WO1995027786A1

WO1995027786A1 - Pharmaceutical formulations for treating japanese cedar pollen allergy

Info

Publication number: WO1995027786A1
Application number: PCT/US1995/004249
Authority: WO
Inventors: Henry M. Franzén; Stephen Palmer Powers; Mei-Chang Kuo; Sean Evans; Ze'ev Shaked; Xian Chen
Original assignee: Immulogic Pharmaceutical Corporation
Priority date: 1994-04-08
Filing date: 1995-04-06
Publication date: 1995-10-19
Also published as: EP0756629A1; JP4179422B2; AU2241995A; JPH09511747A; CA2187336A1

Abstract

The present invention provides novel peptides of Cry j I which have been modified as a part of a preformulation scheme to develop an optimized drug product for therapeutic treatment of humans suffering from allergy to Japanese cedar pollen allergen or a pollen allergen which is immunologically cross reactive Japanese cedar pollen allergen. Such modified peptides possess certain unique characteristics which render them particularly suitable for drug product formulation. The present invention further provides therapeutic compositions and multipeptide formulations which have been optimized to accommodate and maintain the unique characteristics of the modified Cry j I peptides and at the same time provide maximum therapeutic effect when used in therapeutic regimens for the treatment of Japanese cedar pollen allergy in humans.

Description

Pharmaceutical Formulations for Treating Japanese Cedar Pollen Allergy

Background of the Invention

Japanese cedar (Sugi; Cryptomeria japonicά) pollinosis is one of the most important allergic diseases in Japan. The number of patients suffering from this disease is on the increase and in some areas, more than 10% of the population are affected.

The major allergen from Japanese cedar pollen has been purified and the amino acid sequence partially identified, and designated as Sugi basic protein (SBP) or Cry; I (Yasueda et al. (1983) J. Allergy Clin. Immunol. 71: 77-86; and Taniai et al. (1988) FEBS Letters 239: 329-332). Cryj I has been cloned and the full length nucleic acid sequence and the full length amino acid sequence of the Cryj I protein have been identified (WO 93/01213)

The Cryj I allergen found in Cryptomeria japonica has also been found to be cross-reactive with allergens in the pollen from other species of trees, including Cupressus sempervirens. Panzani et al. (Annals of Allergy 57: 26-30 (1986)) reported Λat cross reactivity was detected between allergens in the pollens of Cupressus sempervirens and Cryptomeria japonica in skin testing, RAST and RAST inhibition. A 50 kDa allergen isolated from Mountain Cedar (Juniperus sabinoides, also known as Juniperus ashei) has an NH2-terminal sequence (Gross et al., (1978) Scand. J. Immunol. 8: 437-441) which is the same sequence as the first five amino acids of the NH-2 terminal end of the Cryj I allergen. The Cryj I allergen has also been found to be allergenically cross-reactive with the following species of trees: Cupressus arizonica, Cupressus macrocarpa, Juniperus virginiana, Juniperus communis, Thuya orientalis, and Chamaecyparis obtusa.

Treatment of Japanese cedar pollinosis by administration of Japanese cedar pollen extract to effect hyposensitization to the allergen has been attempted.

Desensitization using Japanese cedar pollen extract, however, has drawbacks in that it can elicit anaphylaxis if high doses are used, whereas when low doses are used to avoid anaphylaxis, treatment must be continued for several years to build up a tolerance to the extract. WO 94/01560 discloses improved compositions and methods for treatment of sensitivity to Japanese cedar pollen or to an immunologically cross reactive pollen allergen which greatly minimizes the potential adverse side effects associated with desensitization therapy using Japanese cedar pollen extract. WO 94/01560 discloses isolated antigenic fragments or peptides derived from Cryj I which when administered to a Japanese cedar pollen-sensitive individual, or an individual allergic to an allergen cross-reactive with Japanese cedar pollen allergen, are capable of down regulating the allergic response of the individual to Japanese cedar pollen or such cross reactive allergen. Such down regulation of the allergic response of the individual results in diminution or alleviation of the classic symptoms of allergy including asthmatic symptoms induced by Japanese cedar pollen. Such antigenic fragments are disclosed as being capable of eliciting a T cell response such as stimulation (i.e. T cell proliferation or lymphokine secretion) and or are capable of inducing T cell non-nonresponsiveness or reduced T cell responsiveness when challenged with Japanese cedar pollen allergen. In addition, WO 94/01560 discloses that the most preferred Cryj I peptides suitable for therapeutic use do not bind IgE specific for Cryj I, or bind IgE to a substantially lesser extent than the native Cry; I allergen, thereby reducing or eliminating the possibility of anaphylaxis in a treatment regimen which includes such peptides. Finally, WO 94/01560 discloses peptides which possess the characteristics described above. As a result of extensive prefόrmulation efforts, the present invention provides novel modified Cryj I peptides and novel combinations and formulations of modified Cry j I peptides which are optimal for preparation of a drug product suitable for use in treating Japanese cedar pollen allergy in humans and other mammals. Such Cryj I peptides and formulations thereof for use as an optimized human drug product have not previously been disclosed or contemplated.

Summary of the Invention

The present invention provides novel peptides of Cry j I which have been modified as a part of a preformulation scheme to develop an optimized drug product for therapeutic treatment of humans suffering from allergy to Japanese cedar pollen allergen or a pollen allergen which is immunologically cross reactive Japanese cedar pollen allergen. Such modified peptides possess-certain unique characteristics which render diem particularly suitable for drug product formulation. The present invention further provides therapeutic compositions and multipeptide formulations which have been optimized to accommodate and maintain the unique characteristics of the modified Cryj I peptides and at the same time provide maximum therapeutic effect when used in therapeutic regimens for the treatment of Japanese cedar pollen allergy in humans.

Description of the Drawings

Fig. 1 shows the complete cDNA sequence for Cryj I (SEQ. ID. NO: 39) which is composed of 1312 nucleotides, including 66 nucleotides of 5' untranslated sequence, an open reading frame starting with the codon for an initiating methionine of 1122 nucleotides, and a 3' untranslated region. Fig. 1 also shows the deduced amino acid sequence of Cryj I (SEQ. ID. NO: 40);

Fig. 2 shows 35 overlapping 20mer peptides which overlap by 10 derived from Cryj I.

Fig. 3 shows the amino acid sequences of the novel "unique" peptides of the invention.

Fig. 4 is a graphic representation depicting responses of T cell lines from twenty- five patients primed in vitro with purified native Cry; I and analyzed for response to the 35 overlapping 20 mer Cryj I peptides shown in Fig. 2, by percent of responses (positive) 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 parenthesis) and the positivity index (Y axis);

Fig. 5 is a graphic representation depicting T cell responses to the overlapping Cry j I peptides shown in Fig. 2, and the novel "unique" peptides of the invention shown in Fig. 3. The mean S.I. shown above each bar (in parenthesis) as well as the percentage of responses, the positivity index (mean S.I. multiplied by percentage of responses) is the Y axis.

Fig. 6a is a pH solubility profile of CJI-24.5 (SEQ. ID. NO: 36) (desalted) in 50mM phosphate buffer with 5% mannitol and 22°C, with 95.2% estimated peptide content and 2.7% acetate.

Fig. 6b is a pH solubility profile of CJI-43.39 (SEQ. ID. NO: 37) in 50mM phosphate buffer with 5% mannitol and 22°C, with 83.5% estimated peptide content and 1.9% acetate. Fig. 6c is a pH solubility profile of CJI-44.8 (SEQ. ID. NO: 38)in 50mM phosphate buffer with 5% mannitol and 22°C, with 86.% estimated peptide content and 1.5% acetate.

Detailed Description of the Invention

The present invention provides novel peptides of Cryj I which have been modified as a part of a preformulation scheme to develop an optimized drug product for therapeutic treatment of humans suffering from allergy to Japanese cedar pollen allergen or an allergen which is immunologically cross reactive with Japanese cedar pollen allergen ( e.g. Cupressus sempervirens, Juniperus sabinoides (also known as Juniperus ashei) Cupressus arizonica, Cupressus macrocarpa, Juniperus virginiana, Juniperus communis, Thuya orientalis, and Chamaecyparis obtusa). Such modified peptides possess certain unique characteristics which render diem particularly suitable for drug product formulation, and may be referred to herein as "unique" peptides.

In accordance with pharmaceutical chemistry, preformulation is the process of optimizing a drug through determination and/or definition of those physical and chemical properties considered important in the formulation of a stable, effective, and safe dosage form. The possible interactions with the various components intended for use in the final drug product are also considered. Preformulation is an intensive effort that includes the study of such parameters as solubility, pH profile of stability, and drug-excipient interactions, which may have a profound effect on a drug's physiological availability and physical and chemical stability. The data obtained from such studies are integrated with those obtained from preliminary pharmacological and biochemical studies of the active drug component thus providing information that permits the selection of the best drug form, and the most desirable excipients for use in its development.

The development of an optimum formulation of active drug component and excipients is complex and many factors influence formulation properties. The high degree of uniformity, the physiological availability and the therapeutic quality expected of pharmaceuticals can only be achieved by considerable effort and expertise. Flexibility is also an important factor in preformulation. Numerous excipients, stabilizers counter ions and the like may have to be tested in order to find those compatible with the active drug component of the formulation. Multiple modifications of die active component may become necessary to successfully formulate a drug product. Such modifications must not effect the overall therapeutic effectiveness of the drug but at the same time, must render the drug more suitable for formulation.

As a part of a preformulation scheme to provide an optimized drug product suitable for use in humans and other mammals for treating sensitivity to Cry; I, it was determined that the active component (a peptide or candidate peptide) in such formulation should possess the following characteristics which would render such peptides "unique" among all of the possible peptides derived from the Cryj I sequence. First, a unique peptide should alone or in combination with other unique peptides comprise a sufficient percentage of the T cell reactivity of the Cry j I protein allergen to induce T cell nonresponsiveness or reduced T cell responsiveness in a substantial percentage of the individuals sensitive to Cryj I protein allergen. Second, the candidate peptide should possess the characteristic of "superior solubility" which is defined herein as solubility of greater than 5 mg/ml at a pH in the pH range of pH 6 to pH 8 in an aqueous buffer. Third, the peptide is stable in an aqueous buffer at a pH in the pH range of pH 6 to pH 8. Candidate peptides which have been determined to be "unique" peptides of the invention are CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36), and CJI- 44.8 (SEQ. ID. NO: 38), all as shown in Fig. 3. In accordance with the first characteristic, those peptides found to elicit a T cell response such as T cell proliferation or lymphokine secretion (i.e. comprise at least one T cell epitope), or induce T cell non-responsiveness or reduced T cell responsiveness are understood to have T cell reactivity. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to a protein allergen which is responsible for the clinical symptoms of allergy. These 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. A T cell epitope is the basic element or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition. It is believed that exposure of Japanese cedar pollen patients to isolated Cryj I peptides which comprise at least one T cell epitope may cause T cell non-responsiveness of appropriate T cell subpopulations such that they become unresponsive or have reduced responsiveness to the protein allergen and do not participate in stimulating an immune response upon such exposure for example, via anergy, tolerance, or apoptosis, the ability to modify the lymphokine secretion profile as compared with exposure to the naturally occurring autoantigen; and /or the ability to cause induction of T suppresser cells.

To determine peptides having T cell reactivity and 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 non-responsiveness. As discussed in the Examples human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to Japanese cedar pollen allergen, (i.e., an individual who has an IgE mediated immune response to Japanese cedar pollen allergen) with a peptide or modified peptide derived from Cryj I and determining whether proliferation of T cells occurs in response to the peptide as measured, e.g., 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 (S.I.) 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. Peptides suitable as candidates for formulation into a final drug product have a mean T cell stimulation index of greater than or equal to 2.0 and preferably higher, (e.g. at least 2.5, more preferably at least 3.5, more preferably at least 4.0, more preferably at least 5, even more preferably at least 7 and most preferably at least about 9).

For therapeutic purposes, candidate peptides are recognized by at least 10%, more preferably at least 20%, more preferably at least 30% and even more preferably at least 40% or more of individuals in a population of individuals sensitive to Japanese cedar pollen. In addition, preferred candidate peptides have a positivity index (P.I.) of at least about 100, more preferably at least about 250 and most preferably at least about 350. 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 Japanese cedar pollen (e.g., preferably at least 15 individuals, more preferably at least 30 individuals or more), who have a T cell stimulation index to such peptide of at least 2.0. Thus, the positivity index represents both the strength of a T cell response to a peptide (S.I.) and the frequency of a T cell response to a peptide in a population of individuals sensitive to Japanese cedar pollen. To determine whether a peptide (candidate peptide) or a combination of candidate peptides are likely to comprise a sufficient percentage of the T cell reactivity of the Cryj I to induce T cell nonresponsiveness in a substantial percentage of a population of individuals sensitive to Cryj I, an algorithm can be used. In accordance with one such algorithm, a set of overlapping peptides is produced by systematically dividing Cryj I into at least two overlapping peptide regions of desired lengths (e.g., of about 12-30 amino acid residues in length, preferably not longer than about 25 amino acid residues in length with about 5-15 amino acid residues of overlap). For example, see Fig. 2. This division into peptide regions can be arbitrary, can be made according to an algorithm, or can be wholly or partially based on regions of Cry; I known to comprise at least one T cell epitope. Preferably, at least 50% of the entire Cryj I protein sequence and more preferably, the entire protein sequence of Cryj I is divided into two or more peptides. A human T cell stimulation index is determined for each of the peptides in an in vitro T cell proliferation assay as described herein for each individual tested in a population of individuals sensitive to the protein antigen. For example, see Fig. 4. A candidate peptide or combination of candidate peptides is selected based, at least in part, on the mean human T cell stimulation index of the candidate peptide in the set of peptides tested and the positivity index of the candidate peptide in the set of peptides tested. For example see, Fig. 5. The human T cell stimulation index for the candidate peptide(s) is summed. For each individual, die human T cell stimulation index for the candidate peptide(s) is divided by the sum of the human T cells stimulation indices of die remaining peptides in the set of peptides tested to determine a percent of T cell reactivity as shown below:

Candidate S.I.

(1) % T Cell Reactivity of a candidate peptide(s) = X 100

Sum of S.I. of the set of Overlapping peptides

Alternatively, the presence of T cell epitopes in the candidate peptide dependent on amino acids residues in an overlapping peptide located at either d e N-terminus or C-terminus of the candidate peptide in the amino acid sequence of the protein antigen, but which epitopes are not present in the candidate peptide can be considered in calculating the percent of T cell reactivity in the candidate peptide by use of the following formula:

(2) % T Cell Reactivity of a candidate peptide(s) =

NT flanking peptide S.I. + Candidate peptide S.I. + C flanking peptide S.I. x 100

Sum of S.I. of the set of overlapping peptides

In this formula, "Nj flanking peptide" refers to a peptide which comprises amino acid residues which overlap with amino acid residues located at d e N- terminus of the candidate peptide in the amino acid sequence of the protein antigen from which the peptide is derived; "C flanking peptide" refers to a peptide which comprises amino acid residues which overlap with amino acid residues located a the C-terminus of the candidate peptide in the amino acid sequence of the protein antigen from which the peptide is derived. In this calculation stimulation indices for the candidate peptide, the N-terminal flanking peptide and the C-terminal flanking peptide are added and divided by the sum total of the stimulation indices for the entire set of overlapping peptides obtain a percent of T cell reactivity for the candidate peptide. If a combination of two or more candidate peptides is selected each of which contains amino acid residues which overlap, this calculation cannot be used to determine a percent of T cell reactivity for each candidate peptide separately. However, a total percent of T cell reactivity for the combination of candidate peptides can be obtained. In this situation, the stimulation indices for all of the candidate peptides which overlap is included in the calculation.

The values obtained for the percentage of T cell reactivity for the candidate peptide or combination of peptides in each individual tested can be expressed as a range of the lower and higher values of the results of the above described calculations. By either of the above calculations, the percent is obtained for at least about twenty (20) and preferably at least about thirty (30) individuals sensitive to the protein antigen and a mean percent is determined. For use in the compositions of the invention, the candidate peptide or combination of candidate peptides has the following criteria: (1) the candidate peptide or combination of candidate peptides has a mean percent of at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40% and more preferably at least about 50-60% or greater; and (2) in the population of individuals tested at least about 60%, preferably at least about 75%, and more preferably at least about 90-100% have positive T call responses (S.I. equal to or greater than 2.0) in response to the candidate peptide or combination of candidate peptides. A candidate peptide or combination of candidate peptides meeting the above criteria is likely to comprise a sufficient percentage of the T cell reactivity to Cryj I to induce T cell non-responsiveness or reduced T cell responsiveness in a substantial percentage of a population of individuals sensitive to Cryj I.

As an illustrative embodiment of the above-described algorithm, a set of overlapping peptides and candidate peptides, CJI-24.5, CJI-23.39 and CJI-44.8, derived from Cryj I were produced and tested. Secondary T cell cultures determined to be reactive with Cry I protein antigen were derived from 36 Cryj I-allergic subjects and analyzed for reactivity to the overlapping set of peptides in an in vitro T cell proliferation assay as described herein. The results are shown in Fig. 5. The highest stimulation index greater than or equal to 2.0 in response to each peptide was recorded for each subject tested. The data were then analyzed by the equations above. The results and calculations of die percent of T cell reactivity for a single Cry; I-allergic subject are shown below using formulas (1) and (2).

T CELL REACTIVITY FOR PATIENT 1308

PEPTIDE STIMULATION INDEX

CJl-1 (SEQ. ID. NO: 1) CJ1-2 (SEQ. ID. NO: 2) CJ1-3(SEQ. ID. NO: 3) CJ1-4 (SEQ. ID. NO: 4) CJ1-5 (SEQ. ID. NO: 5) CJ1-6 (SEQ. ID. NO: 6) CJ1-7(SEQ. ID. NO: 7) CJ1-8(SEQ. ID. NO: 8) CJl-41(SEQ.ID.NO:41 CJl-ll(SEQ.ID.NO: 11 CJl-12(SEQ.JX>.NO: 12 CJ1-13 (SEQ. ID. NO: 13 CJ1-14 (SEQ. ID. NO: 14 CJ1-15 (SEQ. ID. NO: 15 CJ1-42.5 (SEQ. ID. NO: 42) CJ1-18(SEQ. ID. NO: 18 CJ1-19 (SEQ. ID. NO: 19 CJ1-20(SEQ. ID. NO: 20 CJ1-21(SEQ. ED. NO: 21 CJ1-43.39 (SEQ. ID. NO: 36) CJl-23(SEQ.ID.NO:23 CJI-24.5 (SEQ. ID. NO: 37) CJ1-25 (SEQ. ID. NO: 25 CJl-26(SEQ.ID.NO:26 CJl-27(SEQ.ID.NO:27 CJl-28(SEQ.JD.NO:28 CJl-29(SEQ.ID.NO:39 CJ1-30(SEQ.JX>.NO:30 CJI-44.8 (SEQ. ID. NO: 38) CJ1-33 (SEQ. ID. NO: 33 CJl-34(SEQ.ID.NO:34 CJl-35(SEQ.ID.NO:35

SUM OF STIMULATION INDICES: 195.7 (DENOMINATOR) % Reactivity of Peptide 44.8 for patient 1308

(1) CJl - 44.8 (S.I.) 21.5 = X 100 = 11%

195.7 195.7

(2) CJl - 30 + CJI-44.8 + CJ1-33 (S.I.s) 0 + 21.5 + 20.9

X 100 = 21.7%

195.7 195.7

Therefore the estimated range of T cell reactivity for Peptide 44.8 (SEQ.

ID. NO: 38) for this patient is 11 %-21.7% of the total reactivity of the Cry j I protein. The above calculation is repeated for any potential candidate peptides. In the population of 36 Cryj I-allergic subjects tested the following results were obtained:

Candidate Range of mean percentage Frequency of response

Peptides T Cell Reactivity at least one peptide CJ1-24.5 + CJ1-43.3 + CJI-44.8 36-53% 97%

Thus, the combination of the three candidate peptides of the invention are well within the desired range for possessing, in combination, sufficient T cell reactivity of Cryj I, and therefore, meet the first characteristic of a "unique" peptide of the invention. For the treatment of allergy in accordance with the methods of the invention, it is preferred that a peptide used in conjunction tiierewith does not bind immunoglobulin E (IgE) or binds IgE to a substantially lesser extent (i.e. at least 100-fold less binding and more preferably, at least 1, 000-fold less binding) than the Cryj I protein allergen from which the peptide is derived binds IgE. 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 release of mediators (e.g., histamine, serotonin, eosinophil chemotacic factors) in allergic ("atopic") patients. Thus, anaphylaxis in a substantial percentage of a population of individuals sensitive to the allergen being treated could be avoided by the use in immunotherapy of a peptide or peptides which do not bind IgE in a substantial percentage (e.g., at least about 75%) of a population of individuals sensitive to Cryj I, or if the peptide binds 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. IgE binding may be tested, for example by direct ELISA or capture ELISA. Moreover, peptides which have 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 and/or IL-4 production stimulated by the native Cry; I protein allergen. If a peptide binds IgE, it is preferable that such binding does not result in the release of mediators (e.g. histamines) from mast cells or basophils. To determine whether a peptide that binds IgE causes the release of mediators, a histamine release assay can be performed using standard reagents and protocols obtained for example, from Amac, Inc. (Westbrook, ME). Briefly, a buffered solution of a peptide to be tested is combined with an equal volume of whole heparinized blood from an allergic subject. After mixing and incubation, the cells are pelleted and the supernatants are processed and analyzed using a radio immunoassay to determine the amount of histamine released. As described in Example 3, candidate peptides of the invention, 24.5 (SEQ. ID. NO: 37), 43.39 (SEQ. ID. NO: 36), and 44.8 (SEQ. ID. NO: 38), do not appear to bind IgE .

The second characteristic for a unique peptide is that of "superior solubility" which was defined earlier as being solubility of greater than 5 mg/ml at a pH in the pH range of pH 6 to pH 8. Solubility at a pH in a physiologically acceptable pH range (e.g. pH 6 to pH 8) is particularly important when formulating a multipeptide therapeutic for injection. Administration of a soluble drug product in a physiologically acceptable pH range by intravenous or subcutaneous injection provides 100% bioavailability of the drug component to the physiological system into which the drug is being introduced. Thus, it is necessary that a drug product intended for injection be fluid to the extent that easy syringability exists, and the active component be soluble as well if maximum therapeutic effect is to be achieved. Solubility is also useful when formulating compositions to be administered via other modes of administration such as by oral administration (tablet, aerosol, sublingual), or sustained release preparations and formulations. Proteins and peptides may be difficult to formulate into soluble compositions as a peptide may not be soluble in any desirable pH range or may be soluble in only a narrow pH range. It is particularly difficult when multiple peptides are being formulated togetiier into a single multipeptide formulation, as each peptide may be soluble in a pH range which does not overlap with those of the other peptides in die formulation. As a result, it is the requirement of "superior solubility" which requires the most formulation flexibility in that considerable modification of me targeted candidate peptides may be necessary to successfully formulate a multipeptide drug product. The unique peptides of the invention are the product of multiple amino acid modifications of the original targeted candidate peptide sequence ("parent") from which the modified unique peptides of the invention were originally derived for die purpose of finding a peptide which fits the characteristic of "superior solubility". For example, the amino acid sequence of CJI-44.8 (SEQ. ID. NO: 38) was derived from the protein sequence of I (SEQ. ID. NO: 40) by first identifying those regions of the parent protein (Fig. 1) with high T cell reactivity using the set of overlapping peptides 20-mers as discussed in Example 1, and shown in Fig. 2, which covered d e entire sequence. Two of these peptides, CJI- 31 (SEQ. ID. NO: 31) and CJI-32 (SEQ. ID. NO: 32), individually exhibited high T-cell reactivity. Since these peptides were adjacent to each other in die native protein sequence (Fig. 1) and overlapped by 10 residues, a peptide, CJI-44, was synthesized to capture the total T cell reactivity of both peptides. CJI-44 is a peptide 30-mer having the amino acid sequence DEEGAYI^SSGKYEGGNIYTKKEAEFNVE (SEQ. ID. NO: 43) which contains all of the sequence present in the two 20-mers CJI-31 (SEQ. ID. NO: 31) and CJI- 32 (SEQ. ID. NO: 32). However, although CJI-44 (SEQ. ID. NO: 43) possessed T cell reactivity of the two 20-mers, CJI-31 (SEQ. ID. NO: 31) and CJI-32 (SEQ. ID. NO: 32), when the solubility of CJI-44 (SEQ. ID. NO: 43) was tested it had a solubility much lower than the 5 mg/ml solubility required for a "unique" peptide of the invention.

Thus, further attempts were made to increase solubility by truncation at the N-terminus portion of CJI-44 which resulted in CJI-44.1 (NGAYFVSSGKYEGGNIYTKKEAFNVE) (SEQ. ID. NO: 44). Additional truncation of two C-terminal residues yielded 44.2 (NGAYFVSSGKYEGGNIYTKKEAFN) (SEQ. ID. NO: 45). However, although solubility was improved in these sequences it still did not reach the standard of "superior solubility". Thus, 44.2 (SEQ. ID. NO: 45) was further modified by the addition of charged (hydrophilic residues) to the N-terminus and by replacement of the hydrophobic residue Val with the less hydrophobic residue Ala. Two of the resulting analogs, CJI-44.5 (DENGAYFVSSGKYEGGNIYTKKEAFNAE) (SEQ. ID. NO: 46) and CJI-44.6 (DEENGAYFVSSGKYEGGNIYTKKEAFNVE) (SEQ. ID. NO: 47), showed increased solubility to using a "single pH point protocol procedure" (e.g. a protocol procedure wherein determinations of solubility were made at a single pH in 100 mM sodium phosphate buffer without mannitol under constant agitation) Two additional analogs were constructed in which the residue Asn was deleted. Of tiiese two analogs, CJI-44.7

(DEGAYFVSSGKYEGGNIYTKKEAFNAE) (SEQ. ID. NO: 48) and CJI-44.8 (DEEGAYFVSSGKYEGGNIYTKKEAFNVE) (SEQ. ID. NO: 38), CJI-44.8 was very soluble in the "single pH point protocol" and achieved "superior solubility" in the "pH range protocol procedure" (i.e. wherein solubility is measured as a function of pH in 50 mM sodium phosphate containing 5% mannitol with no agitation after initial mixing). CJl -44.8 (SEQ. ID. NO: 38) was stable and soluble at greater than 5 mg/ml over the pH range pH6-pH8 in an aqueous buffer (see, Example 4 and Fig. 6c).

Peptide CJI-44.8 (SEQ. ID. NO: 38) was determined to be a "unique" peptide after confirmation that it also retained a T-cell reactivity similar to its parent peptides, CJI-31 (SEQ. ID. NO: 31), CJI-32 (SEQ. ID. NO: 32)and CJI-44 (SEQ. ID. NO: 43) (see Example 2), and confirmation of its stability as is discussed below. Development of the other "unique" peptides, CJI-24.5 (SEQ. ID. NO: 37) and CJI-43.39 (SEQ. ID. NO: 36), followed a process similar to that described above for CJI-44.8 (SEQ. ID. NO: 38).

The third criteria which the unique peptides of this invention must meet is stability at a physiologically acceptable pH in the range of pH 6 to pH 8. It must be stable under the conditions of manufacture and storage, and under conditions of reconstitution if necessary. Stability testing establishes the time period for which the integrity, quality and purity of the drug product is preserved in its finished dosage form. Stability testing may be performed concurrently with solubility studies as discussed in Example 4. Each of the candidate peptides of d e invention remained stable (e.g. no significant degradation) at a physiologically pH in a pH range from pH 6-pH 8, at room temperature for at least 24 hours. Therefore, candidate peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39

(SEQ. ID. NO: 36), and CJI-44.8 (SEQ. ED. NO: 38) possess each of the three required "unique" characteristics outlined above, indicating that this combination of peptides is suitable for formulation as an optimized therapeutic drug product for administration to humans for treatment of allergy to Japanese cedar pollen allergen. Highly purified peptides of this invention, may be produced synthetically by chemical synthesis using standard techniques. Various methods of chemically synthesizing peptides are known in the art such as solid phase synthesis which has been fully or semi automated on commercially available peptide synthesizers. Synthetically produced peptides may then be purified to homogeneity (i.e. at least 90%, more preferably at least 95% and even more preferably at least 97% purity), free from all other polypeptides and contaminants using any number of techniques known in the literature for protein purification.

In accordance with one procedure for producing highly purified homogenous peptides of the invention, a peptide produced by synthetic chemical means (either anchored to a polymer support "solid phase synthesis" or by conventional homogenous chemical reactions "solution synthesis") may be purified by preparative reverse phase chromatography. In this method, the syn etically produced peptide in "crude" form is dissolved in an appropriate solvent (typically an aqueous buffer) and applied to a separation column (typically a reverse phase silica based media, in addition, polymer or carbon based media may be used). Peptide is eluted from the column by increasing the concentration of an organic component (typically acetonitrile or methanol) in an aqueous buffer (typically TFA, triethylamine phosphate, acetate or similar buffer). Fractions of the eluate will be collected and analyzed by appropriate analytical methods (typically reverse phase HPLC or CZE chromatography). Those fractions having the required homogeneity will be pooled. The counter ion present may be changed by additional reverse phase chromatography in the salt of choice or by ion exchange resins. The peptide may then be isolated as its acetate or other appropriate salt. The peptide is then filtered and the water removed (typically by lyophilization) to give a homogenous peptide composition containing at least 90%, more preferably at least 95% and even more preferably at least 97 "^• of the required peptide component. Optionally, or in conjunction with revers .__se HPLC as described above, purification may be accomplished by affinity chromatography, ion exchange, size exclusion, counter current or normal phase separation systems, or any combination of these methods. Peptide may additionally be concentrated using ultra filtration, rotary evaporation, precipitation, dialysis or other similar techniques.

The highly purified homogenous peptide composition is then characterized by any of the following techniques or combinations thereof: a) mass spectroscopy to determine molecular weight to check peptide identity; b) amino acid analysis to check the identity of the peptide via amino acid composition; c) amino acid sequencing (using an automated protein sequencer or manually) to confirm the defined sequence of amino acid residues; d) HPLC (multiple systems if desired) used to check peptide identity and purity (i.e. identifies peptide impurities); e) water content to determine the water concentration of the peptide compositions; f) ion content to determine the presence of salts in the peptide composition; and g) residual organics to check for the presence of residual organic reagents, starting materials, and/or organic contaminants.

A peptide of the invention may also be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid sequence coding for such peptide. When produced by recombinant techniques, host cells transformed with nucleic acid encoding the desired peptide are cultured in a medium suitable for the cells and isolated 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, ultra filtration, electrophoresis or immunopurification with antibodies specific for the desired peptide. Peptides produced recombinantly may be isolated and purified to homogeneity, free of cellular material, other polypeptides or culture medium for use in accordance with the methods described above for synthetically produced peptides. In certain limited circumstances, peptides of this invention may also be produced by chemical or enzymatic cleavage of a highly purified full length or native protein of which the sites of chemical digest or enzymatic cleavage have been predetermined and d e resulting digest is reproducible. Peptides having defined amino acid sequences can be highly purified and isolated free of any other poly peptides or contaminants present in the enzymatic or chemical digest by any of the procedures described above for highly purified, and isolated synthetically or recombinantly produced peptides.

The present invention also pertains to therapeutic compositions and multipeptide therapeutic formulations comprising the unique peptides of die invention. Therapeutic compositions of the invention may comprise one or more of the unique peptides of the invention which may be administered simultaneously or sequentially as single treatment episode for treatment of allergy to Japanese cedar pollen allergen in a human or other mammal. Such a treatment regimen may not necessarily be a physical mixture of more than one peptide of die invention, but does comprise a combination of such peptides administered simultaneously or sequentially as a single treatment episode in order to achieve the maximum therapeutic effect the combination of the unique peptides, CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36), and CJT-44.8 (SEQ. ID. NO: 38), provide (e.g. solubility and stability at a pH in an acceptable physiological pH range (preferably pH 7.0 to pH 7.5) as well as covering 36-53% T cell reactivity in 97% of the patients tested).

Therapeutic compositions of the invention comprise one or more of peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36), and CJI-44.8 (SEQ. ED. NO: 38) and also comprise one or more pharmaceutically acceptable carriers such as excipients which are compatible with peptide or peptides present in a single composition. When the composition is a multipeptide formulation, the pharmaceutically acceptable carrier must be compatible with all of the peptides in the multipeptide formulation. Preferred excipients include but are not limited to sterile water, sodium phosphate, mannitol, or both sodium phosphate and mannitol or any combination tiiereof. Other suitable excipients include but are not limited to sorbitol, sucrose, dextrose, lactose dextran and PVP. In addition, pharmaceutically acceptable counter ions may be added during the preparation of the multipeptide formulation. Examples of pharmaceutically acceptable counter ions include acetate, HC1, and citrate.

A therapeutic composition of the invention should be sterile, stable under conditions of manufacture, storage, distribution and use and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. A preferred means for manufacturing a therapeutic compositions of the invention in order to maintain the integrity of the composition (i.e. prevent contamination, prolong storage, etc.) is to prepare the formulation of peptide and pharmaceutically acceptable carrier(s) such that the composition may be in the form of a lyophilized powder which is reconstituted in a pharmaceutically acceptable carrier, such as sterile water, just prior to use. In the case of sterile powders for die preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying, freeze-drying or spin drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A preferred multipeptide formulation comprises unique Cry; I peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ID. NO: 38) and sodium phosphate and mannitol. For this embodiment, a suitable counter ion such as acetate may be added during the preparation of the formulation, and the formulation is preferably prepared in the form of a lyophilized powder which is reconstituted in a physiologically acceptable carrier, such as sterile water, prior to use. One, non-limiting example of a preferred multipeptide formulation of the invention is described below. The Cryj I peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ID. NO: 38) will preferably be combined during manufacturing with the appropriate counter ion to produce a vial containing a sterile, pyrogen free, lyophilized powder having the following composition:

Active: Cryj I peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ID. NO: 38)

In concentration of 0.75mg per peptide Inactives: 0.05 M Sodium Phosphate pH 6.0-8.0

5% w/v Mannitol, U.S.P. Diluent: Sterile Water for Injection, U.S.P. (initial reconstitution) 0.9% Sodium Chloride for Injection

(dilution beyond initial reconstitution) Final pH pH 7.0-pH 7.5

The multipeptide formulation of the invention may also be provided in the form of a kit, including instructions for use.

Administration of me therapeutic compositions and multipeptide formulations described above to an individual, preferably in non-immunogenic form, can be carried out using known procedures at dosages and for periods of time effective to cause down regulation of die immune response to Japanese cedar pollen or an allergen immunologically cross reactive with Japanese cedar pollen allergen (i.e., reduce the allergic symptoms caused by Japanese cedar pollen or related allergen including Japanese cedar pollen induced asthma) of the individual. Down regulation of the allergic immune response to Japanese cedar pollen in humans may be determined clinically whenever possible (see e.g., Varney et al, British Medical Journal, 302:265-269 (1990), or may be determined subjectively (i.e. the patient feels as if some or all of the allergic symptoms caused by Japanese cedar pollen have been alleviated).

One of the unique characteristics of each peptides of the invention is tiiat each peptide possesses "superior solubility". Therefore, compositions and multipeptide formulations of the invention are particularly suitable for administration by injection (e.g. subcutaneous, or intravenous). However, optimized compositions and multipeptide formulations of the invention may be administered in any convenient manner wherein solubility of the active drug component is either desirable or acceptable, such as by injection (subcutaneous, intravenous, etc.), oral administration, sublingual, inhalation, transdermal application, rectal administration, or any other route of administration known in the art for administering soluble therapeutic agents. It may be desirable to administer simultaneously or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. Each of such compositions for administration simultaneously or sequentially as a single treatment episode, may comprise only one unique peptide of the invention or may comprise an optimized multipeptide formulation in accordance with the invention.

For subcutaneous injection of one or more therapeutic compositions and multipeptide formulations of the invention, preferably about 1 μg- 3 mg and more preferably from about 20μg-1.5 mg, and even more preferably about 50 ug- 750 μg of each active component (peptide) per dosage unit may be administered. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for human subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. The specification for the novel unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and die particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of human subjects.

To administer a composition of the inver n by other than parenteral administration, (i.e. by oral administration) it may oe necessary to coat the composition with, or co-administer the composition with, a material to prevent its inactivation or enhance its absorption and bioavailability. For example, a peptide formulation may be co-administered 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). When a peptide is suitably protected, 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 shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, solutions, gels, 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% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. In addition, the active compound may be incorporated into sustained-release or controlled release (steady state or pulsatile release) preparations and formulations.

Effective amounts of the optimized drug compositions of the invention will vary according to factors such as the degree of sensitivity of the individual to the antigen, the age, sex, and weight of die individual, and the ability of peptide to cause down regulation of the antigen specific immune response in the individual. Dosage regimen may be adjusted to provide the optimum dierapeutic response. For example, several divided doses may be administered over die course of days, weeks, months or years, or the dose may be proportionally increased or reduced with each subsequent injection as indicated by the exigencies of the dierapeutic situation. In one preferred therapeutic regimen, subcutaneous injections of therapeutic compositions are given once a week for 3-6 weeks. The dosage may remain constant for each injection or may increase or decrease with each subsequent injection. A booster injection may be administered at intervals of about three months to about one year after initial treatment and may involve only a single injection or may involve another series of injections similar to that of the initial treatment.

This invention is further illustrated by the following non-limiting examples. Example 1

Japanese Cedar Pollen Allergic Patient T Cell Studies with overlapping Cry j

I peptides.

Synthesis of Overlapping Peptides

Japanese cedar pollen Cry; I was divided into a set of 35 peptides of 20 amino acids in length and overlapping by 10 amino acids. Overlapping peptides were synthesized using standard Fmoc/tBoc synthetic chemistry and purified by Reverse Phase HPLC. Figure 2 shows the overlapping Cry; I peptides used in these studies. The peptide names are consistent throughout.

T Cell Responses to Cedar Pollen Antigenic Peptides

Peripheral blood mononuclear cells (PBMC) were purified by lymphocyte separation medium (LSM) centrifugation of 60 ml of heparinized blood from Japanese cedar pollen-allergic patients who exhibited clinical symptoms of seasonal rhinitis and were MAST and/or skin test positive for Japanese cedar pollen. Long term T cell Unes were established by stimulation of 2 X 10'*-' PBlVml in bulk cultures of complete medium (RPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 5x1 O^M 2-mercaptoethanol, and 10 mM HEPES supplemented with 5% heat inactivated human AB serum) with 20 μg/ml of partially purified native Cryj I (75% purity containing three bands similar to the three bands in Fig. 2) for 7 days at 37°C in a humidified 5% CO2 incubator to select for Cry; I reactive T cells. This amount of priming antigen was determined to be optimal for the activation of T cells from most cedar pollen 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 _L-4/ml for up to three weeks until the cells no longer responded to lymphokines and were considered "rested". The abihty of the T cells to proliferate to selected peptides, recombinant Cry j I (τCryj I), purified native Cry; I, or recombinant Amb a l.1 (τAmb al.X) or a positive control, phyto- hemaglutinin (PHA) was then assessed. For assay, 2 X 10^* _rested cells were restimulated in the presence of 2 X 10^*^ autologous Epstein-Barr virus (EBV)- trarsformed B cells (prepared as described below) (gamma-irradiated with 25,000 RAD5) with 2-50 μg/ml of selected peptides, Cry; I, purified native Cryj I or xAmb a 1.1 or PHA, in a volume of 200 μl complete medium in duplicate or triplicate wells in 96-well round bottom plates for 2-4 days. The optimal incubation was found to be 3 days. Each well then received 1 μCi tritiated thymidine for 16-20 hours. The counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. Data collected (not shown) indicated the effect of varying antigen dose in assays with recombinant Cry; I, purified native Cryj I, and recombinant Amb a l.l and several antigenic peptides syntiiesized as described above. Some peptides were found to be inhibitory at high concentrations in these assays. 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 (S.I.). The S.I. is the counts per minute (CPM) incorporated by cells in response to peptide, divided by the CPM incorporated by cells in medium only. An S.I. value equal to or greater than 2 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 die group of patients tested. The results (data not shown) demonstrated tiiat patient #999 responds well to recombinant Cryj I, and purified native Cry; I, as well as to peptides CJl -2, 3, 20, and 22 but not to recombinant Amb a l.l. This indicates that Cr j I T cell epitopes are recognized by T cells from this particular allergic patient and tiiat rCry; I and peptides, 3 (SEQ ID NO: 49), 20 (SEQ ID NO: 50), and 22 (SEQ ID NO: 51) contain such T cell epitopes. Furthermore, the epitopes were often not detected with the adjacent overlapping peptides, and therefore probably span the non-overlapping central residues of the reactive peptides. No significant cross- reactivity was found in T cell assays using T cells primed with control antigens or with Cryj I primed T cells against other antigens. 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 die Cry; I protein at an S.I. of 2.0 or greater and d e patient responded to at least one peptide derived from Cry; I at an S.I. of 2.0 or greater. A summary of positive experiments from twenty-five patients is shown in Figure 4. The bars represent the positivity index. Above each bar is the percent of positive responses with an S.I. of at least two to the peptide or protein in the group of patients tested. In parenthesis above each bar are the mean stimulation indices for each peptide or protein for the group of patients tested. All twenty-five T cell lines responded to purified native Cry; I and 68.0% of the T cell lines responded to rCry; I. These twenty-five T cell lines also responded at a significantly lower level to τAmb a l.l indicating that the Amb a I allergens share a degree of homology with Cry; I and that "shared" T cell epitopes might exist between Cryj I and Amb a I. This panel of Japanese cedar allergic patients responded to peptides CJl-1 (SEQ ID NO: 1), CJ1-2 (SEQ ID NO: 2), CJ1-3 (SEQ ID NO: 3), CJ1-4 (SEQ ID NO: 4), CJ1-7 (SEQ ID NO: 7), CJ1-8 (SEQ ID NO: 8), CJ1-9 (SEQ ID NO: 9), CJl-10 (SEQ ID NO: 10), CJl-11 (SEQ ID NO: 11), CJl-12 (SEQ ID NO: 12), CJ1-14 (SEQ ID NO: 14), CJ1-15 (SEQ ID NO: 15), CJ1-16 (SEQ ID NO: 16), CJ1-17 (SEQ ID NO: 17), CJ1-18 (SEQ ID NO: 18), CJ1-19 (SEQ ID NO: 19), CJ1-20 (SEQ ID NO: 20), CJ1-21 (SEQ ID NO: 21), CJ1-22 (SEQ ID NO: 22), CJ1-23 (SEQ ID NO: 23), CJ1-24 (SEQ ID NO: 24), CJ1-25 (SEQ ED NO: 25), CJl-26 (SEQ ID NO: 26), CJl-27 (SEQ ID NO: 27), CJl-28 (SEQ ID NO: 28), CJ1-30 (SEQ ID NO: 30), CJI-31 (SEQ ID NO: 31), CJI-32 (SEQ ID NO: 32), CJ1-33 (SEQ ID NO: 33), CJ1-34 (SEQ ID NO: 34) and CJ1- 35 (SEQ ED NO: 35) indicating that these peptides contain T cell epitopes.

Preparation of (EBV)-transformed B Cells for Use as Antigen Presenting Cells

Autologous EBV-transformed cell lines were γ-irradiated with 25,000 Rad and used as antigen presenting cells in secondary proliferation assays and secondary bulk stimulations. These EBV-transformed cell lines were made by incubating 5 X 10⁶ PBL with 1 ml of B-59/8 Marmoset cell line (ATCC CRL1612, American Type Culture Collection, Rockville, MD) conditioned medium in the presence of 1 μg/ml phorbol 12-myristate 13-acetate (PMA) at 37°C for 60 minutes in 12 X 75 mm polypropylene round-bottom Falcon snap cap tubes (Becton Dickinson Labware, Lincoln Park, NJ). These cells were then diluted to 1.25 X 10⁶ cells/ml in RPMI-1640 as described above except supplemented witii 10% heat-inactivated fetal bovine serum and cultured in 200 μl aliquots in flat bottom culture plates until visible colonies were detected. They were then transferred to larger wells until the cell lines were established.

Example 2

Determination of percentage of total T cell reactivity and patient coverage of the combination of unique peptides of the invention.

Synthesis of unique peptides of the invention Peptides CJI-24.5 (SEQ. ID. NO: 37) and CJl- 44.8 (SEQ. ID. NO: 38) were synthesized using standard Fmoc synthetic chemistry. Peptide CJI-43.39 (SEQ. ID. NO: 36) was synthesized by t-Boc synthetic chemistry. All peptides were purified to 93% or greater purity using the procedures for producing highly purified peptides desribed earlier. Figure 3 shows the unique Cry; I peptides used in these studies. The peptide names are consistent throughout.

T cell reactivity of the unique peptides of the invention:

Peptides CJI-24.5 (SEQ ID NO: 37), CJI-43.39 (SEQ ID NO: 36), and CJI- 44.8 (SEQ ID NO: 38) were tested for T cell reactivity (data not shown) as described in Example 1 and were determined to elicit T cell activity as did each of their "parent" peptides from which they were derived and thus are suitable as candidate peptides for further study to determine if in combination, they possess a sufficient percentage of the total T cell reactivity of Cry; I in a sufficient percentage of the populations sensitive to Cryj I.

T cell studies with overlapping peptides and peptide candidates:

Secondary T cell cultures determined to be reactive with Cryj I protein antigen were derived from 36 Cryj I-allergic subjects and analyzed for reactivity to the overlapping set of peptides (Fig. 2) and the candidates peptides of d e invention (Fig.3) in an in vitro T cell proliferation assay as described in Example 1. The results are shown in Fig. 5. The highest stimulation index greater than or equal to 2.0 in response to each peptide was recorded for each subject tested. The data were then analyzed by the equations described earlier in the specification. The combination of candidate peptides CJI-24.5 (SEQ ID NO: 37), CJI- 43.39 (SEQ ID NO: 36), and CJI-44.8 (SEQ ID NO: 38) had a range of T cell reactivity of abour 36%-53% based on an analysis of 36 patients (Fig. 5), the frequency of response at 97% represents reactivity to at least one of the candidate peptides, indicating that this combination of peptides fits the first criteria for "unique" peptides of the invention in that in combination peptides CJI-24.5 (SEQ ID NO: 37), CJI-43.39 (SEQ ID NO: 36), and CJI-44.8 (SEQ ID NO: 38), comprising a sufficient percentage of the total T cell reactivity of Cryj I in a substantial percentage of the population tested.

Example 3

IgE Binding Studies To analyze IgE reactivity to candidate peptides CJI-24.5 (SEQ ID NO: 37),

CJI-43.39 (SEQ ID NO: 36), and CJI-44.8 (SEQ ID NO: 38) and shown in Fig. 3, a direct ELISA format was used. ELISA wells were coated with the candidate peptides and then assayed for IgE binding. The binding results were generated using two different pools of Cryj allergic patient plasma. Patient plasma pool A (denoted PHP- A) was formulated by mixing equal volumes of plasma from 22 patients that were all shown to be positive for direct IgE binding to native purified Cry; I by ELISA. The second pool (PHP-D) was formulated by the combination of equal plasma volumes from 8 patients that had IgE binding by direct ELISA to botii native and denatured purified Cry; I. This pool was generated to increase the chance of detecting reactivity towards peptides. Both pools in this assay set showed direct binding to the native purified Cry; I (data not shown). There was no detectable IgE binding reactivity to any of the peptides tested at any of the plasma concentrations used. To control for the presence of peptide coating the wells, mouse polyclonal antisera was generated to the peptides. These antisera were then used in direct ELISA binding to demonstrate mat the peptides were coating the wells. The results of these assays (data not shown) indicated tiiat peptides were coating the wells.

Example 4

Simultaneous determination of pH-solubility and pH stability profiles of candidate peptides of the invention

1. Buffer Preparation

50 mM sodium phosphate stock solutions:

Stock solution A: 0.66 g (0.05 mol) of monobasic sodium phosphate monohydrate was dissolved in 100 mL of WFI. The solution was filtered through a 0.2 micron filter

Stock solution B: 0.71 g (0.05 mol) of dibasic sodium phosphate was disolved in 100ml WFI. The solution was filtered through a 0.2 micron filter.

2. Initial peptide dispersions

Dispersion A: 3.0 mg of each peptide CJI-24.5 (SEQ. ID. NO: 37), CJI- 44.8 (SEQ. ID. NO: 38)and CJI-43.39 (SEQ. Ii⁾. NO: 36) was weighed out separately and placed in separate 1.5 mL eppendorf vials with 600 μL of stock solution A. The composition was agitated for 5 seconds to mix well. Dispersion B: 3.0 mg of each peptide, CJI-24.5 (SEQ. ID. NO: 37), CJI-

44.8 (SEQ. ID. NO: 38) and CJI-43.39 (SEQ. ID. NO: 36) was weighed out separately and placed in separate 1.5 mL eppendoff vials with 600 μL of stock solution B. The mixture was agitated for 5 seconds to mix well.

Dispersions A and B were sonicated for 2 minutes for good homogeneity. A small volume was pipetted from each dispersion into a labeled eppendoff vial according to the following volume ratio:

The resultant solutions/suspensions were stored in the dark at 22°C for 24 hours without agitation. 11 micro centrifuge filters were labeled on the retentate cup. The weight of each cup with the cap excluding the filter reservoir was measured. The equilibrated solution/suspension was pipetted from each eppendoff vial into the filer reservoir, and placed into micro centrifuge and spun for 10 minutes. After centrifugation, the filter reservoir was discarded and the weight of each cup/cap with the collected filtrate was recorded. The net weight of the filtrate in each cup was calculated by subtracting the weight of the empty cup/cap from the weight of the cup/cap containing the filtrate. The pH of the filtrates in each cup was measured using a micro combination pH electrode. After each pH reading, the electrode tip was rinsed with 1.0 mL of the selected dilution buffer into each cup. The total of diluted solution in each cup/cap was recorded. The dilution factors were calculated by dividing the weight of the diluted solution by the weight of the filtrate.

The concentration of peptide in the diluted solution was determined by HPLC analysis. The solubility of peptide at each pH was obtained by multiplying the measured concentration with the dilution factor. The extent of degradation of peptides was estimated by calculating the percent of total degradant peak area over the total peak area.

The pH values with respect to the solubility values was plotted and are shown in Fig. 6a-c for each respective peptide. As shown in the solubility curve for each peptide as represented in Fig. 6a-c, peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ID. NO: 38) and CJI-43.39 (SEQ. ID. NO: 36) are each soluble at greater than 5 mg/ml at a pH in the pH range of pH-6 to pH 8.

The pH stability profiles for each peptide CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ID. NO: 38) and CJI-43.39 (SEQ. ID. NO: 36) were calculated and tabulated as a function of total of degradant peak area (data not shown). None of the peptides showed any significant degradation after a 24 hour period.

Therefore, each of peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ID. NO: 38) and CJI-43.39 (SEQ. ID. NO: 36) was determined to possess the appropriate solubility and stability required of a unique peptide of the invention. Although the invention has been described with reference to its preferred embodiments, other embodiments, can achieve the same results. Variations and modifications to the present invention will be obvious to those skilled in the art and it is intended to cover in me appended claims all such modification and equivalents and follow in the true spirit and scope of this invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: IiranuLogic Pharmaceutical Corporation (ii) TITLE OF INVENTION: Pharmaceutical Formulations For Treating Japanese Cedar Pollen Allergy

(iii) NUMBER OF SEQUENCES: 51

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: IrnmuLogic Pharmaceutical Corporation, nc

(B) STREET: 610 Lincoln St

(C) CITY: Waltham

(D) STATE: MA

(E) COUNTRY: USA (F) ZIP: 02154

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: U.S. Serial No. 08/226,248

(B) FILING DATE: April 8, 1994

(A) APPLICATION NUMBER: U.S. Serial No. 08/350,225

(B) FILING DATE: December 6, 1994

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Darlene A. Vanstone

(B) REGISTRATION NUMBER: 35,729 (C) REFERENCE/DOCKET NUMBER: 025.7 PCT(IMI-028CPC)

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 466-6000

(B) TELEFAX: (617) 466-6040

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : sp Asn Pro lie Asp Ser Cys Trp Arg Gly Asp Ser Asn Trp Ala Gin 1 5 10 15 sn Arg Met Lys 20 (2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Asp Ser Asn Trp Ala Gin Asn Arg Met Lys Leu Ala Asp Cys Ala Val 1 5 10 15

Gly Phe Gly Ser 20

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 Leu Ala Asp Cys Ala Val Gly Phe Gly Ser Ser Thr Met Gly Gly Lys 1 5 10 15

Gly Gly Asp Leu 20

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ser Thr Met Gly Gly Lys Gly Gly Asp Leu Tyr Thr Val Thr Asn Ser 1 5 10 15 Asp Asp Asp Pro 20

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :

Tyr Thr Val Thr Asn Ser Asp Asp Asp Pro Val Asn Pro Ala Pro Gly 1 5 10 15

Thr Leu Arg Tyr 20

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Val Asn Pro Ala Pro Gly Thr Leu Arg Tyr Gly Ala Thr Arg Asp Arg 1 5 10 15

Pro Leu Trp lie 20

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 Gly Ala Thr Arg Asp Arg Pro Leu Trp lie lie Phe Ser Gly Asn Met 1 5 10 15

Asn lie Lys Leu 20

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 lie Phe Ser Gly Asn Met Asn lie Lys Leu Lys Met Pro Met Tyr lie 1 5 10 15

Ala Gly Tyr Lys 20

(2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Lys Met Pro Met Tyr He Ala Gly Tyr Lys Thr Phe Asp Gly Arg Gly 1 5 10 15

Ala Gin Val Tyr 20 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Thr Phe Asp Gly Arg Gly Ala Gin Val Tyr He Gly Asn Gly Gly Pro 1 5 10 15

Cys Val Phe He 20 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

He Gly Asn Gly Gly Pro Cys Val Phe He Lys Arg Val Ser Asn Val 1 5 10 15

He He His Gly 20 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Lys Arg Val Ser Asn Val He He His Gly Leu Tyr Leu Tyr Gly Cys 1 5 10 15

Ser Thr Ser Val 20

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Leu Tyr Leu Tyr Gly Cys Ser Thr Ser Val Leu Gly Asn Val Leu He 1 5 10 15

Asn Glu Ser Phe 20

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Leu Gly Asn Val Leu He Asn Glu Ser Phe Gly Val Glu Pro Val His 1 5 10 15 Pro Gin Asp Gly 20

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Gly Val Glu Pro Val His Pro Gin Asp Gly Asp Ala Leu Thr Leu Arg 1 5 10 15 Thr Ala Thr Asn 20

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE : amino acid (D) TOPOLOGY : linear

( ii ) MOLECULE TYPE : peptide

(v) FRAGMENT TYPE : internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 16 : Asp Ala Leu Thr Leu Arg Thr Ala Thr Asn He Trp He Asp His Asn 1 5 10 15

Ser Phe Ser Asn 20

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: He Trp He Asp His Asn Ser Phe Ser Asn Ser Ser Asp Gly Leu Val 1 5 10 15

Asp Val Thr Leu 20

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Ser Ser Asp Gly Leu Val Asp Val Thr Leu Thr Ser Thr Gly Val Thr 1 5 10 15

He Ser Asn Asn 20

(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Thr Ser Thr Gly Val Thr He Ser Asn Asn Leu Phe Phe Asn His His 1 5 10 15 Lys Val Met Leu 20

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Leu Phe Phe Asn His His Lys Val Met Leu Leu Gly His Asp Asp Ala 1 5 10 15

Tyr Ser Asp Asp 20

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Leu Gly His Asp Asp Ala Tyr Ser Asp Asp Lys Ser Met Lys Val Thr 1 5 10 15

Val Ala Phe Asn 20

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Lys Ser Met Lys Val Thr Val Ala Phe Asn Gin Phe Gly Pro Asn Cys 1 5 10 15

Gly Gin Arg Met 20

(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Gin Phe Gly Pro Asn Cys Gly Gin Arg Met Pro Arg Ala Arg Tyr Gly 1 5 10 15

Leu Val His Val 20

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Pro Arg Ala Arg Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp 1 5 10 15

Pro Trp Thr He 20

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Ala Asn Asn Asn Tyr Asp Pro Trp Thr He Tyr Ala He Gly Gly Ser 1 5 10 15

Ser Asn Pro Thr 20

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Tyr Ala He Gly Gly Ser Ser Asn Pro Thr He Leu Ser Glu Gly Asn 1 5 10 15 Ser Phe Thr Ala 20 (2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

He Leu Ser Glu Gly Asn Ser Phe Thr Ala Pro Asn Glu Ser Tyr Lys 1 5 10 15

Lys Gin Val Thr 20 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Pro Asn Glu Ser Tyr Lys Lys Gin Val Thr He Arg He Gly Cys Lys 1 5 10 15

Thr Ser Ser Ser 20 (2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

He Arg He Gly Cys Lys Thr Ser Ser Ser Cys Ser Asn Trp Val Trp 1 5 10 15

Gin Ser Thr Gin 20 (2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : peptide

(v) FRAGMENT TYPE : internal (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 30 :

Cys Ser Asn Trp Val Trp Gin Ser Thr Gin Asp Val Phe Tyr Asn Gly 1 5 10 15 Ala Tyr Phe Val 20

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Asp Val Phe Tyr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu 1 5 10 15 Gly Gly Asn He 20

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Ser Ser Gly Lys Tyr Glu Gly Gly Asn He Tyr Thr Lys Lys Glu Ala 1 5 10 15 Phe Asn Val Glu 20

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33

Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu Asn Gly Asn Ala Thr Pro 1 5 10 15 Gin Leu Thr Lys 20

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Asn Gly Asn Ala Thr Pro Gin Leu Thr Lys Asn Ala Gly Val Leu Thr 1 5 10 15 Cys Ser Leu Ser 20

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Asn Ala Gly Val Leu Thr Cys Ser Leu Ser Lys Arg Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Asp Asp Ala Tyr Ser Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe 1 5 10 15

Asn Gin Phe Gly Asp Glu 20 (2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE peptide

(v) FRAGMENT TYPE internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Asp Lys Glu Pro Arg Ala Arg Tyr Gly Leu Val His Val Ala Asn Asn 1 5 10 15

Asn Tyr Asp Pro Trp Thr He Glu Glu Glu 20 25

(2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Asp Glu Glu Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly

Gly 1 5 10 15

Asn He Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu 20 25

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1337 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: Crytpomeria japonica

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 66..1187

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 129..1187

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

AGTCAATCTG CTCATAATCA TAGCATAGCC GTATAGAAAG AAATTCTACA CTCTGCTACC 60

AAAAA ATG GAT TCC CCT TGC TTA GTA GCA TTA CTG GTT TTC TCT TTT 107

Met Asp Ser Pro Cys Leu Val Ala Leu Leu Val Phe Ser Phe -21 -20 -15 -10

GTA ATT GGA TCT TGC TTT TCT GAT AAT CCC ATA GAC AGC TGC TGG AGA 155 Val He Gly Ser Cys Phe Ser Asp Asn Pro He Asp Ser Cys Trp Arg -5 1 5

GGA GAC TCA AAC TGG GCC CAA AAT AGA ATG AAG CTC GCA GAT TGT GCA 203

Gly Asp Ser Asn Trp Ala Gin Asn Arg Met Lys Leu Ala Asp Cys Ala 10 15 20 25

GTG GGC TTC GGA AGC TCC ACC ATG GGA GGC AAG GGA GGA GAT CTT TAT 251

Val Gly Phe Gly Ser Ser Thr Met Gly Gly Lys Gly Gly Asp Leu Tyr 30 35 40

ACG GTC ACG AAC TCA GAT GAC GAC CCT GTG AAT CCT GCA CCA GGA ACT 299

Thr Val Thr Asn Ser Asp Asp Asp Pro Val Asn Pro Ala Pro Gly Thr 45 50 55

CTG CGC TAT GGA GCA ACC CGA GAT AGG CCC CTG TGG ATA ATT TTC AGT 347

Leu Arg Tyr Gly Ala Thr Arg Asp Arg Pro Leu Trp He He Phe Ser 60 65 70

GGG AAT ATG AAT ATA AAG CTC AAA ATG CCT ATG TAC ATT GCT GGG TAT 395

Gly Asn Met Asn He Lys Leu Lys Met Pro Met Tyr He Ala Gly Tyr 75 80 85

AAG ACT TTT GAT GGC AGG GGA GCA CAA GTT TAT ATT GGC AAT GGC GGT 443

Lys Thr Phe Asp Gly Arg Gly Ala Gin Val Tyr He Gly Asn Gly Gly 90 95 100 105

CCC TGT GTG TTT ATC AAG AGA GTT AGC AAT GTT ATC ATA CAC GGT TTG 491

Pro Cys Val Phe He Lys Arg Val Ser Asn Val He He His Gly Leu 110 115 120

TAT CTG TAC GGC TGT AGT ACT AGT GTT TTG GGG AAT GTT TTG ATA AAC 539

Tyr Leu Tyr Gly Cys Ser Thr Ser Val Leu Gly Asn Val Leu He Asn 125 130 135

GAG AGT TTT GGG GTG GAG CCT GTT CAT CCT CAG GAT GGC GAT GCT CTT 587

Glu Ser Phe Gly Val Glu Pro Val His Pro Gin Asp Gly Asp Ala Leu 140 145 150

ACT CTG CGC ACT GCT ACA AAT ATT TGG ATT GAT CAT AAT TCT TTC TCC 635

Thr Leu Arg Thr Ala Thr Asn He Trp He Asp His Asn Ser Phe Ser 155 160 165

AAT TCT TCT GAT GGT CTG GTC GAT GTC ACT CTT ACT TCG ACT GGA GTT 683

Asn Ser Ser Asp Gly Leu Val Asp Val Thr Leu Thr Ser Thr Gly Val 170 175 180 185

ACT ATT TCA AAC AAT CTT TTT TTC AAC CAT CAT AAA GTG ATG TTG TTA 731

Thr He Ser Asn Asn Leu Phe Phe Asn His His Lys Val Met Leu Leu 190 195 200

GGG CAT GAT GAT GCA TAT AGT GAT GAC AAA TCC ATG AAG GTG ACA GTG 779

Gly His Asp Asp Ala Tyr Ser Asp Asp Lys Ser Met Lys Val Thr Val 205 210 215

GCG TTC AAT CAA TTT GGA CCT AAC TGT GGA CAA AGA ATG CCC AGG GCA 827

Ala Phe Asn Gin Phe Gly Pro Asn Cys Gly Gin Arg Met Pro Arg Ala 220 225 230 CGA TAT GGA CTT GTA CAT GTT GCA AAC AAT AAT TAT GAC CCA TGG ACT 875

Arg Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Trp Thr 235 240 245

ATA TAT GCA ATT GGT GGG AGT TCA AAT CCA ACC ATT CTA AGT GAA GGG 923

He Tyr Ala He Gly Gly Ser Ser Asn Pro Thr He Leu Ser Glu Gly 250 255 260 265

AAT AGT TTC ACT GCA CCA AAT GAG AGC TAC AAG AAG CAA GTA ACC ATA 971

Asn Ser Phe Thr Ala Pro Asn Glu Ser Tyr Lys Lys Gin Val Thr He 270 275 280

CGT ATT GGA TGC AAA ACA TCA TCA TCT TGT TCA AAT TGG GTG TGG CAA 1019

Arg He Gly Cys Lys Thr Ser Ser Ser Cys Ser Asn Trp Val Trp Gin 285 290 295

TCT ACA CAA GAT GTT TTT TAT AAT GGA GCT TAT TTT GTA TCA TCA GGG 1067

Ser Thr Gin Asp Val Phe Tyr Asn Gly Ala Tyr Phe Val Ser Ser Gly 300 305 310

AAA TAT GAA GGG GGT AAT ATA TAC ACA AAG AAA GAA GCT TTC AAT GTT 1115

Lys Tyr Glu Gly Gly Asn He Tyr Thr Lys Lys Glu Ala Phe Asn Val 315 320 325

GAG AAT GGG AAT GCA^■ ACT CCT CAA TTG ACA AAA AAT GCT GGG GTT TTA 1163

Glu Asn Gly Asn Ala Thr Pro Gin Leu Thr Lys Asn Ala Gly Val Leu 330 335 340 345

ACA TGC TCT CTC TCT AAA CGT TGT TGATGATGCA TATATTCTAG CATGTTGTAC 1217 Thr Cys Ser Leu Ser Lys Arg Cys 350

TATCTAAATT AACATCAACA AGAAAATATA TCATGATGTA TATTGTTGTA TTGATGTCAA 1277 AATAAAAATG TATCTTTTAC TATTAAAAAA AAAAATGATC GATCGGACGG TACCTCTAGA 1337

(2) INFORMATION FOR SEQ ID NO: 0:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 374 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Met Asp Ser Pro Cys Leu Val Ala Leu Leu Val Phe Ser Phe Val He -21 -20 -15 -10

Gly Ser Cys Phe Ser Asp Asn Pro He Asp Ser Cys Trp Arg Gly Asp -5 1 5 10

Ser Asn Trp Ala Gin Asn Arg Met Lys Leu Ala Asp Cys Ala Val Gly 15 20 25 Phe Gly Ser Ser Thr Met Gly Gly Lys Gly Gly Asp Leu Tyr Thr Val 30 35 40 Thr Asn Ser Asp Asp Asp Pro Val Asn Pro Ala Pro Gly Thr Leu Arg 45 50 55

Tyr Gly Ala Thr Arg Asp Arg Pro Leu Trp He He Phe Ser Gly Asn 60 65 70 75

Met Asn He Lys Leu Lys Met Pro Met Tyr He Ala Gly Tyr Lys Thr 80 85 90 Phe Asp Gly Arg Gly Ala Gin Val Tyr He Gly Asn Gly Gly Pro Cys 95 100 105

Val Phe He Lys Arg Val Ser Asn Val He He His Gly Leu Tyr Leu 110 115 120

Tyr Gly Cys Ser Thr Ser Val Leu Gly Asn Val Leu He Asn Glu Ser 125 130 135

Phe Gly Val Glu Pro Val His Pro Gin Asp Gly Asp Ala Leu Thr Leu 140 145 150 155

Arg Thr Ala Thr Asn He Trp He Asp His Asn Ser Phe Ser Asn Ser 160 165 170 Ser Asp Gly Leu Val Asp Val Thr Leu Thr Ser Thr Gly Val Thr He 175 180 185

Ser Asn Asn Leu Phe Phe Asn His His Lys Val Met Leu Leu Gly His 190 195 200

Asp Asp Ala Tyr Ser Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe 205 210 215

Asn Gin Phe Gly Pro Asn Cys Gly Gin Arg Met Pro Arg Ala Arg Tyr 220 225 230 235

Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Trp Thr He Tyr

240 245 250 Ala He Gly Gly Ser Ser Asn Pro Thr He Leu Ser Glu Gly Asn Ser

255 260 265

Phe Thr Ala Pro Asn Glu Ser Tyr Lys Lys Gin Val Thr He Arg He

270 275 280

Gly Cys Lys Thr Ser Ser Ser Cys Ser Asn Trp Val Trp Gin Ser Thr

285 290 295

Gin Asp Val Phe Tyr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr 300 305 310 315

Glu Gly Gly Asn He Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu Asn

320 325 330 Gly Asn Ala Thr Pro Gin Leu Thr Lys Asn Ala Gly Val Leu Thr Cys

335 340 345

Ser Leu Ser Lys Arg Cys 350

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 41 :

Lys Met Pro Met Tyr He Ala Gly Tyr Lys Thr Phe Asp Gly Arg Gly

1 5 10 15

Ala Gin Val Tyr He Gly Asn Gly Gly Pro Cys Val Phe He 20 25 30

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : peptide

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

Asp Glu Arg Thr Ala Thr Asn He Trp He Asp His Asn Ser Phe Ser 1 5 10 15

Asn Ser Ser Asp Asp 20

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Asp Val Phe Tyr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu 1 5 10 15

Gly Gly Asn He Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu 20 25 30

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: . ide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn He 1 5 10 15 Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu 95/27786

20 25

(2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn He 1 5 10 15

Tyr Thr Lys Lys Glu Ala Phe Asn 20

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Asp Glu Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly 1 5 10 15

Asn He Tyr Thr Lys Lys Glu Ala Phe Asn Ala Glu 20 25

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Asp Glu Glu Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly 1 5 10 15

Gly Asn He Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu 20 25 (2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

Asp Glu Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn 1 5 10 15

He Tyr Thr Lys Lys Glu Ala Phe Asn Ala Glu 20 25

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

Leu Ala Asp Cys Ala Val Gly Phe Gly Ser Ser Thr Met Gly Gly Lys 1 5 10 15

Gly Gly Asp Leu 20 (2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Leu Phe Phe Asn His His Lys Val Met Leu Leu Gly His Asp Asp Ala 1 5 10 15

Tyr Ser Asp Asp 20 (2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Lys Ser Met Lys Val Thr Val Ala Phe Asn Gin Phe Gly Pro Asn Cys 1 5 10 15

Gly Gin Arg Met 20

Claims

What is claimed is:

1. An isolated peptide of Cryj I having an amino acid sequence selected from the group consisting of: CJI-43.39 (SEQ. ID. NO: 36), CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ID. NO: 38).

2. A therapeutic composition comprising at least one isolated Cryj I peptide selected from the group consisting of: CJI-43.39 (SEQ. ID. NO: 36), CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ID. NO: 38).

3. A method of treating sensitivity to Japanese Cedar Pollen allergen or an allergen immunologically cross reactive with Japanese cedar pollen allergen comprising administering sequentially or simultaneously at least two different compositions of claim 2.

4. A method of treating sensitivity to Japanese cedar pollen allergen or an allergen immunologically cross-reactive with Japanese cedar pollen allergen comprising administering sequentially or simultaneously peptides: CJI-43.39 (SEQ. ID. NO: 36), CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ED. NO: 38) in a pharmaceutically acceptable form. .

5. A multipeptide formulation for pharmaceutical administration comprising at least two peptides of Cry; I selected from the group consisting of: CJI-43.39 (SEQ. ID. NO: 36), CJI-24.5 (SEQ. ID. NO: 37), CJI-44.8 (SEQ. ID. NO: 38); each peptide having T cell activity, each peptide being soluble and stable at a physiologically acceptable predetermined pH; and a pharmaceutically acceptable excipient.

6. The multipeptide formulation of claim 5 further comprising a pharmaceutically acceptable counter ion.

7. The multipeptide formulation of claim 5 wherein said predetermined pH is in the range of about pH 5.5 to pH 7.5. 8. The multipeptide formulation of claim 5 comprising peptides CJI-42.5 (SEQ. ID. NO: 42), CJI-43.39 (SEQ. ID. NO: 36), CJI-24.5 (SEQ. ID. NO: 37), CJI-44.

8 (SEQ. ID. NO: 38).

9. The composition of claim 2 wherein said T cell activity is at least 39%.

10. An optimized multipeptide formulation suitable for dierapeutic treatment of humans suffering from allergy to Japanese Cedar Pollen comprising:

Cryj I peptides CJl -24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ED. NO: 38), each peptide having a concentration of 0.75 mg per peptide;

0.05 M Sodium Phosphate pH 6.0-8.0;

5% w/v Mannitol, U.S.P.; and

Sterile Water for Injection, U.S.P.

11. The optimized multipeptide formulation of claim 10 wherein said formulation is in the form of a lyophilized powder.

12. The optimized multipeptide formulation of claim 10 wherein the final pH of the formulation is in the pH range of pH 7.0 to pH 7.5.

13. Use of a therapeutic composition of claim 2 for the manufacture of a medicament for treatment of sensitivity of an individual to Japanese cedar pollen allergen, or an allergen immunologically cross reactive with Japanese cedar pollen allergen.

14. Use of the multipeptide formulation of claim 5 for the manufacture of a medicament for treatment of sensitivity of an individual to Japanese cedar pollen allergen, or an allergen immunologically cross reactive with Japanese cedar pollen allergen.

15. Use of the optimized multipeptide formulation of claim 10 for the manufacture of a medicament for treatment of sensitivity of an individual to Japanese cedar pollen allergen, or an allergen immunologically cross reactive with Japanese cedar pollen allergen