1994-09-29
ZA941708B (en) 1994-10-05
JPH08509966A (en) 1996-10-22
NO953571L (en) 1995-11-10
FI954269A (en) 1995-09-12
WO1994021675A3 (en) 1994-11-10
EP0688338A1 (en) 1995-12-27
IL108940A0 (en) 1994-06-24
AU684501B2 (en) 1997-12-18
AU6517594A (en) 1994-10-11
NO953571D0 (en) 1995-09-11

Similar Documents

Publication Publication Date Title
CA2157596A1 (en) T cell epitopes of ryegrass pollen allergen
AU678412B2 (en) Allergenic proteins and peptides from Johnson grass pollen
JP3575802B2 (en) Allergenic proteins and peptides from Japanese cedar pollen
WO1994001560A1 (en) Allergenic proteins and peptides from japanese cedar pollen
US5710126A (en) T cell epitopes of ryegrass pollen allergen
AU5668194A (en) Allergenic proteins and peptides from japanese cedar pollen
WO1994011512A9 (en) Allergenic proteins and peptides from japanese cedar pollen
NZ271818A (en) T cell epitopes of ryegrass pollen antigen
NZ252013A (en) T cell receptor epitopes for short ragweed pollen
AU679455B2 (en) T cell epitopes of ryegrass pollen allergen
JPH08502163A (en) Allergenic proteins and peptides derived from cedar pollen
US6335019B1 (en) Methods for treating sensitivity to protein allergen using peptides which include a T cell epitope recognized by a T cell receptor specific for the protein allergen
KR20040004481A (en) Nucleic acids encoding a house dust mite allergen, der p iii, and uses therefor
US6090386A (en) T cell peptides of the CRX JII allergen
US7112333B1 (en) T cell epitopes of ryegrass pollen allergen
WO1992016554A1 (en) Protein allergens of the species cynodon dactylon
AU3438493A (en) Allergenic proteins and peptides from japanese cedar pollen

Legal Events

Date Code Title Description
FZDE Discontinued