Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
  • A61K39/36—Allergens from pollen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, suitable for use in preventing or treating allergy to Japanese cedar pollen and/or Japanese cypress pollen which is up to 30 amino acids in length and comprises:
• the invention further provides a pharmaceutical formulation which comprises a pharmaceutically acceptable carrier or diluent and a polypeptide of the invention, or a pharmaceutically acceptable salt thereof.
• the invention additionally provides a polypeptide, salt or pharmaceutical formulation of the invention for use in a method of treating or preventing allergy to Japanese cedar pollen and/or Japanese cypress pollen.
• the invention also provides use of a polypeptide or salt of the invention for the manufacture of a medicament for the prevention or treatment of allergy to Japanese cedar pollen and/or Japanese cypress pollen.
• the invention further provides a method of preparing a pharmaceutical formulation of the invention, comprising combining a polypeptide or salt described herein with a pharmaceutically acceptable carrier or diluent.
• a Uniprot accession number for the Cry IFR protein is provided in Example 1.
• the present invention is concerned with preventing or treating allergy to Japanese cedar pollen and/or Japanese cypress pollen and provides polypeptides, and pharmaceutically acceptable salts thereof, suitable for this use.
• Said polypeptides or salts may be provided in pharmaceutical formulations.
• a polypeptide of the invention may comprise, consist or consist essentially of an amino acid sequence as shown in SEQ ID NO: 1.
• modifications in a variant sequence do not alter the functional properties of a T cell epitope present in the corresponding original amino acid sequence.
• the functional properties of T cell epitopes are discussed further below.
• sufficient contiguous amino acids of the corresponding original amino acid sequence are retained to contain a T cell epitope.
• a variant sequence retains at least 8, preferably at least 9, contiguous amino acids of the original amino acid sequence.
• the variant sequence may retain from 8 to 12 amino acids or from 9 to 12 amino acids of the original amino acid sequence.
• a variant sequence may have fewer than seven amino acid modifications.
• said variant sequence may have up to six amino acid modifications, such as up to five amino acid modifications, preferably up to four said amino modifications, more preferably up to three amino acid modifications, and most preferably only one or two amino acid modifications. All said modifications are independently a deletion, substitution or insertion.
• a T cell epitope-containing variant sequence has an amino acid modification that is a deletion
• the deleted amino acid is preferably removed from the N- or C-terminus of the corresponding original amino acid sequence. That is, the variant sequence is a truncation of the original amino acid sequence formed by removing one or more contiguous amino acids from the N- and/or C-terminus of the original sequence. Such a variant sequence may optionally have no other deletions or no other modifications.
• a deleted amino acid may less preferably be removed from an internal position in the corresponding original amino acid sequence.
• deletion from an internal position it is meant that a deleted amino acid is not itself at the N- or C-terminus of the original amino acid sequence and nor is it removed as part of a sequence of contiguous amino acids including the N- or C-terminus of the original amino acid sequence. That is, to be considered to be deletion from an internal position, said deletion must occur independently of deletion from the N- or C-terminus of the original amino acid sequence.
• an example variant sequence having an internal deletion of two amino acids could be ADEFGH.
• B and C are removed from internal positions and the original terminal residues A and H are retained.
• a deletion of two contiguous amino acids from the N- terminus of the same original sequence would result in the variant sequence CDEFGH, in which A and B are removed and C is now at the N-terminus.
• the deletion of B in this case is not a removal from an internal position, because it is removed as one of the two contiguous amino acids including the N-terminus of the original sequence.
• the deleted amino acids may be removed from any combination of the N-terminus and/or the C-terminus and/or an internal position.
• Preferred variant sequences have no more than one deletion from an internal position.
• the deleted amino acids are removed from any combination of the N- and/or C-terminus of the original sequence. That is, the deleted amino acids may all be removed from the N-terminus of the original sequence, or they may all be removed from the C-terminus of the original sequence, or some amino acids may be removed from each end of the original sequence.
• a variant sequence is an amino acid sequence of SEQ ID NO: 1 having one, two, three, four, five, six or seven amino acids removed from the C-terminus of said sequence of SEQ ID NO: 1.
• a variant amino acid sequence is an amino acid sequence of SEQ ID NO: 1 having a number of amino acids removed from both the N- and C-terminus of said sequence, provided that said sequence has no more than seven modifications in total.
• a preferred embodiment of such a variant sequence is an amino acid sequence of SEQ ID NO: 1 having one, two or three amino acids removed from the N- and/or C-terminus of said sequence of SEQ ID NO: 1, and optionally no other modifications.
• variant amino acid sequences which have at least one deletion include:
• IKLRRTIEA (SEQ ID NO: 6), which is the amino acid sequence of DLKIKLRRTIEAEGIP (SEQ ID NO: 1) having three amino acids removed from the N-terminus and four amino acids removed from the C-terminus.
• T cell epitope-containing variant sequence has an amino acid modification that is a substitution
• the substitution may occur at any position in the original amino acid sequence. It is preferred that said substitution does not introduce a proline or a cysteine. It is also preferred that said substitution is a conservative substitution.
• amino acids with basic side chains such as lysine, arginine or histidine, may each be independently substituted for each other.
• amino acids with acidic side chains such as aspartate and glutamate
• a glutamate or glutamine may also preferably be replaced with pyroglutamate.
• the amino acids with aliphatic side chains such as glycine, alanine, valine, leucine and isoleucine, may each be independently substituted for each other. Particularly preferred substitutions in this category are limited to the amino acids with smaller aliphatic side chains, that is glycine, alanine, valine, which may preferably each be independently substituted for each other.
• substitutions include the substitution of methionine with norleucine (Nle).
• a neutral amino acid may be substituted with another neutral amino acid
• a charged amino acid may be substituted with another charged amino acid
• a hydrophilic amino acid may be substituted with another hydrophilic amino acid
• a hydrophobic amino acid may be substituted with another hydrophobic amino acid
• a polar amino acid may be substituted with another polar amino acid
• an aromatic amino acid may be substituted with another aromatic amino acid.
• a variant sequence is the amino acid sequence of SEQ ID NO: 1 having a number of amino acids inserted at both the N- and C-terminus of said sequence of SEQ ID NO: 1, and a number of deletions provided that said sequence has no more than seven modifications in total.
• a preferred embodiment of such a variant sequence is the amino acid sequence of SEQ ID NO: 1 having one or two amino acids deleted from the N-and/or C-terminus of said sequence of SEQ ID NO: 1 and one or two substitutions made within the sequence of SEQ ID NO: 1.
• an amino acid may be inserted at the N-terminus and/or C-terminus of the original sequence. That is, the variant sequence is an extension of the original amino acid sequence formed by adding amino acids to the N- and/or C-terminus of the original sequence. Such a variant sequence may optionally have no other insertions or no other modifications.
• an amino acid may be inserted at an internal position.
• insertion at an internal position it is meant that an amino acid is inserted at any position which is C-terminal to the amino acid at the N-terminus of the original sequence, or that an amino acid is inserted at any position which is N-terminal to the amino acid at the C- terminus of the original sequence.
• the added amino acids may be inserted at any combination of the N-terminus and/or the C-terminus and/or an internal position.
• Preferred variant sequences have no more than one insertion at an internal position. In particularly preferred variant sequences there is no insertion at an internal position, and the added amino acids are inserted at any combination of the
• the added amino acids may all be inserted at the N-terminus of the original sequence, or they may all be inserted at the C-terminus of the original sequence, or some amino acids may be inserted at each end of the original sequence. That is, the added amino acids may be considered to extend the original sequence at the N- and/or C-terminus.
• a variant sequence is the amino acid sequence of SEQ ID NO: 1
• a variant sequence is the amino acid sequence of SEQ ID NO: 1 having one, two, three, four, five, six or seven amino acids inserted at the C- terminus of said sequence of SEQ ID NO: 1.
• a variant sequence is the amino acid sequence of SEQ ID NO: 1 having a number of amino acids inserted at both the N- and C-terminus of said sequence of SEQ ID NO: 1, provided that said sequence has no more than seven modifications in total.
• a preferred embodiment of such a variant sequence is the amino acid sequence of SEQ ID NO: 1 having one, two or three amino acids inserted at the N- and/or C-terminus of said sequence of SEQ ID NO: 1, and optionally no other modifications.
• a variant sequence having a charged amino acid inserted at the N- and/or C- terminus is particularly preferred where said charged amino acid will correspond to the N- and/or C- terminus of the polypeptide of the invention which comprises, consists or consists essentially of the variant sequence.
• Charged residues at the N- and/or C- terminus of a polypeptide can improve the solubility of a polypeptide.
• Preferred charged amino acids include lysine, arginine and histidine. Lysine is particularly preferred.
• a particularly preferred variant sequence is an amino acid sequence of SEQ ID NO: 1 having one or more charged amino acids, preferably one or more lysine residues, inserted at the N- and/or C-terminus of said sequence of SEQ ID NO: 1.
• a variant sequence is the amino acid sequence of SEQ ID NO: 1 having a number of amino acids inserted at both the N- and C-terminus of said sequence of SEQ ID NO: 1, and a number of substitutions provided that said sequence has no more than seven modifications in total.
• a preferred embodiment of such a variant sequence is the amino acid sequence of SEQ ID NO: 1 having one or two amino acids inserted at the N-and/or C-terminus of said sequence of SEQ ID NO: 1 and one or two substitutions made within the sequence of SEQ ID NO: 1.
• a polypeptide of the invention is up to 30 amino acids in length and comprises, consists or consists essentially of an amino acid sequence or variant sequence as defined above.
• Said polypeptide may preferably be up to 25 amino acids in length, more preferably up to 20 amino acids in length or up to 17 amino acids in length, and most preferably up to 15 amino acids in length. Put another way, the polypeptide may have a maximum length of 30, 25, 20, 17 or 15 amino acids.
• a polypeptide of the invention is preferably at least 8 amino acids in length, more preferably at least 9 amino acids in length, most preferably at least 12 amino acids in length. Put another way, the polypeptide may have a minimum length of 8, 9, or 12 amino acids.
• a polypeptide of the invention may be of a length defined by any combination of a said minimum and a said maximum length.
• the polypeptide may be 8 to 30, 8 to 25, 8 to 20, 8 to 17 or 8 to 15 amino acids in length.
• the polypeptide may be 9 to 30, 9 to 25, 9 to 20, 9 to 17 or 9 to 15 amino acids in length.
• the polypeptide may be 12 to 30, 12 to 25, 12 to 20, 12 to 17 or 12 to 15 amino acids in length.
• a preferred polypeptide is of 9 to 30 amino acids in length, more preferably 9 to 20 amino acids in length.
• a particularly preferred polypeptide is of 12 to 17 amino acids in length.
• a polypeptide of the invention may comprise an amino acid sequence or variant sequence as defined above. Therefore, said polypeptide may include additional amino acids which are not defined by said amino acid sequence or variant sequence.
• the additional amino acids may be described as flanking said amino acid sequence or variant sequence. That is, the additional amino acids are included at the N-terminus and/or C-terminus of said amino acid sequence or variant sequence.
• a polypeptide of the invention may have a sequence consisting of said amino acid sequence or variant sequence having an N-terminal and/or C- terminal extension of a number of amino acids.
• the maximum number of amino acids in the N-terminal and/or C-terminal extension is determined by the maximum length of the polypeptide, as defined above.
• amino acids in an N-terminal extension of a said amino acid sequence or variant sequence preferably correspond to the amino acids immediately N-terminal to the said amino acid sequence in the native sequence of the protein from which it derives.
• amino acids in a C-terminal extension of a said amino acid sequence or variant sequence preferably correspond to the amino acids immediately C-terminal to the said amino acid sequence in the native sequence of the protein from which it derives.
• the N-terminal and/or C-terminal extension may be the one, two, three, four, five, six, seven, eight, nine or ten amino acids corresponding respectively to the one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acids immediately N- terminal or C-terminal to said amino acid sequence in the sequence of the protein from which it derives.
• the N-terminal and/or C-terminal extension is of from one to ten amino acids corresponding respectively to the one to ten contiguous amino acids immediately N-terminal or C-terminal to the said amino acid sequence in the native sequence of the protein from which it derives.
• the N-terminal and/or C-terminal extension is of from one to six amino acids corresponding respectively to the one to six contiguous amino acids immediately N-terminal or C-terminal to the said amino acid sequence.
• the N-terminal and/or C-terminal extension is of from one to four amino acids corresponding respectively to the one to four contiguous amino acids immediately N-terminal or C-terminal to the said amino acid sequence.
• the N-terminal and/or C-terminal extension is of from one to two amino acids corresponding respectively to the one to two contiguous amino acids immediately N-terminal or C-terminal to the said amino acid sequence.
• polypeptides of the invention which include an N-terminal and/or C-terminal extension to an amino acid sequence or variant sequence include the following:
• DLKIKLRRTIEAEGIP may have a N-terminal extension of one, two, three, four, five, six, seven, eight, nine or ten amino acids corresponding to the one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acids immediately N-terminal to DLKIKLRRTIEAEGIP in the native sequence of Cry IFR, that is the amino acids H, A, V, E, P, M, K, S, M and F.
• the polypeptide of the invention has the amino acid sequence of HAVEPMKSMFDLKIKLRRTIEAEGIP (SEQ ID NO : 2; N-terminal extension underlined).
• DLKIKLRRTIEAEGIP may have a C-terminal extension of one, two, three, four, five, six, seven, eight, nine or ten amino acids corresponding to the one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acids immediately C-terminal to DLKIKLRRTIEAEGIP in the native sequence of Cry IFR, that is the amino acids H, T, Y, V, V, P, H, C, F and A.
• the polypeptide of the invention has the amino acid sequence of DLKIKLRRTIEAEGIPHTYVVPHCF A (SEQ ID NO: 3; C-terminal extension underlined).
• DLKIKLRRTIEAEGIP may have a N-terminal extension of one, two, three, four, five, six, seven or eight amino acids corresponding to the one, two, three, four, five, six, seven or eight contiguous amino acids immediately N-terminal to DLKIKLRRTIEAEGIP in the native sequence of Cry IFR, that is the amino acids V, E, P, M, K, S, M and F. It may also have a C-terminal extension of one amino acid corresponding to the one contiguous amino acid immediately C-terminal to DLKIKLRRTIEAEGIP in the native sequence of Cry IFR, that is the amino acid H. For example, where all eight contiguous amino acids are present in the N-terminal extension and the one contiguous amino acid is present in the C-terminal extension, the polypeptide of the invention has the amino acid sequence of
• VEPMKSMFDLKIKLRRTIEAEGIPH SEQ ID NO: 4; N- and C-terminal extensions are underlined.
• the amino acids in the N-terminal and/or C-terminal extension may not correspond exactly to amino acids in the native sequence of the protein from which the amino acid sequence or variant sequence derives.
• the N-terminal and/or C-terminal extension may include a sequence derived from said native sequence which has been modified, for example to improve stability, solubility or manufacturability of the polypeptide.
• a methionine in the native sequence may be substituted with nor-leucine, and/or one or more charged residues may be added at the N-terminus of a N-terminal extension and/or the C-terminus of a C-terminal extension.
• positively charged residues such as arginine and lysine are added.
• Amino acids selected from histidine, glutamate and aspartate may be added.
• amino acids of an N-terminal and/or C-terminal extension may not correspond to amino acids in native sequence of the protein from which the amino acid sequence or variant sequence derives. They may instead be any suitable amino acids, preferably selected to improve stability, solubility or manufacturability of the polypeptide.
• one or more charged residues may be added at the N and/or C terminus of SEQ ID NO: 1.
• positively charged residues such as arginine and lysine are added.
• Amino acids selected from histidine, glutamate and aspartate may be added.
• suitable polypeptides may have a sequence consisting of an N- or C-terminal extension of a said amino acid sequence of SEQ ID NO: 1 which further comprises one or more deletions.
• the polypeptide may comprise an extension of a said amino acid sequence within the native sequence at one terminus, such as the N-terminus, and a deletion of amino acids at the other terminus, such as the C-terminus.
• the polypeptide may have a sequence consisting of an N-terminal extension of a said amino acid sequence with one, two, three, four, five, six or seven, eight, nine, ten or more amino acids corresponding respectively to the one, two, three, four, five, six, seven, eight, nine or ten or more contiguous amino acids immediately N- terminal to said amino acid sequence in the sequence of the protein from which it derives, and a deletion of one, two, three, four, five, six or seven amino acids from the C-terminus of the said amino acid sequence.
• the polypeptide may have a sequence consisting of a C-terminal extension of a said amino acid sequence with one, two, three, four, five, six, seven, eight, nine, ten or more amino acids corresponding respectively to the one, two, three, four, five, six, seven, eight, nine or ten or more contiguous amino acids immediately C- terminal to said amino acid sequence in the sequence of the protein from which it derives, and a deletion of one, two, three, four, five, six or seven amino acids from the N-terminus of the said amino acid sequence.
• a polypeptide of the invention is up to 30 amino acids in length and comprises, consists or consists essentially of an amino acid sequence or variant sequence as defined above.
• the amino acid sequence and each said variant sequence contains a T cell epitope.
• the T cell epitope is preferably an MHC Class II-binding T cell epitope. It is preferred that the modifications in a variant sequence do not alter the functional properties of a T cell epitope present in the corresponding original amino acid sequence.
• sufficient contiguous amino acids of the corresponding original amino acid sequence are retained to contain a T cell epitope.
• such a variant sequence retains at least 8, preferably at least 9, contiguous amino acids of the original amino acid sequence
• T cell epitope may preferably be confirmed by analysis performed in silico, for example using bioinformatic software as described in Example 1. Alternatively, the presence of a T cell epitope may be confirmed by direct evaluation of its functional properties. Particular functional properties of T cell epitopes include the ability of a polypeptide comprising the epitope to bind to an MHC molecule, preferably an MHC Class II molecule, and/or the ability of a polypeptide comprising the epitope to activate a T cell, preferably when bound to an MHC Class II molecule.
• the ability of a polypeptide to bind to an MHC molecule may be evaluated using any suitable method, such as a competition assay.
• a preferred in vitro assay is described in Example 3.
• a polypeptide to activate a T cell may also be evaluated using any suitable method. Preferred methods include the measurement of one or more parameters associated with T cell activation, such as proliferation or cytokine release. Preferred assays for these parameters are described in Example 4. Relevant cytokines include IFN-gamma, IL-13 and IL-10.
• a polypeptide is typically considered to have activated a T cell if it induces release of one, two, or all of IFN-gamma, IL-13 and IL-10, such as release of both IFN-gamma and IL- 13.
• the polypeptide preferably induces a release of greater than 50 pg/ml of the given cytokine(s). More preferably, the polypeptide induces a release of greater than 100 pg/ml of the given cytokine(s).
• a polypeptide comprising, consisting or consisting essentially of a variant amino acid sequence should have substantially the same MHC class II binding properties and substantially the same T cell activation properties as a polypeptide comprising, consisting or consisting essentially of the corresponding original amino acid sequence.
• a polypeptide has substantially the same MHC Class II binding characteristics as another polypeptide if both polypeptides are capable of binding specifically to one or more MHC Class II molecules belonging to the same MHC Class II allele supertype family.
• MHC Class II allele supertype families include HLA-DR1, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DR11, HLA-DR13, HLA-DR15 and HLA-DR51.
• both polypeptides will bind specifically to the same MHC Class II molecule, that is to an MHC Class II molecule encoded by the same allele.
• a polypeptide has substantially the same T cell activation properties as another polypeptide if both polypeptides specifically activate a T cell expressing the same T cell receptor.
• Suitable polypeptides comprising, consisting or consisting essentially a variant sequence may be derived empirically or selected according to known criteria. Within a single polypeptide there are certain residues which contribute to binding within the MHC antigen binding groove and other residues which interact with hypervariable regions of the T cell receptor (Allen et al (1987) Nature 327: 713-5). Advantageously, peptides may be designed to favour T-cell proliferation and induction of desensitisation. Metzler and Wraith have demonstrated improved tolerogenic capacity of polypeptides in which substitutions increasing polypeptide-MHC affinity have been made (Metzler & Wraith(1993) Int Immunol: 1159-65). That an altered polypeptide ligand can cause long-term and profound anergy in cloned T cells was demonstrated by Sloan-Lancaster et al (1993) Nature 363 : 156-9.
• Suitable T cell epitope-containing variant sequences according to the invention may alternatively be described in terms of their sequence identity to a corresponding original amino acid sequence.
• a variant sequence may have at least 65% identity to the amino acid sequence of SEQ ID NO: 1. More preferably, a variant sequence may have at least 70%, at least 75%, at least 80%>, at least 85%>, at least 90%, or at least 95% amino acid identity to an amino acid sequence of SEQ ID NO: 1.
• Sequence identity is typically evaluated over a number of contiguous amino acids in the original amino acid sequence. For example, sequence identity may be measured over at least 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids in the original amino acid sequence, depending on the size of the peptides of comparison. It is preferred that sequence identity be measured over at least 9 contiguous amino acids in the original amino acid sequence. It is particularly preferred that sequence identity is measured over the entire length of the corresponding original amino acid sequence.
• sequence identity refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:
• the invention encompasses any pharmaceutically acceptable salt of a polypeptide of the invention.
• Said pharmaceutically acceptable salts include, for example, mineral acid salts such as chlorides, hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
• a hydrochloride salt or an acetate salt is preferred.
• a polypeptide of the invention can be prepared by any suitable technique.
• SPPS Solid-phase peptide synthesis
• Suitable protecting groups, reagents, solvents and reaction conditions for SPPS are well known to those skilled in the art and as such conditions can be determined by one skilled in the art by routine optimization procedures.
• compositions of polypeptides can be prepared by any suitable technique. Typically, salification involves reaction of the polypeptide or a salt thereof with a suitable reagent, typically acid, to obtain the pharmaceutically acceptable salt selected.
• a suitable reagent typically acid
• hydrochloride salt of a polypeptide can be prepared by initially cleaving the polypeptide from the solid phase using trifluoroacetic acid.
• polypeptide will thus initially be a trifluoroacetate salt.
• the trifluoroacetate salt can then be converted into a hydrochloride salt by any known technique, such as ion exchange on a suitable column using hydrochloric acid as an eluent.
• polypeptide or polypeptide salt products can be purified, where required, by any suitable technique.
• High pressure liquid chromatography HPLC can be used, for example.
• polypeptide includes not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed.
• Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol.159, 3230-3237. This approach involves making pseudopolypeptides containing changes involving the backbone, and not the orientation of side chains.
• peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will also be appreciated that the peptide may conveniently be blocked at its N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.
• N-terminal amino group of the polypeptides may be protected by reacting with a carboxylic acid and the C-terminal carboxyl group of the peptide may be protected by reacting with an amine.
• modifications include glycosylation and phosphorylation.
• Another potential modification is that hydrogens on the side chain amines of R or K may be replaced with methylene groups (- H 2 — » - H(Me) or -N(Me) 2 ).
• Analogues of polypeptides according to the invention may also include peptide variants that increase or decrease the polypeptide's half-life in vivo.
• analogues capable of increasing the half-life of polypeptides used according to the invention include peptoid analogues of the peptides, D-amino acid derivatives of the peptides, and peptide-peptoid hybrids.
• peptoid analogues of the peptides include peptoid analogues of the peptides, D-amino acid derivatives of the peptides, and peptide-peptoid hybrids.
• polypeptides used according to the invention comprises D-amino acid forms of the polypeptide.
• the preparation of polypeptides using D-amino acids rather than L-amino acids greatly decreases any unwanted breakdown of such an agent by normal metabolic processes, decreasing the amounts of agent which needs to be administered, along with the frequency of its administration.
• polypeptides provided by the present invention may be derived from splice variants of the parent proteins encoded by mRNA generated by alternative splicing of the primary transcripts encoding the parent protein chains.
• the polypeptides may also be derived from amino acid mutants, glycosylation variants and other covalent derivatives of the parent allergen proteins.
• Exemplary derivatives include molecules wherein the polypeptides of the invention are covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Further included are naturally occurring variant amino acid sequences of the parent proteins. Such a variant amino acid sequence may be encoded by an allelic variant or represent an alternative splicing variant.
• Modifications as described above may be prepared during synthesis of the peptide or by post-production modification, or when the polypeptide is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
• polypeptides described herein may also be modified to improve
• original amino acid sequences may be altered to improve their solubility, and accordingly a polypeptide of the invention having a variant sequence will preferably be more soluble than a polypeptide having the corresponding original amino acid sequence under equivalent conditions.
• Methods for evaluating the solubility of polypeptides are well known in the art.
• solubility is advantageous for the tolerisation of subjects to the allergen from which the polypeptide of the invention derives, since administration of poorly soluble agents to subjects causes undesirable, non-tolerising inflammatory responses.
• the solubility of the polypeptides may be improved by altering the residues which flank the region containing a T cell epitope. For example, N and C terminal to the residues of the polypeptide which flank a T cell epitope, at least one amino acid may be added selected from arginine, lysine, histidine, glutamate and aspartate.
• N and C terminal to the residues of the polypeptide which flank a T cell epitope at least one amino acid may be added selected from arginine, lysine, histidine, glutamate and aspartate.
• any two consecutive amino acids comprising the sequence Asp-Gly in the up to four amino acids at the N or C terminus of the native sequence of the polypeptide, which are not comprised in a T cell epitope, are deleted;
• one or more positively charged residues are added at the N and/or C terminus of the native sequence of the polypeptide.
• any polypeptides containing cysteine residues may be engineered to prevent dimer formation such that any cysteine residues are replaced with serine or 2- aminobutyric acid.
• Polypeptide combinations A polypeptide of the invention, or salt thereof, may be provided in combination with one or more further polypeptides, or salts thereof.
• Such a polypeptide combination is suitable for preventing or treating allergy to Japanese Cedar pollen and/or Japanese Cypress pollen by tolerisation.
• Such a polypeptide combination typically includes at least one further polypeptide which is up to 30 amino acids in length and comprises (I) a T-cell epitope containing amino acid sequence from a Japanese Cedar pollen allergen or a Japanese Cypress pollen allergen; or (II) a T cell epitope-containing variant sequence which is a said amino acid sequence (I) having up to seven amino acid modifications, each of which is independently a deletion, substitution or insertion.
• the native T cell epitope-containing sequence of (I) is typically an amino acid sequence of 9 to 20, 9 to 17, 12 to 17, or 12 to 20 amino acids in length which is present as a contiguous sequence within the sequence of the whole allergen and comprises one ore more T cell epitopes.
• the T cell epitope containing variant sequence may be any variant of the corresponding native T cell epitope containing amino acid sequence as described above by reference to SEQ ID NO 1.
• the T cell epitope-containing variant sequence may be a fragment derived by truncation of the Japanese Cedar pollen allergen or a Japanese Cypress pollen allergen which retains the core 8 or 9 amino acids that make up a T cell epitope of the allergen.
• the polypeptide combination may comprise two, three, four, five, six, seven or more further polypeptides as described above.
• the polypeptide combination preferably comprises twelve polypeptides in total or less, more typically ten polypeptides or less, preferably seven or six polypeptides or less.
• the further polypeptides are typically derived from the Japanese Cedar and
• the polypeptide combination will comprise polypeptides which derive from more than one allergen.
• the combination may comprise one or more polypeptides derived from Cry j l and/or Cry2.
• the combination may comprise one or more polypeptides derived from Cha ol and/or Cha 02. Any of the polypeptide combinations described above may be incorporated in a pharmaceutical formulation of the invention as described in more detail below.
• the polypeptide salt, or pharmaceutical formulation of the invention is used for treating or preventing allergy to Japanese Cedar pollen and/or Japanese Cypress pollen. It may prevent or treat the allergy by tolerisation.
• the tolerisation may be to one or more protein allergens of the Cryptomeria and/or Chamaecyparis genus.
• the tolerisation is typically to the pollen allergen Cry IFR (isoflavone reductase) of Cryptomeria Japonica.
• a combination of polypeptides or salts can be used for treating or preventing allergy to Japanese Cedar pollen and/or Japanese Cypress pollen.
• the polypeptides and/or salts in a combination need not be administered together, and/or need not be not part of the same pharmaceutical formulation.
• the invention thus provides a polypeptide or salt of the invention for use in a method of preventing or treating allergy to Japanese Cedar pollen and/or Japanese Cypress pollen as described above, wherein said method further comprises
• the multiple peptides of this method may each be administered simultaneously, sequentially or concurrently. Thus, they may be administered separately.
• the polypeptide, salt or pharmaceutical formulation of the invention may treat or prevent the allergy by desensitising or tolerising to Japanese Cedar pollen and/or Japanese Cypress pollen.
• a polypeptide of the invention may be used to tolerise or desensitise an individual to the allergen from which it is derived. Desensitising an individual to the allergens means inhibition or dampening of allergic tissue reactions induced by the allergens in appropriately sensitised individuals.
• tolerisation refers to an ability to suppress, or abolish a response to an antigen, such as an allergic response to a protein allergen. Tolerisation is also an ability to diminish or abolish an unwanted immune response, or to desensitise a subject to a protein allergen.
• Tolerisation may be determined by in vitro analysis of T cell responses or by observation of a reduction in the symptoms in an individual.
• T cells can be selectively activated, and then rendered unresponsive. Moreover the anergising or elimination of these T-cells leads to desensitisation of the patient for a particular allergen.
• the desensitisation manifests itself as a reduction in response to an allergen or allergen-derived peptide, or preferably an elimination of such a response, on second and further administrations of the allergen or allergen-derived peptide.
• This second administration may be made after a suitable period of time has elapsed to allow desensitisation to occur; this is preferably any period between one day and several weeks. An interval of around four weeks is preferred.
• the individual to whom the polypeptide, salt or pharmaceutical formulation is administered may be asymptomatic.
• a prophylactically effective amount of the polypeptide or pharmaceutical formulation is administered to such an individual.
• a prophylactically effective amount is an amount which prevents the onset of one or more symptoms of allergy.
• the individual to whom the polypeptide, salt or pharmaceutical formulation is administered may be in need thereof. That is, the individual may exhibit one or more symptoms of allergy.
• polypeptide or pharmaceutical formulation is administered to such an individual.
• a therapeutically effective amount is an amount which is effective to ameliorate one or more symptoms of allergy.
• the individual to whom the polypeptide, salt or pharmaceutical formulation is administered is preferably human.
• the individual may be known to be sensitised to Japanese Cedar pollen and/or Japanese Cypress pollen allergens, at risk of being sensitised or suspected of being sensitised.
• the individual can be tested for
• the individual may have a family history of allergy to Japanese Cedar pollen and/or Japanese Cypress pollen.
• proximity to, for example, a tree or pollen or a substance or product derived from a tree or pollen. By proximity is meant 10 metres or less, 5 metres or less, 2 metres or less, 1 metre or less, or 0 metres from the items described above.
• Symptoms of allergy can include an itching nose, sneezing, ocular tearing, an itchy throat, itchy palate, itchy eyes, runny nose, breathing difficulties, bronchospasm, asthma, red itchy skin or rash.
• the individual may be of any age. However, preferably, the individual may be in the age group of 1 to 90, 5 to 60, 10 to 40, or more preferably 18 to 35.
• the individual may have had allergy to Japanese Cedar pollen and/or Japanese Cypress pollen for at least 2 weeks, 1 month, 6 months, 1 year, 5 years or more than 5 years.
• the individual may suffer from a rash, nasal congestion, nasal discharge and/or coughing caused by the allergy.
• the individual may suffer from an itching nose, sneezing, ocular itching, ocular tearing, itchy throat, itchy palate, bronchospasm and/or asthma caused by the allergy.
• the individual may or may not have been administered with other items.
• compositions/compounds which treat Japanese Cedar pollen and/or Japanese Cypress pollen allergy The individual may live in a geographical region which has a temperate, semi-tropical, or tropical climate. The individual typically suffers from allergy to Japanese Cedar pollen and/or Japanese Cypress pollen in a particular season but the allergy may be perennial. Seasonal allergy to Japanese Cedar pollen and/or Japanese Cypress pollen may commonly occur either in late Winter or in Spring in the Far East.
• the allergic individual is allergic to tree pollen from the Japanese Cedar and/or
• the allergic individual may be allergic both to tree pollens of Cryptomeria Japonica and of Chamaecyparis obtusa.
• the polypeptides, salts or pharmaceutical formulations of the invention may be screened in panels of Japanese Cedar pollen and/or Japanese Cypress pollen allergic individuals to confirm their suitability for use.
• the panel of Japanese Cedar pollen and/or Japanese Cypress pollen allergic individuals may comprise individuals known or not known to be allergic to Japanese Cedar pollen and/or Japanese Cypress pollen.
• multiple polypeptides are provided in combination in a pharmaceutical formulation, they may be screened for their ability to cause T cell proliferation in at least 20 % of samples of T cells, wherein each sample is obtained from different Japanese Cedar pollen and/or Japanese Cypress pollen allergic individuals in the population.
• the pharmaceutical formulation will induce T cell proliferation in at least 30 % of samples of T cells obtained from of a panel of Japanese Cedar pollen and/or Japanese Cypress pollen allergic individuals. More preferably, the
• the pharmaceutical formulation will induce T cell proliferation in 35% or more, 40 % or more, 50 % or more, 60 % or more, 70% or more, 80 % or more, or 90 % or more of samples in the panel.
• the number of individuals in a panel of Japanese Cedar pollen and/or Japanese Cypress pollen allergic individuals may be any number greater than one, for example at least 2, 3, 5, 10, 15, 20, 30, 50, 80, or at least 100 individuals.
• polypeptides, salts and pharmaceutical formulations of the invention cause T cell proliferation, but do not lead to the release of histamine from leucocyte samples from a sensitised individual.
• the histamine release profile of a polypeptide, salt or pharmaceutical formulation may thus be confirmed.
• Suitable leucocyte samples include enriched basophils or mast cell preparations. There may be some histamine release, but preferably the amounts released are not significant.
• Significant histamine release may be considered to be the release of 20% or more of the total available leukocyte histamine when a sample of leukocytes from an individual is stimulated with a pharmaceutical formulation in vitro.
• a polypeptide, salt or pharmaceutical formulation of the invention preferably causes the release of less than 5%), less than 4%, less than 3%, less than 2% or less than 1% of the total available leukocyte histamine when a sample of leukocytes from an individual is stimulated with a composition in vitro.
• a normal individual typically has an approximate leukocyte histamine content of 150ng/10 7 cells.
• Each polypeptide or salt of the invention may be provided to an individual in an isolated, substantially isolated, purified or substantially purified form.
• a polypeptide or salt of the invention may be provided to an individual substantially free from other polypeptides or salts thereof. Whilst it may be possible for the polypeptides or salts to be presented in raw form, it is preferable to present them as a pharmaceutical formulation.
• the invention further provides a pharmaceutical formulation which comprises a pharmaceutically acceptable carrier or diluent and a polypeptide, or a pharmaceutically acceptable salt thereof, of the invention.
• the pharmaceutical formulation may comprise any combination of polypeptides or salts of the invention as described above.
• the carrier(s) or diluent(s) present in the pharmaceutical formulation must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• carriers for injection, and the final formulation are sterile and pyrogen free.
• the carrier or diluent is water.
• the carrier or diluent may comprise thioglycerol,thioanisole or methionine.
• a composition containing one or more polypeptides or salts of the invention can be combined with one or more pharmaceutically acceptable excipients or vehicles to produce a pharmaceutical formulation.
• auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle.
• excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity.
• Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol, thioglycerol and ethanol.
• Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
• mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
• organic acids such as acetates, propionates, malonates, benzoates, and the like.
• polypeptides or salts are typically present at 0.1% to 50% by weight in the pharmaceutical formulation, more preferably at 0.1% to 5% by weight.
• the polypeptides or salts may be present at less than 0.1 % by weight in the pharmaceutical formulation.
• the pharmaceutically acceptable carrier or diluent is typically present at 50% to 99.9 % by weight in the pharmaceutical formulation, more preferably at 95% to 99.9 % by weight.
• the pharmaceutically acceptable carrier or diluents may be present at more than 99. 9% by weight in the pharmaceutical formulation.
• compositions include, but are not limited to pharmaceutically acceptable solutions, lyophili sates, suspensions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such pharmaceutical formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
• a lyophilisate may comprise one or more of trehalose, thioglycerol and thioanisole.
• the active ingredient is provided in dry form (e.g., a lyophilisate, powder or granules) for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted pharmaceutical formulation.
• the invention further provides a method of preparing a pharmaceutical formulation of the invention, comprising combining a polypeptide or salt as described above with a pharmaceutically acceptable carrier or diluent.
• said method prepares a pharmaceutical formulation for parenteral administration, and comprises providing said polypeptide, or salt in dry form and reconstituting said polypeptide, or salt with a said pharmaceutically acceptable carrier or diluent.
• the pharmaceutical formulations may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
• This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
• Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
• Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.
• parenterally-administrable pharmaceutical formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems, pharmaceutical formulations for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
• the polypeptides of the present invention may be encapsulated, adsorbed to, or associated with, particulate carriers.
• suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368.
• Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.
• pharmaceutical formulation may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), topically, parenterally, subcutaneously, by inhalation, intravenously, intramuscularly, intralymphatically (such as to lymph nodes in the groin), intrasternally, transdermally, intradermally, epidermally, sublingually, intranasally, buccally or by infusion techniques.
• the administration may be intratonsillar.
• the administration may be as suppositories.
• the administration may be made by
• the administration is intradermal, epidermal or transdermal.
• the administration may be made by a patch, such as a microtine patch.
• a physician will be able to determine the required route and means of administration for each particular individual.
• the pharmaceutical formulations of the invention are preferably provided sealed in a container.
• the pharmaceutically formulations are typically provided in unit dose form, for example single dose form. They may alternatively be provided in multi-dose form.
• the pharmaceutical formulation is a pharmaceutically acceptable solution
• the solution may be provided in an ampoule, sealed vial, syringe, cartridge, flexible bag or glass bottle.
• the pharmaceutical formulation is a lyophilisate, it is preferably provided in a sealed vial.
• the pharmaceutical formulations of the invention will comprise a suitable concentration of each polypeptide to be effective without causing adverse reaction.
• the pharmaceutical formulation is for example a lyophilisate
• the relevant concentration will be that of each polypeptide following reconstitution.
• the concentration of each polypeptide in the pharmaceutical formulation when in solution will be in the range of 0.03 to 200 nmol/ml.
• the concentration of each polypeptide may be more preferably in the range of 0.3 to 200 nmol/ml, 3 to 180 nmol/ml, 5 to 160 nmol/ml, 10 to 150 nmol/ml, 50 to 200nmol/ml or 30 to 120 nmol/ml, for example about 100 nmol.ml.
• the pharmaceutical formulation should have a purity of greater than 95% or 98% or a purity of at least 99%.
• An adjuvant or further therapeutic agent may be used in combination with one or more polypeptides of the invention.
• An adjuvant is preferably administered in an amount which is sufficient to augment the effect of the polypeptide(s) of the invention or vice versa.
• the adjuvant or further therapeutic agent may be an agent that potentiates the effects of a polypeptide of the invention.
• the further therapeutic agent may be an immunomodulatory molecule which enhances the response to the polypeptide of the invention.
• Non-limiting examples of adjuvants include vitamin D, rapamycin and glucocorticoid steroids such as dexamethasone, fluticasone, budesonide, mometasone, beclomethasone, hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, betamethasone and triamcinolone.
• glucocorticoid steroids such as dexamethasone, fluticasone, budesonide, mometasone, beclomethasone, hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, betamethasone and triamcinolone.
• a preferred glucocorticoid is dexamethasone.
• the other therapeutic agents or adjuvants may be administered separately, simultaneously or sequentially. They may be administered in the same or different pharmaceutical formulations.
• a pharmaceutical formulation may therefore be prepared which comprises a polypeptide of the invention and also one or more other therapeutic agents or adjuvants.
• a pharmaceutical formulation of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic compositions as part of a combined treatment. Accordingly, in a method of preventing or treating allergy according to the invention as described below, the subject may also be treated with a further therapeutic agent.
• polypeptide or salt of the invention is to be administered to an individual in a pharmaceutical formulation
• a pharmaceutical formulation can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, epidermal, intradermal, intramuscular, intralymphatic, intraarterial, intraperitoneal, or intravenous injection using a conventional needle and syringe, a microneedle and syringe or using a liquid jet injection system.
• the administration may be made using a patch, such as a microtine patch.
• Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratonsillarly, intratracheally, intestinal, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary
• administration Other modes of administration include oral administration, suppositories, sublingual administration, and active or passive transdermal delivery techniques.
• polypeptides, salts or pharmaceutical formulations of the invention may be by any suitable method as described above. Suitable amounts of the polypeptides or salts to be administered may be determined empirically, but typically are in the range given below. A single administration of each polypeptide or salt may be sufficient to have a beneficial effect for the patient, but it will be appreciated that it may be beneficial if the polypeptide or salt is administered more than once, in which case typical administration regimes may be, for example, once or twice a week for 2-4 weeks every 6 months, or once a day for a week every four to six months. As will be appreciated, each polypeptide or salt in a combination of polypeptides or salts may be administered to a patient singly or in combination.
• Suitable doses of a polypeptide or salt of the invention may be in the order of up to 10 ⁇ g, up to 15 ⁇ g, up to 2C ⁇ g, up to 25 ⁇ g, up to 30 ⁇ g, up to 50 ⁇ g, up to 100 ⁇ g, up to 500 ⁇ g or more per administration.
• Suitable doses may be less than 15 ⁇ g, but at least lng, or at least 2ng, or at least 5ng, or at least 50ng, or least lOOng, or at least 500ng, or at least ⁇ g, or at least 10 ⁇ g.
• the dose used may be higher, for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or higher.
• Doses may be provided in a liquid formulation, at a concentration suitable to allow an appropriate volume for administration by the selected route. It will be understood that the above doses refer to total dose in the case of a combination of peptides or salts. For example, “up to 35 ⁇ ' refers to a total peptide or salt concentration of up to 35 ⁇ g in a composition comprising a combination or more than one peptide or salt.
• the polypeptide of the invention may be administered directly, or may be administered indirectly by expression from an encoding sequence.
• a polynucleotide may be provided that encodes a polypeptide of the invention.
• a polypeptide of the invention may thus be produced from or delivered in the form of a polynucleotide which encodes, and is capable of expressing, it. Any reference herein to the use, delivery or administration of a peptide of the invention is intended to include the indirect use, delivery or administration of such a peptide via expression from a polynucleotide that encodes it.
• the invention provides a polynucleotide which encodes a polypeptide comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO: 1 or a variant derived therefrom.
• the terms "nucleic acid molecule” and “polynucleotide” are used
• polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• mRNA messenger RNA
• cDNA messenger RNA
• recombinant polynucleotides plasmids
• vectors isolated DNA of any sequence
• isolated RNA of any sequence nucleic acid probes, and primers.
• a polynucleotide may be provided in isolated or purified form.
• Polynucleotides can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).
• polynucleotides may be used in vitro, ex vivo or in vivo in the production of a polypeptide of the invention.
• Such polynucleotides may be
• the nucleic acid molecule can be introduced directly into the recipient subject, such as by standard intramuscular or intradermal injection; transdermal particle delivery; inhalation; topically, or by oral, intranasal or mucosal modes of administration.
• the molecule alternatively can be introduced ex vivo into cells that have been removed from a subject.
• a polynucleotide, expression cassette or vector of the invention may be introduced into APCs of an individual ex vivo. Cells containing the nucleic acid molecule of interest are reintroduced into the subject such that an immune response can be mounted against the peptide encoded by the nucleic acid molecule.
• the nucleic acid molecules used in such immunization are generally referred to herein as "nucleic acid vaccines.”
• Antigen presenting cells APCs
• the invention encompasses the use in vitro of a method of producing a population of APCs that present the polypeptide of the invention on their surface. Said population of APCs may be subsequently used in therapy. Said method of production may be carried out ex vivo on a sample of cells that have been obtained from a patient.
• the APCs produced in this way therefore form a pharmaceutical agent that can be used in the treatment or prevention of allergy to Japanese Cedar pollen and/or Japanese Cypress pollen.
• the cells should be accepted by the immune system of the individual because they derive from that individual. Delivery of cells that have been produced in this way to the individual from whom they were originally obtained, thus forms a therapeutic embodiment of the invention.
• an APC is to be administered, it is preferred to administer the APC to a site in the body where it will have the ability to contact, and activate, suitable T cells of the individual.
• In vitro method In vitro method
• the invention further provides an in vitro method of determining whether T cells recognize a polypeptide or salt of the invention, which method comprises contacting said T cells with said polypeptide or salt and detecting whether said T cells are stimulated by said polypeptide or salt.
• the method comprises use of a polypeptide, or salt thereof, comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO: 1 or a variant derived therefrom
• the above method may be carried out to determine whether an individual has, or is at risk of having, an allergy to Japanese Cedar pollen and/or Japanese Cypress pollen.
• the invention is illustrated by the following Examples:
• the aim of this study is to identify a polypeptide having a sequence with strong affinities for the seven most common human MHC Class II HLA-DRB 1 * allotypes.
• EpiMatrix is a matrix-based algorithm that ranks 10 amino acid long segments, overlapping by 9 amino acids, from any polypeptide sequence by estimated probability of binding to each of the selected MHC molecules.
• the procedure for developing matrix motifs was published by Schafer et al, 16 Vaccine 1998 (1998).
• binding potential for HLA DR1, DR2, DR3, DR4, DR7, DR8, DR11, DR13 and DR15 is assessed.
• Putative MHC ligands are selected by scoring each 10-mer frame in a polypeptide sequence. This score is derived by comparing the sequence of the 10-mer to the matrix of 10 amino acid sequences known to bind to each MHC allele.
• Estimated probability of binding to a selected MHC molecule is calculated by EpiMatrix as follows.
• the polypeptides having a given sequence are scored by estimating the relative promotion or inhibition of binding for each amino acid, compared to known MHC binders for a given MHC allele. This information is summed across the polypeptide and a summary score (EMX score) is assigned to the entire polypeptide. After comparing the EMX score to the scores of known MHC ligands, EpiMatrix arrives at an "estimated binding probability" (abbreviated as EBP, but not strictly a probability).
• EBP describes the proportion of polypeptides with EpiMatrix scores as high or higher that will bind to a given MHC molecule. EBPs range from 100% (highly likely to bind) to less than 1% (very unlikely to bind).
• EpiMatrix analyses were performed on the entire sequence of the known isoform of Cry IFR (Uniprot accession no: Q8RYC0). These analyses identified a core polypeptide (and flanking sequences) derived from the above sequence which is predicted to have good MHC class-II binding. The sequence is shown below in Table 2.
• the Epimatrix analysis further identified the peptides LKIKLRRTI (SEQ ID NO: 5) and IKLRRTIEA (SEQ ID NO: 6) as having binding potential for four or more HLA alleles.
• the sequence set out in Table 3 was selected by the inventors as having desirable characteristics, based on the analysis performed in Example 1 and a consideration of solubility and other physicochemical characteristics.
• a polypeptide consisting of the sequence of Table 3 was produced and was particularly preferred for screening in subsequent assays.
• the assay employed is a competitive MHC class II binding assay, wherein each polypeptide is analysed for its ability to displace a known control binder from each of the human MHC class II allotypes investigated.
• the allotypes and control polypeptides used in this study are those shown below:
• Each polypeptide is analysed in the competition assay and screened for relative binding compared to the control polypeptides. Due to the nature of the competitive assay the data for each polypeptide is determined as a ratio of its own IC50 to that of the control polypeptide. Thus, a polypeptide that has an IC50 value that is parity to the control polypeptide has an identical binding affinity, while peptides with a ratio less than one have a higher affinity and those with a ratio greater than one have a lower affinity.
• Solubility in aqueous solution is an essential criterion for a polypeptide to be an effective therapeutic agent. Therefore, as a consequence of the solubility screen very hydrophobic polypeptides with a high frequency of large hydrophobic amino acid residues in multiple binding registers will be eliminated. This is a characteristic of promiscuous HLA-DRB 1 * binders. Polypeptides which bind to one or more of the MHC Class II allotypes are identified. It would be expected that such polypeptides would have the ability to bind similar allotypes that have not been tested through the homology of MHC structures.
• Example 4 The following methods are used to evaluate T cell activation characteristics of polypeptides having the sequences identified in Examples 1 and 2.
• the cell proliferation assay is performed on PBMC's (140xl0 6 cells required for all parameters to be tested). Proliferation is measured by the incorporation of the radiolabelled compound 3H-thymidine.
• ⁇ of the appropriate antigen or polypeptide concentration is distributed into the appropriate wells of 96 well plates. The plates are then placed into a humidified 5% C02 incubator set at 37°C for a maximum of 4 hours.
• PBMC's isolated as described above are prepared to a
• Polypeptides for use in this assay were manufactured at small scale
• polypeptides were prepared in advance and stored at -20°C prior to the day of assay.
• Polypeptides were added to wells in a volume of 100 ⁇ containing polypeptides at a concentration of 200 ⁇ / ⁇ 1, such that subsequent addition of 100 ⁇ of cells would create a final assay concentration of 100 ⁇ / ⁇ 1.
• PBMCs Peripheral blood mononuclear cells
• cytokine bead assays IL-10, IL-13, Interferon gamma (IFN-g)
• Single measurements were performed for each culture supernatant sample.
• individual cytokine levels were determined by interpolation from the standard curve generated in the assay.
• a positive result was taken as a cytokine release of greater than 100 pg/ml in one or more of the IL-13, IL-10 and IFN-g assays.
• the number of responders out of 53 Japanese Cedar pollen allergic subjects tested was calculated for each polypeptide for each of the cytokines.
• the negative control (media only) gave an IL-13 or IFN-g response of greater than 100 pg/ml in 3 subjects tested.
• the cytokine release assay showed only weak responses to the whole allergen positive control, with only 24 of the subjects having an IL-13 or IFN-g response of greater than 100 pg/ml. The poor response is suspected to be due to the PBMC preparations possibly having lost some activity in shipping/storage. The cytokine release assay is thus planned to be repeated with fresh samples of PBMCs.
• peptide CRY26 showed an IL-13 or IFN-g response of greater than 100 pg/ml in 11 of the subjects tested. This response value is well above the negative control value.
• the CRY26 peptide or a variant thereof is therefore preferred for treatment or prevention of Japanese Cedar pollen and/or Japanese Cypress pollen allergy.
• the CRY26 peptide is prepared as follows. Synthesis is performed in a solid phase peptide synthesis (SPPS) reactor and started by suspending the substituted resin in ⁇ , ⁇ -dimethylformamide (DMF). After washing of the resin with DMF, each coupling procedure is performed by addition of the N-a-protected amino acid derivative or the N-a-protected dipeptide to the preceding amino acid in the presence of N-[(1H- Benzotriazol-l-yl)(dimethylamino)methylene]-N-methylmethanaminium
• TBTU tetrafluorob orate N-oxide
• DIPEA ⁇ , ⁇ -diisopropylethylamine
• DIPEA diisopropylcarbodiimide
• HOBt 1-hydroxybenzotriazole
• an Fmoc-deprotection procedure is performed. It consists of washing of the resin with DMF, cleaving the Fmoc-group with 20% (V/V) piped dine in either DMF or l-Methyl-2-pyrrolidone (NMP), and subsequent washings with DMF and isopropanol (IP A). For each single step, the solvents and/or reagents are added, and the reaction mixture is stirred and then filtered to remove the solvents and/or reagents from the resin.
• Acetate or hydrochloride salts of the specified peptides are prepared by the following methods.
• the peptide resin is treated with cold trifluoroacetic acid (TFA) at room temperature for 1.5 to 3 hours in the presence of 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and water.
• TFA 1,2-ethanedithiol
• TMS triisopropylsilane
• the product is precipitated in cold diisopropyl ether (IPE). It is then filtered off, washed with IPE, and dried under reduced pressure.
• the product is then reconstituted and purified by high-performance liquid chromatography (UPLC).
• UPLC high-performance liquid chromatography
• the trifluoroacetate salt is reconstituted in 5% (V/V) aqueous acetic acid and loaded onto an ion exchange resin. The elution is performed with 5% (V/V) aqueous acetic acid. The acetate is filtered through a 0.2 ⁇ membrane filter and lyophilized to yield the final product as a white to off-white powder.
• the trifluoroacetate salt is reconstituted in 0.01 M HCl in purified water and filtered where necessary.
• the solution is loaded onto a preparative HPLC column for ion exchange into the hydrochloride salt.
• the ion exchange is performed by washing the column with a 0.1 M ammonium chloride solution followed by 0.01 M HCl.
• the hydrochloride is filtered through a 0.2 ⁇ membrane filter and lyophilized to yield the final product as a white to off-white powders.
• An exemplary pharmaceutical formulation of the present invention contains the CRY26 peptide.
• the peptide salt is typically an acetate or hydrochloride salt.
• the peptide salt is typically present at a nominal concentration of 40 to 220 ⁇ .
• the pharmaceutical formulation optionally further comprises one or more of L-methionine as an antioxidant (optionally at a nominal concentration of 1 to 15mM, typically 5 mM); phosphoric acid, hydrochloric acid or aqueous ammonia for pH adjustment (as required); and trehalose dihydrate as a tonicity agent (optionally at a nominal concentration of 260 mM).
• the pharmaceutical formulation is prepared in solution prior to being subjected to freeze-drying to produce a lyophilisate.

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