AU684501B2 - T cell epitopes of ryegrass pollen allergen - Google Patents

Description

WO 94/21675 PCT/US94/02537 T CELL EPITOPES OF RYEGRASS POLLEN ALLERGEN Background of the 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 The Antigens, 3:271-359, Academic Press Inc., London, New York)., Hill et al. (1979) Medical Journal ofAustralia, 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, III 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 designated Lol p I, Lol p II, Lolp Ill, and Lol p IV. Another important Lolium perenne allergen which has been identified in the literature 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 allergen's in grasses.

These proteins have been identified in pollen from ryegrass, Loliumperenne, and act as antigens in triggering immediate (Type 1) hypersensitivity in susceptible humans.

Lolp 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 properties have been assessed by direct skin testing of grass pollan-sensitive patients. The results showed that 84% had a skin sensitivity to Lolp I (Freidhoff, et al., (1986) J. Allergy Clin. Immunol., 78:1190-1201) demonstrating the primary importance of this protein as the major allergen.

Furthermore, 95% of patients demonstrated to be grass pollen-sensitive possessed specific IgE antibody that bound to Lolp I, as demonstrated by immunoblotting (Ford and Baldo (1986) International Archives of Allergy and Applied Immunology, 81:193- 203).

Substantial allergenic cros"-reactivity between grass pollens has been demonstrated using an IgE-binding assay, the radioallergo-sorbent test (RAST), for example, 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 demonstrated using both polyclonal and monoclonal antibodies Smart and Knox (1979) International Archives of Allergy and ~C13~Y WO 94/21675 PCT/US94/02537 Applied Immunology, 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 immunochemically similar to Lolp I (Singh and Knox, supra).

Grasses that may be considered immunochemically related to Lol p I and that comprise allergens which may be considered immunologically cross-reactive with antibody to Lolp I include: Pooid (festucoid) grasses of the Poaceae (Gramineae) 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; L.multiflorum, Italian ryegrass; Poa pratensis, Kentucky 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 arundinacea, 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 immunologically related grass allergens, or in treating sensitivities to such allergens, or in assisting in the manufacture of medicaments to treat such sensitivities. The present invention provides materials and methods having one or more of those utilities.

Summary of the Invention 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 Lol 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.

i i WO 94/21675 PCT/US94/02537 The invention also provides modified peptides having similar or enhanced therapeutic or diagnostic properties as the corresponding, naturallyoccurring allergen or portion thereof, but also having advantageous physical or biological properties, such as reduced side effects, reduced IgE binding, improved solubility, increased in vitro or in vivo T cell stimulating ability, increased stability or the like. Preferred peptides of the invention are capable of modifying, in a Lolp I-sensitive individual to whom they are administered, the allergic response of the individual to Lol p I or an allergen immunologically cross-reactive with Lolp I, allergens derived from pollen belonging to the Poaceae (Gramineae) family, such as Dactylis glomerata (Dac g Poa pretensis (Poa p I) and Phleum pratense (Phi p as discussed above.

The present invention also provides non-native recombinant or chemically synthesized) 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 fragments 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 fragments of Dac g I and Poa p I prepared synthetically. The present invention further provides nucleic acid sequences coding for Dac g I, Poa p I and fragments 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 Lolp I.

Methods of treatment and of diagnosis of sensitivity to ryegrass pollen protein, Lolp I, or to pollen proteins that are immunologically related to Lolp I (such as Dac g I, Ph p I and Poa p 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 embodiments of the invention in conjunction with the appended figures.

Brief Description of the Figures WO 94/21675 PCT/US94/02537 Fig. 1 shows the nucleotide sequence of cDNA clone 26.j (SEQ ID NO 1) and its predicted amino acid sequence (SEQ ID NO: 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 LPI-4.1 (SEQ ID NO: 8) and LPI-16.1 (SEQ ID NO: 23)) or homologues of peptides derived from Lolp I LPI-11 (SEQ ID NO: 15), and LPI-12 (SEQ ID NO: 17)).

Fig. 3 is a graphic representation depicting responses ofT cell lines from thirty-five grass-sensitive patients primed in vitro with purified native Lolp I and analyzed for response to various Lol p I peptides by percent of positive responses (with an S.I. of at least two, shown over each bar), the mean stimulation 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).

Fig. 4 shows various peptides of desired lengths derived from Lol p I (SEQ ID NO: 23, 25, 27, 30-50).

Fig. 5 shows the nucleotide sequence of cDNA clone 106.5 (SEQ ID NO: 51) and its predicted amino acid sequence (SEQ ID NO: 52). Clone 106.5 is a PCRgenerated, 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 PCRgenerated, full-length clone of Poa p I.

Fig. 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), Phlp I (SEQ ID NO: 59), and Poa p I (SEQ ID NO: 60), including polymorphisms thereof.

Fig. 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 ID NO: 3-50). The present invention also provides Dac g I and Poa p I protein allergens which are immunologically cross-reactive with Lol p I. The term "peptide" WO 94/21675 PCT/US94/025S37 as used herein refers to any protein fragment of Lol p I that induces an immune response. The terms "fragment" and "antigenic fragment" of a protein as used interchangeably 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 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 medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically. Preferred peptides of the invention include peptides derived 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 Lolp I protein allergen are particularly desirable for increased therapeutic effectiveness.

Peptides that are immunologically related 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 recognized by antibodies specific for a peptide of Lol p I.

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 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 medium suitable for the cells. Peptides can be purified from cell culture medium, host cells, or both, using techniques known in the art for purifying peptides and proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, 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 peptides of the invention, or at least a portion thereof, may be expressed in bacterial IWO 94/21675 PCT/US94/02537 cells such as E. coli, insect cells, yeast, or mammalian 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: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Other suitable expression vectors, promoters, enhancers, and other expression elements are known to those skilled in the art.

Expression in mammalian, 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 YepSecl (Baldari et al. (1987) Embo 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 and mammalian expression systems are also available. For example, a baculovirus system is commercially available (PharMingen, San Diego, CA) for expression in insect cells while the pMSG vector is commercially available (Pharmacia, Piscataway, NJ) for expression in mammalian 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 Biolabs, Beverly, MA); pRIT5 (Pharmacia, Piscataway, NJ); pET-11d (Novagen, Madison, WI) Jameel 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, pSEM and pGEX will lead to the expression of allergen fused to maltose E binding protein (pMAL), protein A (pRIT5), truncated B-galactosidase (PSEM), or glutathione S-transferase (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 Lolp I peptide may then be recovered from the fusion protein through enzymatic cleavage at the enzymatic site and biochemical purification using conventional techniques for purification of proteins and peptides. Suitable enzymatic cleavage sites include those for blood clotting Factor Xa or thrombin for which the appropriate enzymes and protocols for cleavage are commercially available from, for example, Sigma Chemical Company, St. Louis, MO and N.E. Biolabs, Beverly, MA. The different vectors also have different promoter regions allowing constitutive or inducible expression with, for example, IPTG induction (PRTC, Amann et al., (1988)

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WO 94/21675 PCT/US94/02537 supra; pET-lld, Novagen, Madison, WI) or temperature induction Pharmacia, Piscataway, NJ). It may also be appropriate to express recombinant Lol p I peptides in different E. coli hosts that have an altered capacity to degrade recombinantly expressed proteins U.S. Patent 4,758,512). Alternatively, it may be advantageous to alter the nucleic acid sequence to use codons preferentially utilized by E. coli, where such nucleic acid alteration would not affect the amino acid sequence of the expressed protein.

Host cells can be transformed to express the nucleic acid sequences of the invention using conventional techniques such as calcium phosphate or calcium chloride 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 synthesized using standard techniques 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 pETlld (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 into pET lId relies on transcription from the T7 gnlO-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident X prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant Lolp 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 (Gottesman, 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 preferentially utilized in highly expressed E. coli proteins (Wada et al_, (1992) Nuc. Acids Res, 20:2111-2118). Such alteration of nucleic acid sequences of the invention could be carried out by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing -e 41sl WO 94/21675 PCT/US94/02531' polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See 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 fragments of nucleic acid sequences encoding peptides of the invention. As used herein, the term "fragment" 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 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 chemically or mechanically, using known techniques. A functional equivalent of an oligonucleotide sequence of Lol p I is one which is 1) a sequence capable of hybridizing to a complementary oligonucleotide to which the sequence (or corresponding sequence portions) of Lolp 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 Lol p I as shown in Fig. 1 (SEQ ID NO: and/or 3) a sequence which encodes a product a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence pdrtion) of Lol p as shown in Fig. 1 (SEQ ID NO: Whether a functional equivalent must meet one or both criteria will depend on its use 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 Lolp I peptid of the invention, more preferably at least about 60% homology and most preferably at least about 70% homology with a Lolp 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 similarity 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 compared sequence is occupied by the same nucleotide or amino acid, then molecules are 's WO 94/21675 PCT/US94/02537 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.

Preferred nucleic acid fragments encode peptides of at least 7 amino acid residues in length, and preferably 13-40 amino acid residues in length, and more 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 allergens immunologically cross-reactive with Lolp I, such as full length Dac g I and Poap 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 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 culturing a host cell transformed with a nucleic acid sequence encoding a Lol p I peptide of the invention in an appropriate medium to produce a mixture of cells and medium containing said Lolp I peptide; and purifying the mixture to produce substantially pure Lol p I peptide. Host cells transformed with an expression vector containing DNA coding for a Lol pI peptide of the invention are cultured in a suitable medium for the host cell. Lolp I peptides of the invention can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying peptides and proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, 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 reactive with a Lol p I peptide. Such antibodies may be used to standardize 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 immunized with an immunogenic form of an isolated Lol p I peptide of the invention capable of eliciting an antibody response. Techniques for conferring immunogenicity on a peptide include conjugation 9 -I i; .WO 94/21675 PCT/US94/02537 to carriers or other techniques well-known in the art. The Lolp I peptide can be administered in the presence of adjuvant. The progress of immunization 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 ,ntibodies.

Following immunization, anti-Lol p I peptide antisera can be obtained and, if desired, polyclonal anti-Lolp I peptide antibodies from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard scmatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Hybridoma cells can be screened immunochemically for production of antibodies reactive with the Lol p I peptides of the invention. These sera or monoclonal antibodies can be used to standardize allergen extracts.

Through use of the peptides and antibodies of the present invention, preparations of consistent, well-defined composition and uniform biological activity can be made. Compositions having therapeutic activity may be administered for therapeutic purposes 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 Phlp Administration of such peptides may, for example, 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 immunotherapy 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 sensitivity to grass allergens crossreactive to ryegrass allergens, because the components include T cell epitopes recognizing the allergens.

The present invention also pertains to T cell clones which specifically recognize Lol p I peptides of the invention. These T cell clones may be suitable for isolation and molecular cloning of the gene for the T cell receptor which is specifically reactive with 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. Saunders 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 specifically reactive with such a T cell receptor can also be produced according to the WO 94/21675 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 are described in Immunology: A Synthesis, 2nd Ed., Edward S. Golub et al., Sinaur Assoc., Sunderland, 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, enhancing therapeutic or preventive efficacy, increasing stability shelf life ex vivo or resistance to proteolytic degradation in vivo), decreasing adverse side effects, and tl v 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 other component.

For example, a peptide can be modified so that it maintains the ability to induce T cell anergy and to bind MHC proteins but reduces the ability to induce a strong proliferative response, or possibly any proliferative response, when administered in immunogenic form. In this instance, critical binding residues for the T cell receptor can be determined using known techniques substitution of each residue and determination of the presence or absence of T cell reactivity). Those residues shown to be essential to interact with the T cell receptor can be modified by replacing the essential amino acid with another preferably similar amino acid residue (a "conservative substitution") whose presence is shown to enhance, diminish but not eliminate, or not affect T cell reactivity. In addition, those amino acid residues that are not essential for T cell receptor interaction can be modified by replacement with another amino acid whose incorporation may enhance, diminish or not affect T cell reactivity but does not eliminate binding to relevant MHC.

Additionally, peptides of the invention can be modified by replacing an amino acid shown to be essential to interact with the MHC protein complex with anotLer, preferably similar amino acid residue (conservative substitution) whose presence is shown to enhance, diminish but not eliminate 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 protei complex can be modified by replacement with another amino acid whose incorporation may enhance, not affect, or diminish but not eliminate T cell reactivity. Preferred amino acid substitutions for nonc- *WO 94/21675 PCT/US94/02537 essential artdno acids include, but are not limited to substitutions with alanine, glutamic 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 sequence 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 protein or peptide conjugated with PEG. In addition, PEG can be added during chemical synthesis of a protein or peptide of the invention. Modifications of peptides or portions thereof can also include reduction/ alyklation (Tarr in: Methods of Protein Microcharacterization, J.E. Silver ed. Humana Press, Clifton, NJ, pp 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate 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 Immunology, 41:199-215 (1971)).

To facilitate purification and potentially increase solubility of peptides of the invention, it is possible to add reporter group(s) to the peptide backbone. For example, poly-histidine can be added to a peptide to purify the peptide by immobilized metal ion affinity chromatography (Hochuli, E. et al., Bio/Technology, 6:1321-1325 (1988)). In addition, specific endoprotease cleavage sites can be introduced, if desired, 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 an individual to a protein antigen, it may be necessary to increase the solubility of a peptide by adding functional groups to the peptide or by not including hydrophobic T cell epitopes or regions containing hydrophobic epitopes in the peptides or hydrophobic regions of the protein or peptide. Functional groups such as charged amino acid pairs 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 LP1-16.1 (SEQ ID NO: 23) (Fig. such modified peptides include: LPI-16.2 (SEQ ID NO: 31), LP1-16.3 (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), all as shown in Fig. 4.

WO 94/21675 PCT/US94/02537 To potentially aid proper antigen processing of T cell ep s within a peptide, canonical protease sensitive sites can be recombinantly or syntheucally 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 peptide during recombinant construction of the peptide or added to the amino or carboxy terminus of a synthetically produced peptide. The resulting peptide can be rendered sensitive to cathepsin and/or other trypsin-like enzymes cleavage to generate portions of the peptide containing 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 mutagenesis 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. Comm., 161:1056-1063 (1989)). To enhance bacterial expression, the aforementioned methods can be used in conjunction with other procedures to change the eucaryotic codons in DNA constructs encoding protein or peptides of the invention to ones preferentially processed in E. coli, yeast, mammalian 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 Phi p I, with an isolated peptide(s) of Lol p I, under conditions appropriate for binding of components in the blood antibodies, T-cells, B cells) with the peptide(s) and determining the extent to which such binding occurs. Other diagnostic methods for allergic diseases in which 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), radioimmunoassays (RIA), immuno-radiometric assays (IRMA), luminescence immunoassays (LIA), histamine 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 administering to the individuals an Immediate Type Hypersensitivity test and a Delayed Type Hypersensitiity test. The individuals

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WO 94/21675 PCTiUS94/02537 are administered an Immediate Type Hypersensitivity test (see Immunology (1985) Roitt, Brostoff, Male, D.K. (eds), C.V. Mosby Co., Gower Medical Publishing, London, NY, pp. 19.2-19.18; pp. 22.1-22.10) utilizing the protein allergen or a portion thereof, or a modified form of the protein allergen or a portion thereof, each of which binds IgE specific for the allergen. The same individuals are administered a Delayed Type Hypersensitivity test prior to, simultaneously with, or subsequent to administration of the Immediate Type Hypersensitivity test. Of course, if the Immediate Type Hypersensitivity test is administered prior to the Delayed Type Hypersensitivity test, the Delayed Type Hypersensitivity test would be given to those individuals exhibiting a specific Immediate Type Hypersensitivity reaction. The Delayed Type Hypersensitivity test utilizes 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 stimulating activity and each of which does not bind IgE specific for the allergen in a substantial percentage of the population of individuals sensitive to the allergen at least about Those individuals found to have both a specific Immediate Type Hypersensitivity reaction and a specific Delayed Type Hypersensitivity reaction may be treated with a therapeutic composition comprising the same modified form of the protein or portion thereof, the recombinantly produced protein allergen, or the peptide, each as used in the Delayed Type Hypersensitivity test.

Isolated peptides of the invention, when administered in a therapeutic regimen to a Lolp 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 alergic response of the individual to Lol p I ryegrass pollen allergen (or 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-cell response of the individual 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 non-responsiveness or diminution in symptoms to the allergen, as determined by stafidard clinical procedures (See, Varney et al, British Medical Journal, 302:265-269 (1990)) including diminution in ryegrass polleninduced asthmatic symptoms. As referred to herein, a diminution 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 invention. This diminution may be subjective the patient feels more comfortable I- IWO 94/21675 PCT/US94/02537 in the presence of the allergen), or diminution 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 preferred. In referring to an epitope, the epitope will be the basic element or smallest unit of recognition by a receptor, particularly immunoglobulins, histocompatibility antigens 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 Lolp I can also be used. T cell epitopes are believed to be involved in initiation and perpetuation of the immune 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 appropriate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation. These events lead to T cell proliferation, lymphokine secretion, local inflammatory 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 fundamentally important to the development of allergic symptoms, and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted.

Exposure of ryegrass pollen-sensitive patients or patients sensitive to an immunogically cross-reactive protein allergen such as Dac g I, Poa p I and Phi 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 protein allergen, may tolerize or anergize appropriate T cell subpopulations such that they become unresponsive to the protein allergen and do not participate in stimulating an immune response upon such exposure.

In addition, administration 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 Lol p I protein allergen or portion thereof 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 nasal mucosa, skin, and lung) towards the site(s) of therapeutic administration of the fragment or protein allergen. This redistribution of T cell WO 94/21675 PCT/US94/02537 subpopulations can have the effect of ameliorating or reducing the ability of an individual's immune system to stimulate the usual immune response at the site of normal exposure to the allergen, resulting in a dimunution in allergic symptoms.

The isolated Lol p peptides of the invention can be used in methods of diagnosing, treating or preventing allergic reactions to Lolp I allergen or an immunogically related protein allergen such as Dac g I, Poap I and Phl p I. Thus, the present invention provides compositions useful in allergery diagnosis and/or useful in allergy therapy comprising isolated ol p I peptides or portions thereof. Such compositions will typically also comprise a pharmaceutically acceptable carrier or diluent when intended for in vivo administration. Therapeutic compositions of the invention may include synthetically prepared Lolp I peptides.

Administration of the therapeutic compositions of the present invention to an individual to be desensitized can be carried out using known techniques. Lol p I peptides or portions thereof may be administered to an individual in combination with, for example, an appropriate diluent, a carrier and/or an adjuvant. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutically 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 preferably administered in nonimmunogenic form, it does not contain adjuvant.

The therapeutic compositions of the invention are administered to ryegrass pollensensitive individuals or individuals sensitive to an allergen which is immunologically cross-reactive with ryegrass pollen allergen Dactylis glomerata, or Sorghum halepensis, etc.). Therapeutic compositions of the invention may also be used in the manufacture of medicaments 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 desensitized can be carried out using known procedures at dosages and for periods of time effective to reduce sensitivity to reduce the allergic response) of the individual to the allergen. Effective amounts of the therapeutic 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 protein or fragment thereof) may be administered in any convenient manner such as by injection (subcutaneous, intravenous, etc.), oral I c i i .WO 94/21675 PCT/US94/02537 administration, inhalation, transdermal application, or rectal administration.

Depending on the route of administration, the active compound may be coated within a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.

For example, preferably about 1 3 mg and more preferably from about 750 lpg of active compound protein or fragment thereof) per dosage unit may be administered by injection. Dosage regimen may be adjust to provide the optimum therapeutic response. For example, sever ,/ded doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

To administer a peptide by other than parenteral administration, it may be necessary to coat the protein with, or co-administer the protein with, a material to prevent its inactivation. For example, the peptide or portion thereof may be coadministered 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 conventional liposomes (Strejan et al., (1984), J. Neuroimmunol., 7:27).

The active compound may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where the peptides are water soluble) or dispersions and sterile powders for 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 necessary to provide easy syringability. It should preferably be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, 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 maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal I c WVO 94/231675 PCT/US94/02537 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 about, including in the composition, an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound protein or peptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile indectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient protein or peptide) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When a peptide of the invention is suitably protected, as described above, the peptide may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The peptide and other ingredients may also be enclosed in a hard or soft gelatin capsule, compressed into tablets, or incorporated directly into the individual's food. For oral therapeutic administration, the active compound may be formulated with conventional excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the composition and preparations 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 preparations according to the present invention are prepared so that an oral dosage unit contains from about 10 gg to about 200 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients sn-ch as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of WO 94/21675 PCTIUS94/02537 wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, 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 or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservative, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.

In addition, the active compound may be incorporated into sustained-release preparations and formulations.

As used herein "pharmaceutically 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 pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also 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 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, Lol p I is divided into nonoverlapping peptides of desired length or overlapping peptides of desired lengths as discussed 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 tolerization). To determine peptides comprising at least 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 stimulating activity.

As discussed in Example 4, human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to Lol p I allergen, an L' WO 94/21675 PCT/US94/02537 individual who has an IgE-mediated immune response to Lolp 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, by cellular uptake of tritiated thymidine. Stimulation indices for responses by T cells to peptides can be calculated as the maximum counts-per-minute (CPM) in response to a peptide divided by the control CPM. A stimulation index equal to or greater than two times the background level is considered "positive". Positive results are used to calculate the mean stimulation index for each peptide for the group of patients tested. Preferred peptides of this invention comprise at least one T cell epitope and have a mean T cell stimulation index of greater than or equal to 2.0. 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 at least 10% of patients tested) is considered useful as a therapeutic agent. Preferred 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 stimulation index of at least 5, as shown in Fig. 3, include LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: 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-19 (SEQ ID NO: 26), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 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: 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: In addition, preferred peptides have a positivity index 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 determined by multiplying the mean T cell stimulation index by the percent of individuals, in a population of individuals sensitive to ryegrass pollen 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 2.0. Thus, the positivity index represents both the strength of a T cell response to a peptide 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, Lolp 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 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: 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), and LPI-23 (SEQ ID NO: In order to determine 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 determined by T cell biology techniques is modified by addition or deletion of amino acid residues at either the amino or carboxy terminus of the peptide and tested 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 determined by T cell biology techniques, additional peptides can be produced comprising all or a portion of such peptides and these additional peptides can be tested by a similar procedure. Following this technique, peptides are selected and produced recombinantly 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; modified 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: 50) all as shown in Fig. 4.

Peptides are selected for diagnostic or therapeutic uses based on various factors, including the strength of the T cell response to the peptide 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 df these selected peptides solubility, stability) are examined 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 selected peptides or selected modified peptides to stimulate human T cells induce proliferation, lymphokine secretion) is determined.

I WO 94/21675 PCTIUS94/02537 The most preferred T cell epitope-containing peptides of the invention do not bind immunoglobulin E (IgE) of an allergic individual or bind IgE to a substantially lesser extent at least 100 fold less and more preferably, at least 1000 fold less) than the protein allergen from which the peptide is derived. The major complications of standard immunotherapy are IgE-mediated responses such as anaphylaxis.

Immunoglobulin E is a mediator 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 mediators histamine, serotonin, eosinophil chemotacic factors). Anaphylaxis in a substantial percentage of a population of individuals sensitive to Lol p I could be avoided by the use in immunotherapy of a peptide which do not bind IgE in a substantial percentage at least about 75%) of a population of individuals sensitive to Lolp I allergen, or, if the peptides do bind IgE, such binding does not result in the release of mediators from mast cells or basophils. The risk of anaphylaxis could be reduced by the use in immunotherapy of a peptide or peptides which have reduced IgE binding. Moreover, peptides having minimal IgE stimulating activity are desirable for therapeutic effectiveness. Minimal IgE stimulating activity refers to IgE production that is less than the amount of IgE production stimulated by the native Lolp I protein allergen. Similarly, IL-4 production can be compared, with reduces IL-4 production indicating lessened IgE stimulating activity.

Preferred T cell epitope-containing peptides of the invention, when administered to a ryegrass pollen-sensitive individual or an individual sensitive to an allergen which is immunologically 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 preferred Lol p I 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 administered to an Ildividual sensitive to ryegrass pollen are capable of modifying T cell response of the individual to the allergen, and they will thus be useful as therapeutics in addressing sensitivity to grasses.

A preferred 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 approximately seven amino acid residues. For purposes of therapeutic effectiveness, preferred therapeutic compositions of the invention preferably comprise at least two T cell epitopes of Lol p I, and accordingly, the peptide comprises at least approximately eight amino acid residues and preferably at least fifteen amino acid I IS""I WO 94/21675 PCT/US94/02537 residues. Additionally, therapeutic compositions comprising preferred isolated peptides of the invention most preferably comprise a sufficient percentage of the T cell epitopes of the entire protein allergen so that a therapeutic regimen of administration of the composition to an individual sensitive to ryegrass pollen results in T cells of the individual being tolerized to the protein allergen. Synthetically produced peptides of the invention comprising up to approximately forty-five amino acid residues in length, and most preferably up to approximately 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 exceeding 45 amino acids will be more easily produced recombinantly.

Peptides derived from the Lolp I protein allergen which exhibit T cell stimulatory properties and thus are believed to be useful therapeutics and/or intermediatea in developing tolerizing peptides comprise all or a portion of the following peptides: LPI-1 (SEQ ID NO: LPI-1.1 (SEQ ID NO: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 8), (SEQ ID NO: 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-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 stimulation 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 Lolp I protein allergen comprise all or a portion of the following peptides: LPI-1.1 (SEQ ID NO: 4), LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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-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 more preferred peptides derived from the Lolp I protein comprise the following peptides: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-10 (SEQ ID NO: 14), LPI-I1 (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

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WO 94/21675 PCT/US94/02537 NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: 30), all as shown in Fig. 2. Additional preferred peptides believed to T cell stimulating activity comprise the following peptides: 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-16.1 (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: One embodiment of the present invention features a peptide or portion thereof of Lol p I which comprises at least one T cell epitope of the protein allergen and has a formula Xn-Y-Zm. According to the formula, Y is an amino acid sequence selected from the group consisting of LPI-1 (SEQ ID NO: LPI-1.1 (SEQ ID NO: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-5 (SEQ ID NO: 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-20 (SEQ ID NO: 27), LPI-21 (SEQ ID NO: 28), LPW-2 (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) and preferably selected from the group consisting of LPI-1.1 (SEQ ID NO: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: 7), LPI-4.1 (SEQ ID NO: 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 IDi NO: 25), LPI-19 (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 (SEQ ID NO: 31), LPI-16.3 (SEQ ID NO: 32), LPI-16.4 (SEQ ID

~I

WO 94/21675 PCTIUS94/02537 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) and more preferably selected from the group consisting of LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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), and most preferably selected from the group consisting of LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), (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: LPI-lf.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). In addition, Xn are amino acid residues contiguous to the amino terminus 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 Izi; amino acid sequence of the protein allergen. In the formula, n is 0-30 and m is 0-30. Preferably, the peptide or portion thereof has a mean T cell stimulation index equivalent to greater than the mean T cell stimulation index of Y as shown in Fig. 3. Preferably, amino acids comprising the amino terminus of X and the carboxy terminus of Z are selected from charged amino acids, arginine lysine histidine glutamic acid (E) or aspartic acid amino acids with reactive side chains, cysteine asparagine or glutamine or amino acids with sterically small side chains, e.g., I, I IWO 94/21675 PCT/US94/02537 alanine or glycine Preferably n and m are 0-5; most preferably n m is less than 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 accordingly each region comprises at least approximately seven amino acid residues, These peptides comprising at least two regions can cumprise 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 joined by a linker to increase sensitivity to processing by antigen-presenting cells.

Such linker can be any non-epitope amino acid sequence or other appropriate linking or joining agent. To obtain preierred 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 containing 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 containing 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 containing T cell epitopes can be arranged in a nonsequential order in an order different from the order of the amino acids of the native protein allergen from which the region containing 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 Lol p I.

The individual peptide regions can be produced and tested to determine which regions bind immunoglobulin E specific for Lol p I and which of such regions would cause the release of mediators histamine) from mast cells or basophils. Those peptide regions found to bind immunoglobulin E and to cause the release of mediators 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 preferred peptides of the invention.

Examples of preferred peptide regions which do not bind to IgE (data not shown) include: LPI-1 (SEQ ID NO: LPI-1.1 (SEQ ID NO: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 8), WO 94/21675 PCT/US94/02537 (SEQ ID NO: 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-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 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), the 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 preferred regions, or a portion thereof. Preferred peptides comprising a combination of two or more regions (each region having an amino acid sequence as shown in Fig. 2 or Fig. include the following: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-10 (SEQ ID NO: 14), and LPI-11 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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: LPI-4.1 (SEQ ID NO: 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); (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16.1 (SEQ ID NO: 23);

I

WO 94/21675 'NO 9421675PCTIUS94/02537 (SEQ ED 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 (SEQ ID NO: 27); (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 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: (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), and LPI-20 (SEQ ED NO: 27); (SEQ ED 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: LPI-18 (SEQ ID NO. 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: LPI-18 (SEQ ID NO: 25) and LPI-20 (SEQ ID NO: 27); LPI-18 (SEQ ED NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-23 (SEQ ID NO: LPI-18 (SEQ ED NO: 25), LPJ-20 (SEQ ID NO: 27) and LPI-16.1 (SEQ ID NO: 23); LPI-18 (SEQ ED 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: 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 (SEQED NO: 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 IID NO: WO 94/21675 PCT/US94/02537 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 peptides comprising various combinations of two or more of the above discussed preferred regions include: LPI-16.2 (SEQ ID NO: 31), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.3 (SEQ ID NO: 32), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.4 (SEQ ID NO: 33), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.5 (SEQ ID NO: 34), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.6 (SEQ ID NO: 35), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.7 (SEQ ID NO: 36), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.9 (SEQ ID NO: 37), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: 30); and ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID 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: In yet another aspect of the present invention, a composition is provided comprising at least two peptides 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 additionally with a pharmaceutically acceptable carrier of diluent for therapeutic uses, or with conventional non-pharmaceutical excipients for reagent use. When used therapeutically, an effective amount of one or more of such compositions can be administered simultaneously or sequentially to an individual sensitive to ryegrass pollen.

In another aspect of the invention, combinations of Lol p I peptides are provided which can be administered simultaneously or sequentially. Such combinations may comprise therapeutic compositions comprising only one peptide, or I WO 94/21675 PCTIUS94/02537 more peptides if desired. Such compositions may be used simultaneously or sequentially in preferred combinations.

Preferred compositions and preferred combinations of Lolp I peptides which can be administered or otherwise used simultaneously or sequentially (comprising peptides having amino acid sequences shown in Fig. 2) include the following combinations: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-10 (SEQ ID NO: 14), and LPI-11 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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: LPI-4.1 (SEQ ID NO: 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); (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16.1 (SEQ ID NO: 23); (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 (SEQ ID NO: 27); (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: (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 25), and LPI-20 (SEQ ID NO: 27); (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: LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: LPI-18 (SEQ ID NO: 25) and LPI-20 (SEQ ID NO: 27); e I WO 94/21675 PCT/US94/02537 LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27) and LPI-23 (SEQ ID NO: 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: 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: 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 preferred combinations of Lol p I peptides which can be administered or used simultaneously or sequentially (comprising peptides having amino acid sequences shown in Figs. 2 or 4) include the following 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: 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:

I

I WO 94/21675 PCT/US94/02537 LPI-16.4 (SEQ ID NO: 33), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.5 (SEQ ID NO: 34), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.6 (SEQ ID NO: 35), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.7 (SEQ ID NO: 36), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: LPI-16.9 (SEQ ID NO: 37), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: 30); and LPI-16.10 (SEQ ID NO: 38), LPI-18 (SEQ NO: 27), and LPI-23 (SEQ ID NO: ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID ID NO: 25), LPI-20 (SEQ ID In each of the above preferred compositions, peptides LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: 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 with peptides LPI-18.5 (SEQ ID NO: 39), LPI-18.6 (SEQ ID NO: 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 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; 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: 50), all as shown in Fig. 4.

The present invention is further illustrated by the following non-limiting Figures and Examples.

EXAMPLES

Example 1 Isolation and Cloning of Nucleic Acid Sequence Coding for Lolp I Total mRNA was extracted from mature ryegrass pollen by the phenol method of Herrin and Michaels, supra. Double-stranded cDNA was synthesized from lg of I WO 94/21675 PCT/US94/02537 total mRNA using a commercially available kit (cDNA SYNTHESES SYSTEM PLUS KIT, 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 et al. (1990), Proc. Natl. Acad. Sci. USA, 85:8998-9002; and Roux et al. (1990), BioTech., 8: 48-57. Oligonucleotide AT has the 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 (GeneAmp® DNA Amplification kit, Perkin Elmer Cetus, Norwalk, CT) whereby 10 pl 10x buffer containing dNTPs were mixed with 1 pg 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 CCCTGCAGATTATTTGAGATCTTGAG-3' (SEQ ID NO: 74), cDNA (3-5 pl of a pl linkered cDNA reaction mix), 0.5 pl Amplitaq DNA polymerase, and distilled water to 100 pl.

Nucleotides 1 through 8 (5'-CCCTGCAG) of LpA-5 correspond to a Pst I site added for cloning purposes; the remaining nucleotides correspond to the non-coding strand sequence complementary 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 of denaturation at 94 0 C for 1 minute, annealing of primer to the template at 45 0 C for minutes, and chain elongation at 70 0 C for 2 minutes. The final 20 rounds of amplification consisted of denaturation as above, annealing at 55°C for 1.5 minutes, and elongation as above. Five percent (5 pl) of this initial amplification was then used in a secondary amplification whereby 10 pl 10x buffer containing dNTPs was mixed with 1 pg each of primer AP and primer LpA-3, which has the sequence CCCTGCAGTCATGCTCACTTGGCCGAGTA-3' (SEQ ID NO: 75), 0.5 pl Amplitaq DNA polymerase, ard distilled water to 100 pl. The secondary PCR reaction was performed 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 correspond to the complementary sequence for a nw stop codon, and the remaining nucleotides correspond to the non-coding strand sequence complementary I WO 94/21675 PCT/US94/02537 to nucleotides 793 through 810 of the full length clone of Lolp I as shown in Fig. 1, including translated sequence of Lol p I (Fig. the native stop codon and 3' untranslated sequence.

Amplified DNA was recovered by sequential chloroform, phenol, and chloroform extractions, followed by precipitation at -20 0 C with 0.5 volumes of ammonium acetate and 1.5 volumes of isopropanol. After precipitation and washing with 70% ethanol, the DNA was simultaneously digested with Xba I and Pst I in a p1 reaction and electrophoresed through a preparative 3% GTG NuSieve low melt gel (FMC, Rockport, ME). The appropriate sized DNA band was visualized by EtBr staining, excised, and ligated into appropriately digested M13mp18 for sequencing by the dideoxy chain termination 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 GAGTACGGCGACAAGTGGC-3' (SEQ ID NO: 76), which corresponds to nucleotides 121 through 139 as shown in Fig. 1. LpA-12 has the sequence TTCGAGATCAAGTGCACC-3t (SEQ ID NO: 77), which corresponds to nucleotides 310 through 318 as shown in Fig. 1. LpA-9 has the sequence GTGACAGCCTCGCCGG-3' (SEQ ID NO: 78), which corresponds to the noncoding strand sequence complementary 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 remaining 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 complementary to nucleotides 503 through 518 of Fig. 1. LpA-10 has the sequence 5'-CCGTCGACGTACTTCA-3' (SEQ ID NO: 81), which corresponds to non-coding strand sequence complementary 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 reactions were sequenced. The nucleotide (SEQ ID NO: 1) and deduced amino acid sequences (SEQ ID NO: 2) of a representative clone of Lolp I, clone 26.j are shown in Fig. 1. As shown in Fig.l, the ,WO 94/21675 PCT/US94/02537 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 of 263 amino acids with a predicted molecular weight of 28.4 kD and a pi of 5.55. The initiating methionine is numbered amino acid -23, with amino acid numbered +1 corresponding to the NH 2 -terminus of the mature protein, as defined by amino acid sequencing (Cottam et al. (1986), Biochem. 234: 305-310). Amino acids -23 through -1 (Fig.1), 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 Nlinked glycosylation site at amino acid 9.

Amino acids 1 through 30 of clone 26.j (Fig. 1) correspond exactly to the published sequence of the NH 2 terminus of Lol p I (Cottam et al., supra). Amino acids 213 through 240 of clone 26.j (Fig. 1) correspond exactly to the published internal amino acid sequence of Lol p I (Esch and Klapper (1989), Mol. Immunol., 26: 557- 561).

Example 2 Identification of Polymorphisms in Lolp I A number of polymorphisms in the nucleotide sequence coding for Lolp I were discovered during the amplification and sequencing of different Lol p I clones. Some of the polymorphisms cause an amino acid change relative to that of clone 26.J, while others are silent polymorphisms that do not cause an amino acid change. The polymorphisms found in the sequence coding for Lolp I are summarized in Table 1.

The nucleotide base numbers are those of the sequence of clone 26.j shown in Fig 1.

I WO 94/21675 WO 9421675PCTfUS94/02537 Table 1 Polymtorphisms Detected in Lolp I Nucleotide Polvmornhism Amino Acid Polvmorifhism 1 GGC 2 1 5-4GGA/GGT Nn 2 G 234

AC

2 1 6 -4GAT D 45

N

3 GTT 2 39 GTC None 4 CGT 3 5 1-4CGC None

GGC_

56 -4GGT None 6 AACIS 9 AAT None 7 CCC 3 2 6 CCT None 8 CAT 41 1-4CAC None 9 GCC 4 34 GCA None

GAC

5 30 o-*GAT None 11 GG 532 C-*GAC G] 1 4-4D 12 CCG, 54 2 -4CCA None 13 ACA, 45 -*ACG None 14 G 5 6 T:±qGT _Aj-G CTCiB 1 >CTG None 16 GCG 626 C-4~GCC None 17 ATC 7 8 7 -*AIT None 18 CCT 7 g~j--4CCC None All confirmed nucleotide polymorphisms (polymorphisms observed in the sequence analysis of clones from two independent PCR reactions) are shown relative to the sequence of clone 26.j (Fig.l1) (SEQ ID NO: The polymorphic residues in 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 obser-ved in clones from single PCR reactions. Seventeen of these 28 potential polymorphisms are silent mutations and do not result in an amino acid pol norphism; the remaining 11 potential polymorphic sites would result in the following amino acid changes, specifically: T 1

I

*WO 94/21675 PCT/US94/02537

A

49

R

6 7 S, K 79

V

9 0 I, Q 133 R, 1 16 2 T, V 17 3 -4E, I 1 8 7

T,

V

2 23 F and K 2 3 2 The potential polymorphism at amino acid 223 (V 2 2 3

F)

has been previously reported. (Perez et al., supra) Example 3 Human IgE reactivity to Purified Recombiant and Native Lolp I Cloned DNA encoding Lol p I and Lolp IX was expressed in E. coli and purified on a Ni-chelating affinity column. Monoclonal antibodies were also used to affinity purify and distinguish isoforms of these and native grass proteins. The recombinant Lol p I was compared to biochemically purified native Lol p I and Lol p IX 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 competition assays, the native Lolp I and Lol p IX proteins could completely inhibit IgE binding to Lol p soluble pollen extract (SPE), whereas the recombinant form of Lol p I and Lol p IX could only partially inhibit IgE binding to the extract. However, the recombinant Lol p I and Lol p IX was still active in these competition assays. These asays were then extended to western blot inhibition studies; both methods confirm the previous finding that Group I and Group IX constitute one of the major allergenic proteins of Lolium perenne grass pollen. Furthermore, the Lol p I and Lol p IX native and recombinant allergens showed ihibition of grass allergic patient IgE binding to soluble pollen extracts of other grass species (Dac g, Phlp and Poa The degree to which Lol p and Lol p IX proteins successfully compete for IgE binding to these other grasses implies a hierarchy of homology between the species. These studies confirm and extend the findings of shared IgE epitopes between temperate grass allergens.

The procedures used for the foregoing examples were as follows: 91 WO 94/21675 PCT/US94/02537 Extraction and Depigmentation of Allergens Defatted Lol p I pollen was extracted twice, overnight at 4 0 C in phosphate buffer, 15mM NaC1, pH 7.2 and protease inhibitors (PMSF, Luepeptin, SPTI and pepstatin). The extract was then depigmented by batch absorption with DE- 52 (Whatman) in 50mM phosphate buffer, 0.3M NaC1, pH 7.2.

Biochemical Purification of Lol I Allergen Depigmented Lolp I extract was dialyzed into H 2 0, pH 8.0 by addition of

NH

4 0H. This mateial was loaded onto a DE-52 column and eluted stepwise with 1mMd, 4.5mM and 7.5mM NaH 2

PO

4 The majority of the Group I allergens was eluted with 4.5mM NaH 2 P04. A further separation of Group I was accomplished by running this DE-52 enriched fraction over A (26/60) superdex 75 column (Pharmacia).

Immunoaffinity Purification of Lol p IX Allergen 1B9 ascites wvs precipitated by 50% (NH 4 2

SO

4 followed by purification over Q-sepharose (Pharmacia). Purified 1B9, an anti-Lol p IX antibody, was then coupled to Affigel-10 (Biorad), according to the manufacturer's instructions. Either depigmented pollen extract or DE-52 enriched material was circulated over the 1B9 affigen column overnight at 4 0 C. The column was washed with PBS, PBS MaCl and then eluted with 0.1M Glycine, pH2.7. Eluted Lolp IX fractions were neutralized with 1M tris-base, pH 11.

Expression and Purification of Recombinant Lol p I Lolp I cDNA's encoding from the first amino acid of the mature protein to the stop codon were ligated into pETlldAHR containing a leader which encodeu 6 hisdidines. The HIS 6 was used for purification over a nickel-NTA agarose column (Qiagen). rLol p I was expressed in E. coli.

SDS-PAGE. Electroblotting and Immunoblotting Electrophoresis was performed using 12.5% polyacrylamide 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 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 NaCI, and 0.05% Tween 20) for 1 hour. The human plasma samples were pre-absorbed with blank I WO 94/21675 PCT/US94/02537 nitrocellulose for 1.5 hours prior to incubation. Blot sections were incubated with 1st antibodies diluted in 1% milk/Tween solution overnight at room temperature (RT).

The blot sections were washed three times and inucbated in the appropriate biotinylated 2nd AB (1:2500) for 2 hours at RT. The blot sections were washed three times and finally incubated with 125 I-streptavidin 1 hour at RT. The sections were washed extensively to remove unbound label and exposed to film. Autoradiography was carried out at -80 0

C.

Direct. Competition and Depletion ELISA Microtiter plates were coated with 2.5-10.0pg/mL of coating antigen (grall soluble pollen extract (SPE), Lolp I, Lolp IX, Lol p IX, recombinant Lolp I, and/or recombinant Lol p IX) in PBS at 100L/well and incubated overnight at 4 0 C. The plates were washed three times between each step with PBS-T (Phosphate buffered saline 0.05% Tween 20). The unbound antigen was removed and the plate blocked with 300L/well of 1MG/ML PVP in 0.5% gelatin/PBS for one hour at room temperature All subsequent reagents were added at 100plwell for direct ELISA, serially diluted human plasma was added to duplicate wells and incubated overnight at 4 0 C. 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 substrate and H202 were freshly mixed and added; th. color was allowed to develop for 2-5 minutes. The reaction was stopped by the addition of 1M 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 equal volume of serially diluted antigen or with PBS-T (as a control). These samples were incubated overnight at 4°C before addition to the microtiter plate and performing the remaining 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 then performed as outlined above.

I II JWO 94/21675 PCT/US94/02537 EXAMPLE 4 Human T Cell Studies with Lol p Synthesis of Overlapping Peptides Ryegrass Lolp I overlapping peptides were synthesized using standard Fmoc/tBoc synthetic chemistry and purified by Reverse Phase HPLC. Fig. 2 shows Lolp I peptides used in these studies (SEQ ID NO: 3-30). The peptide names are consistent throughout.

IgE Binding Studies with overlapping peptides None of the peptides shown in Fig. 2 bound a detectable amount of IgE from pooled human plasma when analyzed 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 Cell Responses to Rvegrass Antigen Peptides Peripheral blood mononuclear cells (PBMC) were purified by lymphocyte separation medium (LSM) centrifugation of 60 ml of heparinized blood from grassallergic 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 stimulation of 2x10 6 PBUml in bulk cultures of complete medium IRPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 5x10- 5 M 2-mercaptoethanol, and mM HEPES, supplemented with 5% heat-inactivated human AB serum) with mg/ml of purified native Lolp I (95% pure with a single band on protein gel) for 6 days at 37 0 C in a humidified 5% CO 2 incubator to select for Lolp I reactive T Cells.

This amount of priming antigen was determined 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 medium, 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 Lol p I, recombinant Lol p IX (rLol p IX), or Der p I (rDerp I) was then assessed. For assay, 2x104 rested cells were restimulated in the presence of 2x10 4 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 1, or rLol p IX, in a volume of 200 ml complete medium in duplicate wells in 96-well round- WO 94121675 PCT/US94/02537 bottom plates for three days. Each well then received 1 mCi tritiated thymidine for 16hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. The varying antigen dose in assays with rLolp I, purified native Lol p I, and recombinant Lolp IX and several antigenic peptides peptides that induce an immune response, or, specifically, a positive T cell response in these assays) synthesized as described above were determined. The titrations were used to optimize the dose of peptides in T cell assays. The maximum response in a titration of each peptide is expressed as the stimulation index The S.I. is the counts per minute (CPM) incorporated oy 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 times the background level is considered "positive" and indicates that the peptide contains a T cell epitope. The positive results were used in calculating mean stimulation indices for each peptide for the group of patients tested. The results (not shown) demonstrate that one patient responds well to rLol p I and purified native Lolp I, as well as to Lol p I peptides but not to recombinant Derp I. This indicated that Lolp IT cell epitopes are recognized 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 IX, suggesting a presence of Lol p IX antigen in the purified native Lol p I prep that was used to prime T cells.

The above procedure was followed with a number of other patients. 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 patient responded to at least one peptide derived from Lol p I at an S.I. of 2.0 or greater. A summary of positive experiments from 35 patients is shown in Fig. 3. All 35 T cell lines responded to purified native Lolp I and rLol p I. The numbers enclosed in the parentheses denote percentage of patients responding to that particular peptide. The bar represents the positivity index for each peptide of patients responding multiplied by mean Preparation of EBV-transformed B Cells for Use as Antigen-presenting Cells Autologous EBV-transformed cell lines were derived by incubating 5x10 6

PBL

with 1 ml ofB-59/8 Marmoset cell line (ATCC CRL1612, American Type Culture Collection, Rockville, MD) conditioned medium in the presence of 1 mg/ml phorbol 12-myristate 13-acetate (PMA) at 37 0 C for 60 minutes in 12x75 mm polypropylene round-bottom Falcon snap cap tubes (Becton Dickinson Labware, Lincoln Park, NJ).

WO 94/21675 PCT/US94/02537 These cells were then diluted to 1.25x10 6 cells/ml in the RPMI-1640 medium 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 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 modifications. The invention also includes all steps, features, compositions and compounds referred to or indicated in this specification, 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 A. Cloning of Dac g 1.

RNA was obtained from the pollen of Dactylis glomerata using a standard acid phenol extraction procedure (Sambrook et al. (1989), Molecular 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 prepared 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 selfannealed oligonucleotides AT (SEQ ID NO: 71) and AL (5'-AATGATCGATGCT-3') (SEQ ID NO: 72) (Rafnar et al. (1991), J. Biol. Chem., 266:1229-1236).

The amino portion of the gene encoding Dac g 1, including 5' untranslated 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 GGGTCTAGAGGTACCGTCC-3') (SEQ ID NO: 83) and LpA-7 GTGCCGTCCGGGTACT-3') (SEQ ID NO: 80) were used in a primary amplification. Oligonucleotide primers AP-2 and LpA-9 GTGACAGCCTCGCCGG-3') (SEQ ID NO: 78) were used in a secondary amplification using 10% of the primary amplification as template cDNA. PCRs were 3' carried out using the GeneAmp DNA Amplification kit (Perkin Elmer, Norwalk, CT) I WO 94/21675 PCT/US94/02537 using a programmable thermal controller from MJ Research, Inc. (Cambridge, MA).

Samples were amplified for 24 cycles by heating to 94C for 1 min, 54 0 C for 1.5 min and 700C for 1 min.

The resulting PCR product was blunted with T4 DNA polymerase (Sambrook et al. (1989) supra) and digested with the restriction endonuclease Xbal. Unless otherwise stated, all endonucleases and polymerases were obtained from New England BioLabs (Beverly, MA). A band of approximately 400 base pairs was isolated from a low melting temperature agarose gel (FMC, Rockland, ME) and ligated into appropriately digested pUC19. The clones 22.2 and 22.5 were subsequently identified 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 containing internal nucleotide sequence of the gene encoding Dac g 1 was amplified using the primers DGI-3 TTGGATCCTACGGCAAGCCGACCGGC-3') (SEQ ID NO: 84) and LpA-10 CCGTCGACGTACTTCA-3') (SEQ ID NO: 81). A 300 base pair cDNA containing internal Dac g 1 sequence was amplified using the primers DGI-4 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°C for 45 sec, 60 0 C for 45 sec and 72 0 C for 1 min. These PCR products were blunted with T4 DNA polymerase as above, digested with BamHI and ligated into appropriately digested pUC19. 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 the 3' untranslated region, was cloned using oligonucleotide primers AP 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 TTGGATCCTGGCGCTGCTGGTGAAGTA-3') (SEQ ID NO: 87) in a secondary PCR. Material was amplified for 25 cycles of heating to 94 0 C for 1 min, 60 0 C for sec and 74 0 C for 1 min. The 700 base pair PCR product was digested with BamHI and Asp718 (Boehringer Mannheim, Indianapolis, IN), isolated and digested into appropriately digested pUC19 as described above. The clones 119.2, 119.4, 119.6, 119.9 and 119.12 were isolated, sequenced and found to contain sequence of the gene encoding Dac g 1.

'WO 94/21675 PCT/US94/02537 cDNA clones encoding the mature Dac g 1 protein were obtained by PCR with the oligonucleotide primers DGI-7Eco TIGAATTCATCCCGAAGGTGCCCCCG-3' (SEQ ID NO: 88), wherein G at position 14 can also be A) and PhA-1.2 TTGGTACCTCACTTGGACTCGTAGCT-3') (SEQ ID NO: 89). The cDNAs were amplified for 24 cycles of heating to 94 0 C for 1 min, 540C for 1.5 min and 70 0 C for 1 min. The amplified cDNA was digested with EcoRI and Asp718, isolated, and ligated into the appropriately digested pUC19. The cDNA clones 106.5, 106.6, 106.9 and 106.12 were identified as containing Dac g 1 sequence by dideoxysequencing. The 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 106.5 was isolated and ligated into appropriately digested expression vector pET-11d (Novagen, Madison, WI: Jameel et al. (1990), J.

ViroL, 64:3963-3966). The pET-11d vector had been modified to contain a sequence encoding 6 histidines (His 6) immediately 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 HindMII endonuclease restriction sites, had previously been removed by digestion with EcoRI and Hindu, blunted and religated.

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 centrifugation (7,930 G, 10 min) and lysed in 90 ml of 6 M Guanidine-HC1, 0.1 M Na2HPO4, pH 8.0 for 1 hour with vigorous shaking. 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).

B. Cloning of Poa I.

RNA was isolated froa 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 amplified using oligonucleotide primers Phl-7 (5'-CCGAATTCGTGGAGAAGGGGTCCAA-3') (SEQ ID NO: 90) and Poa-1 TTAGGATCCTCACTTATCATAIGACGTATC-3' (SEQ ID NO: 91), wherein C at *WO 94/21675 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 All Poa p 1 clones were amplified by 20 cycles of heating to 94 0 C for 1 min, 55 0 C for 1 min and 72oC for 1 min. The amplified material was finally heated to 720C for 5 min. Three clones, 11, 15 and 17, were isolated that contained part of the nucleotide sequence for the gene that encodes Poap 1. The Dac g 1 sequence encoded by clones 11, 15 and 17 corresponds to amino acids 151 240 of Fig. 6.

Clones containing partial nucleotide sequences of the gene encoding Poa p 1 were derived from PCRs that used oligonucleotide primers AP and Poa-3 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 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-1 and Poa-5 (5'-ATGAATTCATCGCAAAGGTTCCCCCC-3' (SEQ ID NO: 96), wherein A at position 14 can also be G or C or 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. The nucleotide (SEQ ID NO: 53) and deduced 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 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 The codon containing nucleotide 93 (GGN) encodes a Glycine at residue 31. The codon containing 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 Inserts from clones 11 and 114 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.

I WO 94121675 PCT/US94/02537 C. Cloning of Phi 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 PhAI.1 (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 These clones, 20 and 22, corresponded to the portion of the gene that encoded amino acids 1 240 of Phi p 1 (see Fig. The nucleotide (SEQ ID NO: 55) and deduced amino acid (SEQ ID NO: 56) sequences of clone are shown in Fig. 7.

Clones containing partial nucleotide sequence of the gene encoding Phl p 1 were derived from a PCR using oligonucleotide primers Phl-7 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-11d (Novagen, Madison, WI: Jameel et al. (1990) J.

Virol. 4:3963-3966). Recombinant proteins were expressed as descibed in section A, above.

Example 6 Comparison of Dac g 1, Phl p 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 Lolp 1 (SEQ ID NO: 57). The amino acid sequences of these Group 1 allergens had 95% (Dac g 91% (Phlp 1) and 91% (Poap 1) identity, respectively, with Lolp 1. This comparison is shown schematically 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 indicated 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 containing peptides of Lol 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 ~I ~L I WO 94/21675 PCTIUS94/02537 Example 4 (Fig, 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 containing peptides of Lolp 1 are likely to be the major T cell epitope containing regions in the related grasses. Comparison of the sequences of the Lol p 1 peptides with the homologous peptides containing Dac g 1, Phl p 1 and Poa p 1 polymorphisms are shown in Fig. 9 (SEQ ID NO: 23, 25, 27, 30, 61-70).

~ee~LIB~ WO 94/21675 PCT/US94/02537 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: IMMULOGIC PHARMACEUTICAL CORPORATION STREET: 610 LINCOLN STREET CITY: WALTHAM STATE: MASSACHUSETTS COUNTRY: USA POSTAL CODE (ZIP): 02154 TELEPHONE: (617) 466-6000 TELEFAX: (617) 466-6010 (ii) TITLE OF INVENTION: T CELL EPITOPES OF RYEGRASS POLLEN

ALLERGENS

(iii) NUMBER OF SEQUENCES: 98 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: US ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: ASCII text (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/106,016 FILING DATE: 31-AUG-1993 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/031,001 FILING DATE: 12-MAR-1993 (viii) ATTORNEY/AGENT INFORMATION: NAME: Amy E. Mandragouras REGISTRATION NUMBER: 36,207 REFERENCE/DOCKET NUMBER: (IMI-040PC) (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617) 227-5941 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1124 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear s I WO 94/21675 WO 9421675PCT/US94/02537 (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 16. .804 (ix) FEATURE: NAME/KEY: nat-peptide LOCATION: 85. .804 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CAAATTCAAG ACAAG ATO GCC TCC TCC TCG TCG GTG CTC CTG GTG OTO GCG 51 Met Ala Ser Ser Ser Ser Val Leu Leu Val Val Ala -23 -20 CTG TTC CCC OTO TTC CTO GGC AGC GCO CAT GGC ATC C AAG GTA CCA LeU Phe Ala Val Phe Leu CCG GOC CCC. AAC ATC ACG 147 Pro Gly Pro Asn Ile Thr Gly Ser Ala His Gly Ile Ala -5 1 Lys Val Pro GCC GAG TAC Ala Glu Tyr AAG AGC ACC 195 Lys Ser Thr 0CC 0CC OCG 243 Oly Oly Ala ATO ACC C 291 Met Thr Gly TOO TAT GOC AAG CCG Trp Tyr Gly Lys Pro TOC 000 TAC AAG GAC Cys Gly Tyr Lys Asp 45 TOC 0CC AAC ACC CCC Cys Gly Asn Thr Pro 60 TTC GAG ATC AAG TOC Phe GluIle Lys Cys 75 ACC GTC ACA ATC ACC Thr Val Thr Ile Thr 0CC GAC AAG TOG CTG GAC GCG Oly Asp Lys Trp Leu Asp Ala 0CC 0CC GOT CCC AAG GAC AAC Gly Ala Gly Pro Lys Asp Asn GAC AAG C CCG TTC AAC C Asp Lys Ala Pro Phe-Asn Gly

C

339 Gly

GAG

387 Glu

TCC

Ser

OCT

Ala ATC TTC AAG GAC Ile Phe Lys Asp ACC AAO CCC GAO Thr Lys Pro Glu

C

Gly COT 0CC TGC Arg Gly Cys

TGC

Cys

OTC

TCC TOC TCC Ser Cys Ser

GAC

Asp CCC TAC CAT TTC 435 Pro Tyr His Phe 105 AAG 0CC GAO GAG 483 Lys Cly Olu Glu 120

GAC

Asp

CAG

Gin CTC TCG 0CC CAC Leu Ser Cly His 110 AAO CTC CCC AOC Lys Leu Arg Ser GAC AAC GAO GAO CCC ATC OCA Asp Asn Glu Glu Pro Ile Ala 95 100 OCO TTC COG TCC ATO C AAO Ala Phe Cly Ser Met Ala Lys 115 0CC C GAG CTC GAG CTC CAG Ala Gly Clu Leu Glu Leu Gin 130 125 WO 94/21675 WO 9421675PCT/US94/02537 TTC AGG 531 Phe Arg 135 COG GTC AAG TG AAG TAG CCG GAC GG ACC AAG CCG ACA TTC

CAC

579 His 150

TAG

627 Tyr

GTG

Val

GTG

Val Arg Val Lys Gys GAG AAO GGT TGG Glu Lys Ala Ser 155 GAG GGG GACG GT Asp Gly Asp Gly Lys 140

AAC

Asn Tyr Pro Asp Gly GGG AAG TAG GTG Pro Asn Tyr Leu 160 Lys Pro Thr Phe ATT CG GTO AAG Ile Leu Val Lys 165 ATG AAG GAG AAG Ile Lys Glu Lys 170 GG AAG OAT AAG TG 675 Oly Lys Asp Lys Trp 185 AGO ATG GAG AGG GGG 723 Arg Ile Asp Thr Pro 200

ATG

Ile

GAT

Asp GACGOTO OTO Asp Val Val GAG GTG AAO Glu Leu Lys 190 AAO CG AG Lys Leu Thr

GO

Ala 175 180

OTO

Val

GAG

Asp GAG TG TGO OGA GGA GTG TGG 01U Ser Trp Oly Ala Val Trp 195 GOC GGA TTG AGCGOTG GG TAG Gly Pro Phe Thr Val Arg Tyr 210 OTG GAG OAT OTG ATG GGT GAG Val Olu Asp Val Ile Pro Olu 205 ACG AGG GAG G 771 Thr Thr Olu Gly 215 GC TOO AAG GG 824 Gly Trp Lys Ala 230 GG AGG AAA Oly Thr Lys 220 GAG AGG TOG Asp Thr Ser 235

TGG

Ser

TAG

OAA

Glu 225 TG GG AAO TGAGGAAGAA GTGGAGTGAT Tyr Ser Ala Lys 240 GTTCTTGGAA TGAGTTAAT TTTGAGTGAA 884 CGAGGCGGTG AOAGATAGAA GTGGTGGAT 944 AGAAAGATG CGAATAAGA OTTTGAGGTC 1004 ACGGAATCG GCTGGATGTT TGTTTGGTG 1064 AGGCGAGATT AAGGTAGTAT GTAATATATO 1124 OATGTGAAAT AATCGAGGG GAGATATATA GAOTATATTG ATTGATGGG TATAGAGAGO GAGAGGTTOT GAGAAGTGTA TATAGGAGGA GAGGTGTAG TGTAAGOTT ATGTTGTAAG GAAGGTATGG TGATTTTCGG TAAAAAAAAA INFORMATION FOR SEQ ID NO:2: SEQUENGE GHARAGTERISTIGS: LENGTH: 263 amrino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENGE DESGRIPTION: SEQ ID NO:2: Met Ala Ser Ser Ser Ser Val Leu Leu Val Val Ala Leu Phe Ala Val WO 94/21675 WO 9421675PCT(LUS94/02537 -23 Phe Leu Ile TIyr Gly Gly Giu Val Asp Gin Lys Ala Asp 170 Trp Pro Giy~ Asp Thr Ala Giy Tyr Asn Ile Thr Leu Lys Cys Ser 155 Giy Ile Asp rhr ['hr Lys Lys Thr Lys Ile Ser Leu Lys 140 Asn Asp Giu Lys Lys 220 Ser -20 Ser GiU Pro Asp Pro Cys Thr Gly Arg Tyr Pro Val Leu Leu 205 Ser C Tyr E Aia His Giy Tlyr Gly Asp 15 Thr Giy Ala 30 Vai Asp Lys Ile Phe Lys Thr Lys Pro 80 Asp Asp Asn 95 His Ala Phe 110 Ser Aia Gly Pro Asp Gly Psn Tyr Leu 160 lai Aia Vai 175 ys Giu Ser L9 0 ['hr Gly Pro iu Vai Giu er Ala Lys Ile Lys Giy Ala Asp 65 Glu Glu Giy Giu Thr 145 Ala Asp I'rp Ala Trp Pro Pro 50 Gly Ser Giu Ser Leu 130 Lys Ile Ile Gly *Lys Val Pro Leu Asp Aia 20 Lys Asp Asn 35 Phe Asn Gly Arg Gly Cys Cys Ser Gly Pro Ile Aia 100 Met Ala Lys 115 Giu Leu Gin Pro Thr Phe Leu Val Lys 165 Lys Giu Lys 180 Ala Val Trp 195 Val Arg Tyr Ile Pro Giu Pro Lys Gly Met Gly GlU Pro Lys Phe His

ISO

Tyr Gly Arg rhr Gly 230 Gly Ser Gly Thr Ser Ala Tyr Gly Aazg 135 Val Val Lys Ile rhr 215 Pro Thr Ala Gly Cys Val His GlU 120 Arg Giu Asp Asp Asp 200 Glu Asn Trp Cys Cys Phe Thr Phe 105 Glu Val Lys Gly Lays 185 rhr ily Phe Thr 210 Asp Val 225 Trp Lys Ala 240 WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Lys Trp Leu Asp INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Lys Trp Leu Asp INFORMATION FOR SEQ I* SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Ala Glu Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr 1 5 10 Gly Lys Pro Thr INFORMATION FOR SEQ ID NO:6:

I

WO 94/21675 PCT/US94/02537 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Asn Gly Gly Ala INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Asp Lys Ala Pro INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Asp Lys Ala Pro INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: II Ip -r 1 111"-- WO 94/21675 PCT/US94/02537 LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Cys Gly Asn Thr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID Phe Asn Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys Asp Gly 1 5 10 I I- WO 94/21675 PCT/US94/02537 Arg Gly Cys Gly INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Pro Ile Phe Lys Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys 1 5 10 Cys Thr Lys Pro INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro Glu Ser Cys Ser Gly Glu 1 5 10 Ala Val Thr Val INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Glu Glu Pro Ile ul l'-l l-ag WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Leu Ser Gly His INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ser Met Ala 1 5 10 Asp Asp Gly Glu ii WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Lys Lys Gly Glu INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Ala Gly Glu Leu INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu Gin Lys Leu Arg Ser 1 5 10 Ala Gly Glu Leu INFORMATION FOR SEQ ID NO:19: WO 94/21675 PCT/US94/02537 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Glu Gin Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gin Phe Arg Arg 1 5 10 Val Lys Cys Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Leu Gin Phe Arg Arg Val Lys Cys Lys Tyr Pro Asp Asp Thr Lys 1 5 10 Pro Thr Phe His INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Pro Asn Tyr Leu INFORMATION FOR SEQ ID NO:22: I IIL I -WO 94/21675 PCT/US94/02537 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Val Asp Gly Asp INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Val Asp Gly Asp INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Asp Ile Lys Glu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: I-I Ire U WO 94/21675 PCT/US94/02537 LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp 1 5 10 Ile Glu Leu Lys INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Trp Arg Ile Asp INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Gly Pro Phe Thr INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids I I l~~rlg~ IWO 94/21675 PCT/US94/02537 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Gly Gly Thr Lys WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Ile Pro Glu Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser 1 5 10 Tyr Ser Ala Lys INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Lys Tyr Val Asp Gly Asp INFORMATION FOR SEQ ID NO:32: I WO 94/21675 PCT/US94/02537 SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Lys Tyr Val Asp Gly Asp INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Lys Lys INiORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid WO 94/21675 PCT/US94/02537 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile Leu Asp Glu 1 5 10 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 Lys Lys INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal WO 94/21675 WO 9421675PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Asp Glu Pro Asn Tyr Leu Ala Ile Leu Val Lys Tyr Val Asp Glu 1 5 10 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu 1 5 10 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys 1 5

L

WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:7 SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr 1 5 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro Phe Thr 1 5 I I- WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide .0 FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: G.LU Ser Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys 1 5 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 7 Ala Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr 1 5 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10

II

WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Ser Ala Lys 1 5 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 723 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..720

Y

I WO 94/21675 WO 9/2165 1PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: ATC CCC AAG GTG CCC CCG GGC CCG AAC ATC ACO GCG ACC TAC GOT GAC Ile 1

AAG

Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp TOG CTG GAC Lys Trp Leu Asp CCC CCC AAG CAC 144 Gly Pro Lys Asp C AAG AGC ACA TGO TAC COC AAG Ala Lys Ser Thr Trp Tyr Gly Lys 25 CCG ACG CCC GCC Pro Thr Cly Ala GCG CCG 192 Ala Pro GAC CG 240 Asp Gly GAG TCG 288 Giu Ser

TTC

Phe AAC GOC CCC GCG TC Asn Gly Gly Ala Cys 40 GGC ATG ACC 0CC TGC Cly Met Thr Cly Cys

GGG

C ly

C

Gly TAC AAG GAC GTG GAC AAG Tyr Lys Asp Val Asp Lys AAC ACC CCC ATC TTC AAG Asn Thr Pro Ile Phe Lys

AAC

Asn 55 CCC CCC TGC COT Arg Gly Cys Oly 70 TGC TCC OGC GAG Cys Ser Cly Giu TCC TCC TTC GAG Ser Cys Phe Glu 0CC GTC ACC CTC Ala Val Thr Val 90

ATC

Ile 75

AAC

Lys TG ACG AAG CCC Cys Thr Lys Pro GAG GAG 336 CiU CiU CCC ATC Pro Ile 100 COT TCC ATG 0CC 384 Cly Ser Met Ala 115 GAG CTC GAG 432 Glu Leu Giu 130 ACC AAC GTG 480 Thr Lys Val

CTG

Leu

ACC

Thr CC CCC TAC CAC TTC Ala Pro Tyr His Phe 105 AAC AAG 0CC GAG GAG Lys Lys Cly Ciu Ciu .120 CAG TTT AGO CCC GTO Gin Phe Arg Arg Val 135 TTC.CAC GTC GAG AAG Phe His Val Giu Lys

GAC

Asp

CAG

Gin CAC ATC ACC GAC GAC AAC His Ile Thr Asp Asp Asn CTT TCC GGC CAC CC TTC Leu Ser Gly His Ala Phe 110 AAG CTG CCC AGC CC GGC Lys Leu Arg Ser Ala Gly AAG TCC AAG Lys Cys Lys 140 COT TCC AAC Gly Ser Asn 155 125 TAC CCC GAG 0C Tyr Pro Glu Gly CCC AAC TAC CTG Pro Asn Tyr Leu 160 145

GCG

528 Ala

CAT

576 Asp

TG

624 15.0 CTG CTG GTG AAG Leu Leu Val Lys 165 ATC MAG GAG AAG Ile Lys Ciu Lys 180

TAC

Tyr

C

Gly OTC GAC 0C Val Asp Gly AAC GAC AAG Lys Asp Lys

CAC

Asp 170

TG

Trp 0CC CAC GTC GTC CC GTG Gly Asp Val Val Ala Val 175 ATC CCG CTC AAC GAG TCA Ile Ala LeU Lys Clu Ser 190 CAC MAG CTG ACG GGC CCA 185 GOA CCC ATC TGG AGO GTC GAC ACC CCC IWO 94/21675 PCTIUS94/02537 Trp Gly Ala Ile Trp Arg Val. Asp Thr Pro Asp Lys Leu Thr Gly Pro 195 200 205 WO 94/21675 WO 9421675PCT/US94/02537 TTC ACC GTT CGC TAC ACC ACC GAG GGA GGC ACC AAG TCC GAA GTT GAG 672 Phe Thr Val Arg Tyr Thr Th,: Glu Gly Gly Thr Lys Ser Glu Val Glu 210 215 220 GAC GTC ATC CCC GAG GGC TGG AAG GCC GAC GCC AGC TAC GAG TCC AAG 720 Asp Val Ile Pro Giu Gly Trp Lys Ala Asp Ala Ser Tyr Glu Ser Lys 225 230 235 240

TGA

723 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) 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 Lys Gly Al.a Asp 65 Glu Glu Gly Glu Thr 145 Ala Asp Trp Leu Asp Pro Lys Asp Pro Phe Asn Gly Arg Gly Ser Cys Ser Glu Pro Ile 100 Sar Met Ala 115 Leu Glu Leu 130 Lys Val Thr Leu Leu Val Ile Lys Giu 180 Ala Asn Gly Cys Gly Ala Lys Gin Phe Lys 165 Lys Lys Gly Met G iy 70 Glu Pro Lys Phe His 150 Tlyr Ser Gly Thr 55 Ser Ala Gly Arg 135 Val Val Thr Trp 25 Ala Cys 40 Gly Cys Cys Phe Val Thr His Phe Glu Glu 120 Arg Val Glu Lys Asp Gly Tyr Gly Lys Gly Tyr Lys Gly Asn Thr Glu Ile Lys 75 Val His Ile 90 Asp Leu Ser Gin Lys Leu Lys Cys Lys 140 Giy Ser Asn 155 Asp Gly Asp 170 Trp Ile Ala Pro Asp Pro Cys Thr Gly Arg 125 Tyr Pro Val Leu Thr Val 11ie Thr Asp His 110 Ser Pro Asn Val Lys 190 Gly Asp Phe Lys Asp Ala Ala Giu Tyr Ala 175 Glu Ala Lys Lys Pro Asn Phe Gly Gly Leu 160 Val Ser Gly Lys Asp Lys 185 Trp Gly Ala 195 Ile Trp Arg Val lie Tp Ar Val Thr Pro Asp Lys ThGyPr Thr Gly Pro WO 94/21675 PCT/US94/02537 Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ser Glu Val Glu 210 215 220 Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Ala Ser Tyr Glu Ser Lys 225 230 235 240 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 723 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..720 (ix) FEATURE: NAME/KEY: Modified-sit LOCATION: 32 OTHER INFORMATION: /nc (xi) SEQUENCE DESCRIPTION: SEQ I ATC GCG AAG GTT CCC CCC GGC CCG AAC Ile Ala Lys Val Pro Gly Pro Asn te ote= "Xaa is Ser, Pro or Ala" [D NO:53: ATC ACG GCG ACC TAC GGC GAC Ile Thr Ala Thr Tyr Gly Asp TAC GGC AAG CCG ACC GGN BCC Tyr Gly Lys Pro Thr Gly Xaa GGA TAC AAG GAC GTG GAC AAG Gly Tyr Lys Asp Val Asp Lys GGC AAC ACC CCC ATC TTC AAG Gly Asn Thr Pro Ile Phe Lys AAG TGG CTT 96 Lys Trp Leu GGT CCC AAG 144 Gly Pro Lys

GAC

Asp

GAC

Asp GCG AAG AGC ACC Ala Lys Ser Thr AAC GGC GGC GCG Asn Gly Gly Ala 40 CCC CCG 192 Pro Pro

TCC

240 Ser

GAG

288 Glu

GGC

Gly

TCC

Ser TTC AGC GGC ATG ACC Phe Ser Gly Met Thr 55 CGC GGC TGC GGC TCC Arg Gly Cys Gly Ser 70 TGC TCC GGG GAG CCC Cys Ser Gly Glu Pro

GGC

Gly

TGC

Cys TGC TTC GAG ATC Cys Phe Glu Ile 75 GTC CTG GTC CAC Val Leu Val His 90 CAC TTC GAC CTC His Phe Asp Leu 105 AAG TGC ACC AAG CCC Lys Cys Thr Lys Pro ATC ACC GAC GAC AAC Ile Thr Asp Asp Asn TCC GGC AAG GCG TTC Ser Gly Lys Ala Phe 110 GAG GAG CCC ATC 336 Glu Glu Pro Ile 100 GCC GCC TAC Ala Ala Tyr I I WO 94/21675 WO 9421675PCT[US94/02537 GGG GCC ATG CO AAG AAG GGT GAG GAG CAG AAG CTG CGC AGC GCC GCC Gly Ala Met 115 GAG OTG 432 Glu Leu 130 ACC AAG 480 Thr Lys

GAG

Glu

GTT

Val Ala Lys Lys Gly CTC AAG, TTC CC Leu Lys Phe Arg 135 ACC TTC CAC GTC Thr Phe His Val Glu 120

CGC

Arg

GAG

Glu Glu Gin Lys Leu GTC AAG TGC GAG Val Lys Cys Ul1u 140 AAG GGG TCC AAO Lys Giv Ser Asn Arg Ser Ala Gly 125 TAO CCG AAG GO Tyr Pro Lys Cly CCC AAC TAC OTT Pro Asn Tyr Leu 160 GTG GTG GOG GTG Val Val Ala Val 145

GCG

528 Ala 150 OTGC TG GTG AAG Leu Leu Val Lys 165 TAO GTC Tyr Val 155 GAO GGO GAO CCC GAO Asp Gly Asp Gly Asp 170 175 GAO ATO AAG CAG 576 Asp Ile Lys Gin 180 TGG GGA GC GTO 624 Trp Gly Ala Val

AAG

Lys GGO AAG GAO AAG Gly Lys Asp Lys 185

TGG

Trp ATO GAG OTO AAG GAG TOG Ile Giu Leu Lys Glu, Ser 190 TTO ACC 672 Phe Thr 210 GAO GTO 720 Asp Val 225

TGA

723 195 GTO OGO Val Arg ATO 000 TOG AGG ATO GAO Trp Arg Ile Asp 200 TAO ACC ACC GAG Tyr Thr Thr Glu 215 GAG GGC TGG AAG Thr

GC

Glv 000 GAO AAG OTO ACO CCC 000 Pro Asp Lys Leu Thr Gly Pro 205 CCC ACC AAG CO CAA CO GAG Cly Thr Lys Ala Glu Ala Giu 220 CO GAO ACC CO TAO GAG CCC AAC Ile Pro Glu Gly 230 Trp Lys Ala Asp Thr Ala Tyr Glu Ala INFORMATION FOR SEQ ID NO:54: SEQUENCE OHARAOTERISTIOS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 94/21675 WO 94/ 1675PCT/US94/02537 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 32 OTHER INFORMATION: /note= "Xaa (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: is Ser, Pro or Ala" Ile Ala Lys Val Lys Trp Leu Gly Pro Ser Giu Giu Gly Giu Thr 145 Ala Asp Trp Phe Asp 225 Pro Pro dly Ser Glu Ala Leu 130 Lys Leu Ile Gly Thr 210 Val Lys 35 Phe Arg Cys Pro Met 115 Glu Val Lemu Lys Ala 195 Val Ile Asp Asp Ser Gly Ser Ile 100 Ala Leu Thr Val Gin 180 Val Arg Pro Pro Ala Asn Gly Cys Gly Ala Lys Lys Phe Lys 165 Lys Trp Tyr Giu Gly Met Gly 70 Glu Ala Lys Phe His 150 Tyr Gly Arg I'hr Gly 230 Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Gly Thr 55 Ser Pro Gly Arg 135 Val Vai Lys Ile Thr 215 Trp Lys Ser Thr Ala 40 Gly Cys Val His Glu 120 Arg GiU Asp Asp Asp 200 Giu Lys Trp 25 Cys Cys Phe Leu Phe 105 Glu Val Lys Gly Lys 185 Thr Gly Ala Tyr Gly Lys Gly Tlyr Lys Gly Asn Thr Glu Ile Lys 75 Val His Ile 90 Asp Leu Ser Gin Lys Leu Lys Cys Giu 140 Gly Ser Asn 155 Asp Gly Asp 170 Trp Ile Giu Pro Asp Lys Gly Thr Lys 220 Asp Thr Ala 235 Pro Asp Pro Cys Thr Gly Arg 125 Tyr Pro Val rLeu Leu 205 Ala T'yr Thr Val Ile Thr Asp Lys 110 Ser Pro Gly

ASP

Phe Lys Asp Ala Ala Lys Xaa Lys Lys Pro Asn Phe Giy Gly Asn Val L.ys 190 rhr Glu 3iu Tyr Leu 160 Ala Val 175 Glu Ser Gly Pro Ala Giu Ala Lys 240 IWO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 723 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..720 (xi) SEQUENCE DESCRIPTION: SEQ ID ATC GCG AAG GTG CCC CCG GGT CCG AAC ATC 48 Ile 1 Ala Lys Val Pro Pro Gly Pro 5 AAG TGG CTC 96 Lys Trp Leu GGT CCC AAG 144 Gly Pro Lys GAC GCG AAG AGC ACA Asp Ala Lys Ser Thr GAC AAC GGC GGC GCT Asp Asn Gly Gly Ala AGC GGC ATG ACC GGC Ser Gly Met Thr Gly 55 GGC TGC GGC TCC TGC Gly Cys Gly Ser Cys Asn Ile 10 TGG TAC Trp Tyr 25 TGC GGG Cys Gly ACG GCG ACC TAC GGC GAC Thr Ala Thr Tyr Gly Asp GGC AAG CCG ACG GGG GCC Gly Lys Pro Thr Gly Ala TAC AAG GAC GTG GAC AAG Tyr Lys Asp Val Asp Lys CCC CCG 192 Pro Pro TCC GGC 240 Ser Gly

CGT

Arg TGC GGC AAC Cys Gly Asn TTT GAG ATC Phe Glu Ile 75

ACC

Thr CCC ATC TTC AAG Pro Ile Phe Lys

GAG

288 Glu 70 AAG TGC ACG AAG CCC Lys Cys Thr Lys Pro ATC ACC GAC GAC AAC Ile Thr Asp Asp Asn GCC TGC TCC GGC Ala Cys Ser Gly 70 GAG GAG CCC ATC 336 Glu Glu Pro Ile 100 GGG GCG ATG GCC 384 Gly Ala Met Ala

GCC

Ala

AAG

Lys GAG CCC GTG GTA Glu Pro Val Val CCC TAC CAC TTC Pro Tyr His Phe 105 AAG GGC GAT GAG Lys Gly Asp Glu

GTC

Val 90

CAC

His GAC CTC TCC GGC CAC GCG TTC Asp Leu Ser Gly His Ala Phe 110 CAG AAG CTG CGC ACG GCC GGC Gin Lys Leu Arg Thr Ala Gly 125 AAG TGC AAG TAC CCG GAG GGG Lys Cys Lys Tyr Pro Glu Gly 140 GAG CTG 432 Glu Leu 130 115 GAG CTC CAG TTC Glu Leu Gln Phe 120 CGG CGC GTC Arg Arg Val 135 WO 94/21675 PCT/US94/02537 ACC AAG 480 GTG ACC TTC CAC GTG GAG AAG GGG TCC AAC CCC AAC TAC CTG Thr 145

GCG

528 Ala Lys Val Thr Phe CTG CTT GTG AAG Leu Leu Val Lys 165 Val Glu Lys Gly GTT AAC GGC GAC Val Asn Gly Asp 170 AAG GAC AAG TGG Lys Asp Lys Trp Ser Asn 155 GGA GAC Gly Asp Pro Asn Tyr GTG GTG GCG GTG Val Val Ala Val 175 GAC ATC AAG GAG AAG GGC 576 Asp Ile Lys Glu Lys Gly 180 TGG GGA GCC ATC TGG AGG 624 Trp Gly Ala Ile Trp Arg ATC GAG CTC AAG GAG TCG Ile Glu Leu Lys Glu Ser 190 185 ATC GAC ACT Ile Asp Thr TTC ACC 672 Phe Thr 210 GAC GTC 720 Asp Val 225

TGA

723 195 GTC CGC Val Arg ATC CCT Ile Pro CCC GAC AAG CTC ACG GGC CCC Pro Asp Lys Leu Thr Gly Pro 205 GGC ACC AAG ACC GAA GCC GAG Gly Thr Lvs Thr Glu Ala Glu TAC ACC ACC Tyr Thr Thr 215 GAG GGC TGG Glu Gly Trp 230

GGC

Gly

AAG

LVs 220 GCC GAC ACC AGC Ala ASD Thr Ser TAC GAG TCC AAG Tyr Glu Ser Lys 240 235 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: Ile 1 Ala Lys Val Pro Pro Gly Pro Asn Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr Trp 20 25 Gly Pro Lys Asp Asn Gly Gly Ala Cys 1 Pro Pro Phe Ser Gly Met Thr Gly Cys 55 Ser Gly Arg Gly Cys Gly Ser Cys Phe 70 Glu Ala Cys Ser Gly Glu Pro Val Val Glu Glu Pro Ile Ala Pro Tyr His Phe Gly Lys Pro Gly Tyr Lys Gly Asn Thr Glu Ile Lys Thr Gly Ala Val Asp Lys lie Phe Lys Cys Thr Lys Val His Ile Thr Asp Asp Asn Ala Phe Leu Set Gly His WO 94/21675 PCT/US94/02537 Gly Glu Thr 145 Ala Asp Trp Phe Asp 225 (2) 100 105 Ala Met Ala Lys Lys Gly Asp Glu 115 120 Leu Glu Leu Gin Phe Arg Arg Val I 130 135 Lys Val Thr Phe His Val Glu Lys C 150 Leu Leu Val Lys Tyr Val Asn Gly A 165 1 Ile Lys Glu Lys Gly Lys Asp Lys T 180 185 Gly Ala Ile Trp Arg Ile Asp Thr P 195 200 Thr Val Arg Tyr Thr Thr Glu Gly G 210 215 Val Ile Pro Glu Gly Trp Lys Ala A 230 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal 31n Lys Lys Cys ;ly Ser 155 %sp Gly .70 'rp Ile ro Asp ly Thr sp Thr 235 Leu Lys 140 Asn Asp Glu Lys Lys 220 Ser Arg 125 Tyr Pro Val Leu Leu 205 Thr Tyr Thr Pro Asn Val Lys 190 Thr Glu Glu Ala Gly Glu Gly Tyr Leu 160 Ala Val 175 Glu Ser Gly Pro Ala Glu Ser Lys 240 (ix) FEATURE:

NAME/KEY:

LOCATION: OTHER INFORMATION: (ix) FEATURE:

NAME/KEY:

LOCATION: 144 OTHER INFORMATION: (ix) FEATURE:

NAME/KEY:

LOCATION: 154 OTHER INFORMATION: (ix) FEATURE:

NAME/KEY:

LOCATION: 187 OTHER INFORMATION: (ix) FEATURE:

NAME/KEY:

LOCATION: 223 OTHER INFORMATION: /note= "Xaa is Asn or Asp" /note= "Xaa is Asp or Gly" /note= "Xaa is Gly or Ala" /note= "Xaa is Ile or Thr" /note= "Xaa is Val or Phe" WO 94/21675 WO 9421675PCTfUS94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Ile Ala Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Glu Tyr Gly Asp 1 Lys Gly Ala Asp Glu Glu Gly Glu Thr 145 Ala Asp Trp Phe Asp 225 Trp, Pro Pro Gly Ser Glu Ser Leu 130 Lys Ile Ile Gly Thr 210 Val Leu Lys Phe Arg Cys Pro Met 115 Glu Pro Leu Lys Ala 195 Val Ile Asp Asp Asn Gly Ser Ie 100 Ala Leu Thr Val Giu 180 Val Arg Pro Ala Asn Gly Cys Gly Ala Lys Gin Phe Lys 165 Lys Trp Tyr Glu Lys Gly Met Gly 70 Giu Pro Lys Phe His 150 Gly Arg Thr Gly 230 Ser Gly Thr 55 Ser Ala Tyr Gly Arg 135 Val Val Lys Ile Thr 215 Trp Thr Ala 40 Gly Cys Val His Glu 120 Arg Giu Asp Asp Asp 200 Glu Lys Trp 25 Cys Cys Phe Thr Phe 105 Glu Val Lys Gly Lys 185 Thr G ly Ala 10 Tyr Gly Gly Tyr Gly Asn Giu Ile 75 Val Thr 90 Asp Leu Gin Lys Lys Cys Xaa Ser 155 Asp Gly 170 Trp Xaa Pro Asp Gly Thr Asp Thr 235 Lys Lys Thr Lys Ile Ser Leu Lys 140 As n Asp Glu Lys Lys 220 Ser Pro Thr Xaa Val Pro Ile Cys Thr Thr Asp Gly His 110 Arg Ser 125 Tyr Pro Pro Asn Val Val Lou Lys 190 Lou Thr 205 Ser GlU Tyr Ser Gly Ala Asp Lys Phe Lys Lys Pro Asp Asn Ala Phe Ala Gly Asp Xaa Tyr Leu 160 Ala Val 175 Giu Ser Gly Pro Xaa Giu Ala Lys 240 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (ix) FEATURE:

NAME/KEY:

WO 94/21675 PCT/tIS94/02537 LOCATION: 199 OTHER INFORMATION: /note= "Xaa (ix) FEATURE:

NAME/KEY:

LOCATION: 235 OTHER INFORMATION: /note= "Xaa (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Ile Pro Lys Val Pro Pro Gly Pro Asn Ile 1 5 10 Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr 25 Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly 40 Ala Pro Phe Asn Gly Met Thr Gly Cys Gly 55 Asp Gly Arg Gly Cys Gly Ser Cys Phe Glu 70 Glu Ser Cys Ser Gly Glu Ala Val Thr Val 90 Giu Giu Pro Ile Ala Pro Tyr His Phe Asp 100 105 Gly Ser Met Ala Lys Lys Gly Glu Glu Gin 115 120 is Val or Ile" is Ala or Thr" Thr Gly 1'yr Asn Ile His Leu Lys Ala Lys Lys Thr Lys Ile Ser Leu Thr Pro Asp Pro Cys Thr Gly Arg 125 Thr Val Ile Thr Asp His 110 Ser Gly dly Asp Phe Lys Asp Ala Ala WO 94/21675 PCT/US94/02537 Glu Leu Glu Leu Gin Phe Arg Arg 130 135 Thr Lys Val Thr Phe His Val Glu 145 150 Ala Leu Leu Val Lys Tyr Val Asp 165 Asp Ile Lys Glu Lys Gly Lys Asp 180 Trp Gly Ala Ile Trp Arg Xaa Asp 195 200 Phe Thr Val Arg Tyr Thr Thr Glu 210 215 Asp Val Ile Pro Glu Gly Trp Lys 225 230 INFORMATION FOR SEQ ID NO:59: Val Lys Gly Lys 185 Thr Gly Ala Lys Cys Gly Ser 155 Asp Gly 170 Trp Ile Pro Asp Gly Thr Asp Xaa 235 Tyr Pro Val Leu Leu 205 Ser Tyr Pro Asn Val Lys 190 Thr Glu Glu Glu Gly Tyr Leu 160 Ala Val 175 Glu Ser Gly Pro Val Glu Ser Lys 240 SEQUENCE CHARACTERISTICS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: Ile Ala Lys Val Pro Pro Gly Pro Asn Ile 1 5 10 Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr 25 Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly 40 Pro Pro Phe Ser Gly Met Thr Gly Cys Gly 55 Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu 70 Glu Ala Cys Ser Gly Glu Pro Val Val Val 90 Glu Glu Pro Ile Ala Pro Tyr His Phe Asp 100 105 Gly Ala Met Ala Lys Lys Gly Asp Glu Gin 115 120 Glu Leu Glu Leu Gin Phe Arg Arg Val Lys 130 135 Thr Gly Tyr Asn Ile 75 His Leu Lys Cys Ala Lys Lys Thr Lys Ile Ser Leu Lys 140 Thr Pro Asp Pro Cys Thr Gly Arg 125 Tyr Tyr Gly Asp Thr Gly Ala Val Asp Lys Ile Phe Lys Thr Lys Pro Asp Asp Asn His Ala Phe 110 Thr Ala Gly Pro Glu Gly WO 94/21675 PCT/US94/02537 Thr 145 Ala Lys Leu His Val 150 Tyr Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp 180 185 Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro 195 200 Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly 210 215 Asp Val Ile Pro Glu Gly Trp Lys Ala Asp 225 230 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 240 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (ix) FEATURE:

NAME/KEY:

LOCATION: 88 OTHER INFORMATION: /note= "Xaa (ix) FEATURE:

NAME/KEY:

LOCATION: OTHER INFORMATION: /note= "Xaa (ix) FEATURE:

NAME/KEY:

LOCATION: 180 OTHER INFORMATION: /note= "Xaa (xi) SEQUENCE DESCRIPTION: SEQ ID Ile Ala Lys Val Pro Pro Gly Pro Asn Ile 1 5 Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly 40 Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu 70 Ser 155 Gly Ile Asp Thr Thr 235 Asn Asp Glu Lys Lys 220 Ser Pro Val Leu Leu 205 Thr Tyr Asn Val Lys 190 Thr Glu Glu Glu Ser Gly Pro Ala Glu Ser Lys 240 is Val or Ile" is Val or Ile" is Gin or Glu" Thr Pro Asp Pro Cys Asp Xaa Lys Lys Pro Glu Ser Cys Ser Gly Glu Pro Xaa Leu Xaa His Ile Thr Asp Asp Asn I- II I WO 94/21675 PCT/US94/02537 90 Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Lys Ala Phe 100 105 110 Gly Ala Met Ala Lys Lys Gly Glu Glu Gln Lys Leu Arg Ser Ala Gly 115 120 125 Glu Leu Glu Leu Lys Phe Arg Arg Val Lys Cys Glu Tyr Pro Lys Gly 130 135 140 Thr Lys Val Thr Phe His Val Clu Lys Gly Ser Asn Pro Asn Tyr Leu 145 150 155 160 Ala Leu Leu Val Lys Tyr Val Asp Gly Asp Gly Asp Val Val Ala Val 165 170 175 Asp Ile Lys Xaa Lys Gly Lys Asp Lys Trp Ile Glu Leu Lys Glu Ser 180 185 190 Trp Gly Ala Val Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly Pro 195 200 205 Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Ala Glu Ala Glu 210 215 220 Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ala Lys 225 230 235 240 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Val Asp Gly Asp INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: lm-- -m II I WO 94/21675 PCT/US94/02537 Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr 1 5 10 Val Asn Gly Asp INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Ile Ala Leu Lys INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: Gly Asp Val Val Ala Val Asp Ile Lys Gin Lys Gly Lys Asp Lys Trp 1 5 10 Ile Glu Leu Lys INFORMATION FOR SEQ ID SEQUL,.JE CHARACTERISTICS: LENGTH: 20*amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID e- r WO 94/21675 PCT/US94/02537 Glu Ser Trp Gly Ala lie Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr 1 5 10 Gly Pro Phe Thr INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Gly Pro Phe Thr INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: Thr Glu Ala Glu Asp Val 1le Pro Glu Gly Trp Lys Ala Asp Thr Ser 1 5 10 Tyr Glu Ser Lys INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (li) MOLECULE TYPE: peptide 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 WO 94/21675 PCT/US94/02537 1 5 10 Tyr Glu Ala Lys INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 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 1 5 10 Tyr Glu Ser Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: arpino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Glu Val Glu Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser 1 5 10 Tyr Glu Ser Lys INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: GGGTCTAGAG GTACCGTCCG ATCGATCATT I II a so WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: AATGATCGAT GCT 13 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: GGGTCTAGAG GTACCGTCCG INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: CCCTGCAGAT TATTTGAGAT CTTGAG 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID IWO 94/21675 PCT/US94/02537 CCCTGCAGTC ATGCTCACTT GGCCGAGTA 29 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: GAGTACGGCG ACAAGTGGC 19 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: TTCGAGATCA AGTGCACC 18 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA WO 94/21675 PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: GTGACAGCCT CGCCGG 16 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: GGGAATTCCA TGGCGAAGAA GGGC 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GTGCCGTCCG GGTACT 16 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: CCGTCGACGT ACTTCA 16 WO 94/21675 PCT/US94/02537 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: GGAGTCGTGG GGAGCAGTC 19 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: GGGTCTAGAG GTACCGTCC 19 INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: TTGGATCCTA CGGCAAGCCG ACCGGC 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID

I

'WO 94/21675 PCT/US94/02537 TTGGATCCAT CCCGAAGGTG CCCCCGGG 28 INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: ACGTGACCTT CCACGTCG 18 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: TTGGATCCTG GCGCTGCTGG TGAAGTA 27 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA WO 94/21675 PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: TTGAATTCAT CCCGAAGGTG CCCCCG 26 INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: TTGGTACCTC ACTTGGACTC GTAGCT 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CCGAATTCGT GGAGAAGGGG TCCAA INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 22 OTHER INFORMATION: /note= "Xaa is losine" 1 I WO 94/21675 PCT/US94/02537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: TTAGGATCCT CACTTATCAT ANGACGTATC INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: TTGAATTCCT TGTCATTGCC CTTCTG 26 INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STPRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: AAGAATTCCT TCTGCTTGAT GTCCAC 26 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: ATGAATTCGA GTCGTGGGGA GCCGTC 26 WO 941/21675 PCT/US94/02537 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDA (xi) SEQUENCE DESCRIPTION: SEQ ID ATGAATTCGT CTGGAGGATC GACACC 26 INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:96: ATGAATTCAT CGCAAAGGTT CCCCCC 26 INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: TTTGGATCCT CACTTGGACT CGTAGCT 27 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: WO 94/21675 TTGAATTC"'C GCGAAGGTGC CCCCG PCTIUS94/02537

Claims (39)

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: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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 Lolp 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: 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), 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. 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. i: I I WO 94/21675 PCT/US94/02537 6. An isolated peptide or portion thereof of claim 1 or 2 which, when administereJ 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 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. 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. I WO 94/21675 PCT/US94/02537 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 a positivity 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. n1 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 An isolated peptide or portion thereof of claim 17 wherein said mean T cell stimulation index is at least about

21. A peptide or portion thereof of claim 17 wherein said peptide is selected from the group consisting of: LPI-2 (SEQ ID NO: LPI-11 (SEQ ID NO: 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: 1t cet\\ ewherope-ina

22. An isolated peptide of Ll p I, or a portion thereof wherein said peptide is selected from the group corisisting of: LPI-1.1 (SEQ ID NO: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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- I I.WO 94/21675 PCT/US94/02537 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:

23. Amiodifyedpeptide or a-odifi.,portion of a peptide of claim 22 moi-AeA fco ;inc.\u-<Ae. cvn-r,\c\ a~c.cd 'iiuckot-Jon. oddtion or de\e'on.

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. 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 in release of mediators from mast cells or basophils in a substantial percentage of individuals 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 pI 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 Lolp 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: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-8 (SEQ ID NO: 12), (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-13 (SEQ ID NO: 19), (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 <4 y 99 WO 9/21675 PCT/US94/02537 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:

28. An isolated peptide of claim 27 wh a said regions compzise an amino acid sequence selected from the group consisting of: LPI-3 (SEQ ID NO: 6), LPI-4.1 (SEQ ID NO: LPI-10 (SEQ ID NO: 14), LPI-11 (SEQ ID NO: (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. (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 Lolp 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: LPI-4.1 (SEQ ID NO: 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: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-10 (SEQ ID NO: 14), and LPI-11 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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); WO 94/21675 WO 9421675PCT[US94/02537 LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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); (SEQ ID NO: 14), LPI-11 (SEQ ID NO: 15), LPI-15 (SEQ ID NO: 21), and LPI-16.1 (SEQ ID NO: 23); (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: (SEQ ID NO: 21), LPI-16.1 (SEQ ID NO: 23), LPI-18 (SEQ ID NO: and LPI-20 (SEQ ID NO: 27); (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: LPI-18 (SEQ ED NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: 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: 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: LPI-16.1 (SEQ ID NO: 23) and LPI-1 1 (SEQ ID NO: LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO: 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: 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: LPI- 16.1 (SEQ ID NO: 23), LPI-1I1 (SEQ ID NO. 15) and LPI-4.1 (SEQ ID NO: 8); WO 94/21675 PCT/US94/02537 LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO: 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: 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). 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: 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: 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: 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: 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: 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: 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:

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. WO 94/21675 PCTIUS94/02537

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. 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. 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 I, said peptide or port4pn 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: LPI-1.1 (SEQ ID NO: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-5 (SEQ ID NO: 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 lp' II Ills__C~II P:\0PERUMS\65175-94.CLM 8/9/97 -104- 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) wherein X, 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 contiguous 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- S:42. A portion of an isolated peptide of claim 41 wherein the portion comprises at least S fifteen amino acid residues. S43. A composition comprising at least one isolated peptide or a portion thereof of claim 22, 23 or 24 and a pharmaceutically acceptable carrier or diluent. A therapeutic composition comprising an isolated peptide or portion thereof of claim 27, 28 or 29 and a pharmaceutically acceptable carrier or diluent.

46. A method for treating sensitivity to Lolp I protein allergen or an allergen which is immunologically cross reactive with Lolp I protein allergen, which comprises administration of a composition of claim 43 to a patient in need thereof.

47. A method for treating sensitivity to Lolp I protein allergen or an allergen which is immunologically cross reactive with Lol p I protein allergen, which comprises administration of at least two compositions of claim 43 to a patient in need thereof. ,i P:\OPERJMS(65l75-94.CLM- 819/97 -105-

49. The method of claim 40, wherein said immunologically cross reactive allergen is Dac g I, Poap I or Phlp I. A method of detecting sensitivity to Lol p 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 the extent to which such binding occurs as indicative of sensitivity in the individual to ryegrass pollen allergen 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 cel' 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: LPI-2 (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4 (SEQ ID NO: LPI-4.1 (SEQ ID NO: 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: (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: 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) and wherein said composition comprises a sufficient percentage of the T cell epitopes of said protein allergen such that T cells of an individual sensitive to Lolp I protein pollen or an immunologically cross reactive allergen, are tolerized to said at least one protein allergen. !':AO1fl)RJMS\65 i75-94.CLM 19197 -106-

53. A composition of claim 45 comprising a combination of peptides selected from the group consisting of: LPI-3 (SEQ ID NO: LPI-4. 1 (SEQ ID NO: LPI- 10 (SEQ ID NO: 14), LPI-1 1 (SEQ ID NO: 15), LPI- 15 (SEQ ID NO: 2 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: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-l 0 (SEQ ID NO: 14), and LPI-I I (SEQ ID NO: LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI- 10 (SEQ ID NO: 14), LPI-1I1 (SEQ ID NO: 15), LPI- 15 (SEQ ID NO: 21), and LPI-1 6 (SEQ ID NO: 22); LPI-3 (SEQ ID NO: LPI-4.1 (SEQ ID NO: LPI-10 (SEQ ID NO: 14), LPI-1I1 (SEQ ID NO: 15), LPI- 15 (SEQ ID NO: 2 and LPI- 16.4 (SEQ'ID NO: 23); *LPI- 10 (SEQ ID NO: 14), LPJII (SEQ ID NO0: 15), LPI- 15(SEQ ID NO: 2 LPI- 16.1 (SEQ ED NO: 23), LPI- 18 (SEQ ID NO: 25),'LPI-20 (S8Q ID NO: 27), LPI-22 (SEQ ID NO: 29) and LPI-23 (SEQ ID NO: 15 (SEQ ID NO: 2 LPl- 16.1 (SEQ ID NO: 23), LPI- 18 (SEQ IY) NO: and LPI-20) (SEQ ID NO: 27); LPI- 15 (SEQ ID NO: 2 LPI- 16.1 (SEQ ID NO: 23), LPI- 18 (SEQ ID NO: LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-23 (SEQ ID NO: :LPI- 18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-22 (SEQ ID NO: 29), and LPI-2.3 (SEQ ID NO: LPI- 18 (SEQ ID*YNO: 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: LPI- 18 (SEQ MD 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: and LPI- 16.1 (SEQ ID NO: 23); WO 94/21675 PCT/US94/02537 LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO: LPI-16.1 (SEQ ID NO: 23) and LPI-11 (SEQ ID NO: LPI-18 (SEQ ID NO: 25), LPI-20 (SEQ ID NO: 27), LPI-23 (SEQ ID NO: 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: 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: 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: 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: 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 pjptides 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: 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: 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: 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: 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: 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: WO 94/21675 PCT/US94/02537 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: 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: recombinantly or synthetically producing peptides of Lol p I; examining said peptides for their ability to influence B cell and/or T cell responses in ryegrass pollen sensitive individuals; selecting appropriate peptides which contain epitopes recognized by the cells, and 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 mcdif t the individual's allergic reaction to ryegrass pollen comprising the steps of: recombinantly or synthetically producing peptides of Lol p I; examining said peptides for their ability to influence B cell and/or T cell responses in ryegrass pollen sensitive individuals; and 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. An isolated nucleic acid having a nucleotide sequence coding for Dac g I, or the functional equivalent of said nucleotide sequence. I -I-ip I II~- PAOPERUMS\65175-94.C -B1997 -109-

61. An isolated nucleic acid sequence of claim 60 wherein said nucleotide sequence comprises the nucleotide sequence of Fig.

62. An expression vector comprising a nucleotide sequence coding for Dac g I, or the functional equivalent of said nucleotide sequence.

63. A host cell transformed to express a protein encoded by the nucleic acid of claim

64. Isolated Dac g I protein prod.,ced in a host cell transformed with the nucleic acid of claim An isolated nucleic acid having a nucleotide sequence coding for Poa p I, or the 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. S i: 67. An expression vector comprising a nucleotide sequence coding for Poa p I, or the functional equivalent of said nucleotide sequence.

68. A host cell transformed to express a protein encoded by the nucleic acid of claim

69. Isolated Poa p I protein produced in a host cell transformed with the nucleic acid of claim An isolated protein allergen which is immunologically related to Lol p I, wherein the protein allergen is either Dac g I or Poap I. Dated this 8th day of September, 1997. ImmuLogic Pharmaceutical Corporation By its Patent Attorneys Davies Collison Cave VTO III