PEPTIDE WITH REDUCED DIMER FORMATION
The present invention relates to peptide which are formulated or engineered to prevent or reduce the formation of dimers.
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The present invention relates to peptides which are engineered or formulated to prevent or reduce the formation of dimers.
BACKGROUND OF THE INVENTIONT-cell antigen recognition requires antigen presenting cells (APCs) to present antigen fragments (peptides) on their cell surface in association with molecules of the major histocompatibility complex (MHC). T cells use their antigen specific T-cell receptors (TCRs) to recognise with high specificity the antigen fragments presented by the APC. Such recognition acts as a trigger to the immune system to generate a range of responses to eradicate the antigen which has been recognized.
Most of the specificity of T cell recognition of the antigen fragments is provided by a smaller subsequence of amino acids within the fragments. This subsequence is known as the T cell epitope. In the case of extracellular allergens and auto- or allo-antigens, the peptides are presented on MHC Class II molecules, which are recognized by CD4 T cells. Accordingly, interest in allergic and auto- or alloimmune disorders has focused on MHC Class II-binding T cell epitopes.
Given their role in the immune system, there is considerable interest in such epitopes for use as therapeutic agents to modulate the immune systems of subjects. For example, administration of peptide epitopes to subjects has been demonstrated to result in the induction of tolerance to the antigen from which the epitope derives. Therapeutic agents based on such an effect have great potential in the prevention and treatment of allergy, and auto- or allo-immune diseases where the down-regulation of an immune response is desirable.
Further progress in this area is hindered by a number of problems. Firstly, epitope sequences from allergens and auto- and allo-antigens are often poorly soluble, and are therefore problematic both to manufacture and to administer to subjects. Secondly, the majority of epitopes have typically been poorly defined. Most epitopes known in the art are loosely identified as being a core sequence present somewhere within a longer sequence, typically of approximately twenty amino acids. The core sequence itself is often not identified. In the absence of a clear definition of the core sequence an epitope, it has not been possible to modify known T cell epitopes to improve their solubility, since this risks eliminating the core residues required for T cell recognition.
SUMMARY OF THE INVENTIONPeptides comprising T cell epitopes may be prone to the formation of dimers in solution. This can result in a loss of active species and in the case of mixtures of different peptides can result in novel degradants or heterodimers that may increase IgE or IgG binding on the surface of mast cells. Dimerisation can also lead to the aggregation of peptides as insoluble precipitates. Thus, peptides comprising T cell epitopes are often unsuitable for tolerising a subject because they provoke undesirable immune responses and/or cannot be stored for long periods without forming aggregates and/or are problematic both to manufacture and to administer to subjects.
The minimal amino acid sequence of a T cell epitope required for binding to MHC Class II-binding can be precisely identified and generally comprises approximately nine amino acids. The present inventors have made the finding that by modifying specific residues within the minimal sequence of an epitope particularly prone to dimer formation, or modifying specific residues which flank the minimal sequence, it is possible to reduce dimer formation. It is also possible to reduce dimer formation by adding certain specific agents to a composition comprising the unmodified sequence of such a peptide. Thus, a composition comprising a peptide modified as above, or comprising a peptide and an agent which inhibits dimer formation, is a composition in which the peptide is present in predominantly monomeric form, and therefore has improved solubility without reducing the ability of the peptide to stimulate specific T cells and without becoming large enough to possess significant tertiary structure that would enable it to retain the conformation of an IgG or IgE-cross-linking epitope. Consequently the downstream immune responses caused by such cross-linking do not occur, and the compositions are well suited to tolerising an individual to the protein from which the peptide derives. Furthermore, the reduced dimer formation of the compositions of the invention has further advantages for the tolerisation of individuals, since peptide dimers may be more immunogenic, possibly due to cross-linking by immunoglobulins. Accordingly, the present invention provides a composition comprising:
- a) i) at least one peptide of 9 to 25 amino acids in length wherein the peptide comprises a region comprising at least one MHC Class II-binding T cell epitope; and
- ii) at least one agent which inhibits dimer formation;
or
- ii) at least one agent which inhibits dimer formation;
- b) i) at least one peptide as defined in a)i) wherein the amino acid sequence of the region has additionally been engineered to reduce dimer formation; and optionally
- ii) at least one agent which inhibits dimer formation,
wherein a minimal proportion of the peptide of the composition is present in solution as a dimer. The at least one peptide of a)i) is typically suitable for tolerisation therapy.
- ii) at least one agent which inhibits dimer formation,
It is to be understood that references to inserting, deleting, replacing amino acids herein does not require the actual physical insertion, deletion or replacement of amino acids, and instead a peptide can be synthesized comprising sequence which represents (or is the end result of) the insertion, deletion or replacement having occurred.
Amino AcidsThe table below shows the properties of amino acids. Molecular weights are shown beneath the 3-letter code for each amino acid. The molecular weights given are those of the neutral, free amino acids; residue weights can be obtained by subtraction of one equivalent of water (18 g/mol). Figures were obtained from The Merck Index, (Budavari, S., ed.) Merck & Co., Rahway, (1989).
The MHC Class II-binding T cell epitope comprised in the peptides of the invention is typically the minimal amino acid sequence that is capable of binding to Class II molecules and capable of stimulating T cells when presented to T cells in association with Class II on the cell surface. The epitope is typically one that binds to a human MHC class II molecule, such as any such molecule mentioned herein.
An MHC Class II molecule consists of two proteins, α and β, each of which is encoded by a different gene. In humans, there are three clusters of genes encoding different α and β proteins. These are the Human Leukocyte Antigen (HLA) clusters, DR, DQ and DP. Each cluster comprises multiple different A genes encoding different variant of the α protein and multiple different B genes encoding different variants of the β protein. The resulting MHC Class II heterodimers are therefore extremely diverse, and correspondingly so are the T cell epitopes that they bind.
The binding site of MHC Class II molecules is composed of two separate proteins which form a cleft. The cleft is open-ended, which in theory allows a peptide of any length to bind. However, only 9 amino acids can occupy the cleft itself. The identities of the up to 9 amino acids which occupy the cleft define whether or not a given peptide will bind to a given MHC Class II molecule and be available for presentation to T cells. These up to 9 amino acids therefore represent the minimal sequence that is required for MHC Class II-binding. It is generally assumed that such a sequence will be capable of stimulating T cells when presented to T cells in association with Class II on the cell surface. However, this may be confirmed experimentally by methods standard in the art.
Such methods may typically comprise contacting the epitope with T cells in a sample taken from a subject, under conditions which allow the epitope and the T cells to interact; and then determining whether or not any of the T cells are stimulated. Determining whether or not the T cells are stimulated may be achieved by any suitable method, for example by detecting the production of cytokines by the T cells, wherein cytokine production indicates that T cells have been stimulated. Suitable cytokines include interferon gamma, interleukin 4 and interleukin 13. Cytokine production may be detected by any suitable method, for example an ELISA, ELISPOT assay or a flow cytometric assay. The T cells in a sample from a subject are typically present in a population of peripheral blood mononuclear cells (PBMCs) isolated from a blood or serum sample taken from the subject.
The MHC Class II-binding T cell epitope of the invention typically consists of 8 or 9 amino acids, but may consist of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. The amino acid sequence of the epitope may be broadly defined by further reference to the binding site of MHC Class II molecules. This binding site has specific binding pockets, which corresponding to primary and secondary anchor positions in the sequence of the binding peptide epitope. The binding pockets are defined by amino acid positions in the sequence of the MHC Class II molecule, and are generally not absolutely discriminatory for a specific amino acid in the epitope. Therefore the peptide binding specificity of any given MHC molecule is relatively broad. Thus, peptides binding to the same MHC allotype exhibit some degree of similarity, but there is no requirement for identity.
For the most common human MHC Class II type, HLA-DR, the key anchor positions for binding to the binding pockets are at positions 1, 4, 6, 7 and 9 of the peptide epitope (counting from the most N terminal residue occupying the cleft to the most C terminal). Different HLA-DR alleles which have similar amino acids in their binding pockets therefore typically bind peptides with similar amino acids at positions 1, 4, 6, 7 and 9. Accordingly, the region containing an MHC Class II binding T cell epitope preferably has amino acids at positions corresponding to positions 1, 4, 6, 7 and 9 that allow binding to the widest range of HLA-DR alleles. Examples of characteristic binding properties of different HLA-DR alleles are set out below:
DR alleles with Glycine at position 86 of the β chain show strong preferences for large hydrophobic side chains (Trp, Tyr, Phe) at peptide position 1, whereas Valine at position 86 restricts the pocket size and alters the preferences to small hydrophobic side chains (Val and Ala) at this position. Medium sized hydrophobic amino acids Leu and Ile are well accepted in all DR alleles.
DR alleles with Gln at position 70, Lysine at position 71, and Arginine or Gln at position 74 of the β chain have an overall positive charge within pocket 4, which requires negatively charged amino acids Asp and Glu at position 4 of the binding peptide (as in for example, DRB1*0301). DR alleles with this motif are associated with two autoimmune diseases: systematic lupus erythematosus and Hashimoto's thyroiditis.
DR alleles with Gln or Arg at position 70, Arg or Lys at position 71 and Glu or Ala at position 74 of the β chain bind similar peptides to those directly above since the only significant difference is at position 74. However, when Ala is present at position 74, pocket 4 increases in size and can accommodate larger amino acids such as Phe, Trp, and Ile (as in for example DRB1*0401, 04, 05). Alleles bearing Glu at position 74 are expected to allow small polar residues, like Ser and Thr at position 4 of the binding peptide. DR alleles with this motif are associated with a susceptibility to rheumatoid arthritis.
DR alleles with Asp at position 70, Glu or Arg at position 71, and Leu or Ala at position 74 of the β chain exclude peptides with negatively charged amino acids at peptide position 4 (for example DRB1*0402). This is due to the presence of Asp at position 70. DR alleles with this motif are associated with the autoimmune diseases Juvenile rheumatoid arthritis (JRA), pemphigus vulgaris, and allergic bronchopulmonary disease/syndrome.
Polymorphisms at position 9 of the β chain define the size of binding pocket 9 in all DR alleles. Alleles with Trp at this position accept only small amino acids in position 9 of the binding peptide, e.g. Ala, Val, Gly, Ser, Thr, Pro (as in for example DRB1*0101 and *1501). Glu at position 9, in combination with Asp at position 57, makes pocket 9 negatively charged, facilitating the accommodation of positively charged amino acids, such as Lys (as in for example DRB1*0401 and *0404) and Histine (as in for example DRB1*0402). In most MHC class II alleles, Asp at position 57 makes a salt-bridged hydrogen bond with Arg at position 76, allowing the pocket to also accommodate aliphatic and polar amino acids. In cases where Asp at position 57 is replaced by Ser (for example DRB1*0405) or Ala (DQ8), the hydrogen bonding network is destroyed and Arg at position 76 can strongly attract negatively charged amino acids such as Asp or Glu at position 9 of the binding peptide (as in for example DRB1*0405).
An example of a preferred sequence for an epitope therefore has Trp, Tyr, Phe, Val or Ala at position 1; Asp, Glu, Ser or Thr at position 4; and Ala, Val, Gly, Ser, Thr, Pro at position 9. A further example of a preferred sequence for an epitope has a large aromatic or hydrophobic amino acid at position 1, for example Tyr, Phe, Trp, Leu, Ile or Val, and a small, non-charged amino acid at position 6, for example Ser, Thr, Ala, Pro, Val, Ile or Met. Approximately 87.5% of peptides binding to all or a combination of the MHC Class II molecules encoded by the DRB1*0101, *0401 and *0701 alleles contain this motif. Furthermore, since T cell epitopes derived from allergens and autoimmune antigens do not typically contain a large number of repeats of a given amino acid or amino acids, preferred epitopes of the invention typically comprise at least 5, 6, 7 or 8 different amino acids.
The precise amino sequence of an epitope may be predicted by computer-based algorithms and confirmed by in vitro biochemical analysis. Suitable commercially available algorithms include the EpiMatrix algorithm (EpiVax Inc.). Other algorithms are available at, for example http://www.imtech.res.in/raghava/propred/ and http://www.imtech.res.in/raghava/mhc2pred/. Analysis with these algorithms typically comprises parsing a larger polypeptide sequence into multiple overlapping small peptides. The sequences of these small peptides are then analysed using the algorithm to identify those which are predicted to bind MHC Class II molecules. The overlapping small peptides are typically 9-mers.
The candidate peptides which score most highly in this analysis are then assessed for the ability to bind a panel of MHC Class II molecules encoded by different Class II alleles in vitro using standard binding assays. For example a competitive MHC class II binding assay may be used, wherein each peptide is analysed for its ability to displace a known control binder from each of the human MHC class II allotypes investigated. In such an assay each peptide is assigned an IC50 value (the concentration at which 50% inhibition of control peptide binding is achieved). The lower the IC50 the higher the affinity of a peptide for a given MHC class II allotype.
The epitope or epitopes in a polypeptide are taken to be those peptides which show the highest binding affinity to MHC Class II molecules. Particularly preferred epitopes show high affinity binding to different Class II molecules encoded by more than one preferably two, more preferably three, four or five MHC Class II alleles.
Particularly preferred epitopes are those which are comprised in regions which are prone to dimer formation, as defined below.
Regions Containing at Least One MHC Class II-Binding T Cell EpitopeBiochemical assays for the identification of a T cell epitope are not typically able to define the position of the minimal epitope sequence within a larger sequence more accurately than to within approximately 12 amino acids, and more typically 15, 20 or more amino acids. The reason for this is that a large sequence must be physically fragmented into smaller overlapping peptides, or smaller overlapping peptides must be manufactured de novo prior to in vitro assessment of the ability of these peptides to bind MHC Class II molecules. The skilled person will recognise that the smaller the overlapping peptide fragments used, the more time-consuming and labour intensive is the process of manufacture. Hence epitopes are often identified as being contained within a larger polypeptide region. It is envisaged that the peptides of the invention may comprise such a larger region.
Accordingly, in the peptides of the invention, the region containing an MHC Class II-binding T cell epitope is typically 8 or 9 amino acids in length, but may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.
The region of the invention is typically a sequence which is prone to dimer formation. This will be understood to include both homodimer formation (i.e. association of peptide monomers with other identical peptide monomers) and heterodimer formation (i.e. association of peptide monomers with different peptide monomers). It will also be understood that by a sequence prone to dimer formation, it is also intended to refer to sequences which are prone to form higher order oligomers, such as trimers, tetramers and the like. The region of the invention may comprise or consist of any sequence which is prone to dimer formation. The particular amino acid sequence within a given region which promotes dimer formation may be comprised within the minimal MHC class II-binding sequence of the T cell epitope, or may be comprised within the residues which flank this sequence. The sequence prone to dimer formation may thus consist entirely of the minimal MHC class II-binding sequence of the T cell epitope.
Particularly preferred sequences comprise at least one cysteine residue. The skilled person will appreciate that any peptide that contains a single cysteine residue may form dimers, either with itself, or with other cysteine containing peptides with which it may be contacted. Peptides that contain two or more cysteines have the potential to form long chains which may then aggregate. Such dimer/aggregate formation leads to the risk of IgE or IgG binding and thus having a local inflammatory response. Accordingly, a preferred region of the invention typically derives from a protein with a high proportion of cysteine residues. For example, the region of the invention may derive from a protein having greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35% cysteine residues as a proportion of the total number of amino acid residues in the protein. The region of the invention is preferably selected from a sequence within such a protein that has a lower proportion of cysteine residues. Accordingly, the region may comprise up to a maximum of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% cysteine residues as a proportion of the total number of amino acid residues in the region. The cysteine residues may be comprised in the minimal MHC Class II-binding sequence of the epitope, or may be comprised in the residues which flank this sequence.
Other sequences prone to dimer formation may be identified by in silico analysis using suitable computational methods, or by in vitro analysis using suitable laboratory methods which quantify the proportion of a sequence which is present in monomeric or dimeric form as set out below. For a sequence that is prone to dimerisation the proportion of sequence present as a dimer may be minimal, i.e. less than about 0.5% or 1% in the solid state, but this will typically increase over time to at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% for material stored in solution for a suitable period of time under suitable conditions. Suitable periods of time and conditions include ranges of time and conditions under which a skilled practitioner might reasonably expect to keep a sequence in solution prior to use. For example, periods of time of about 24 hours, about 48 hours, or about 72 hours are typical, although some solutions may be kept for longer periods for example, at least a week, a month, 6 months, 1 year, 2 years, 3 years or more. Storage conditions may typically be room temperature and relative humidity, or typically 25° C. and 60% relative humidity, but could include any standard storage conditions encountered by the skilled person, for example approximately 4° C., −20° C., or −80° C.
The sensitivity of the immune system is such that only a small proportion of dimer is considered likely to trigger an undesirable immune response.
For the assessment of the proportion of a sequence present in a given form a suitable method is, for example, analytical gel electrophoresis under non-denaturing conditions. In such a method, a solution of the sequence is run in a polyacrylamide gel, alongside a set of standard molecular weight markers. If the sequence forms dimers, a protein band will be observed in the gel corresponding to a species with a molecular weight approximately twice that calculated for the sum of the amino acids of the sequence. (Similarly, any trimers or tetramers present will be observed as bands corresponding to species with molecular weights approximately three or four times that calculated for the sum of the residue weights of an amino acids of the sequence). Since it is rare that 100% of a sequence is present in oligomeric form, a second band may also be observed corresponding to a species with approximately the molecular weight calculated for the sum of the amino acids of the sequence—this represents the sequence in monomeric form. The relative intensities of the bands may be used to quantify the proportion of the sequence which is present in each form. Similar methods may assess molecular weight by alternative means, for example, analytical centrifugation, mass spectrometry or size exclusion chromatography. Alternatively, oligomers may be quantified using reverse phase high performance liquid chromatography (RP-HPLC) where the dimers and higher oligomeric species are separated from the monomers based on differences in their hydrophobicities. Identification of the species is achieved using mass spectrometric detection. The same methods may be adapted to assess whether a given peptide shows a tendency to heterodimerise with any other peptide or molecule.
Additionally, the region of the invention may have a solubility of less than 3.5 mg/ml in aqueous solution at pH 2.0 to 12.0, or pH 2.0 to 11.0, pH 2.0 to 10.0, pH 2.0 to 9.0, pH 2.0 to 8.0 or pH 2.0 to 7.0; and/or comprise 1, 2, 3 or 4 cysteine residues; and/or have an isoelectric point lower than 4.5; and/or have a GRAVY score above +0.25. These parameters may be assessed by any suitable method. For example, solubility may be assessed by standard in vitro methods, GRAVY and isoelectric point may be assessed in silico using suitable computational methods, such as the ProtParam tool (Gasteiger E. et al pp. 571-607 The Proteomics Protocols Handbook, Humana Press (2005); John M. Walker (ed)) which is available at http://www.expasy.ch/tools/protparam.html.
PeptidesThe peptide of the invention may comprise or consist of the native sequence of the region as defined above or may comprise or consist of the native sequence of the region engineered to reduce dimer formation. The region is engineered by the modification of its native sequence. Particularly preferred modifications are wherein:
at least one cysteine residue in the native sequence of the region is replaced with serine, 2-aminobutyric acid, alanine or glycine; and/or
at least one cysteine residue in the native sequence of the region is cysteinylated to create a cystine residue; and/or
The residue or residues which are modified may be comprised in any part of the sequence of the region. In one embodiment the residue or residues which are modified are not comprised in the minimal MHC class II-binding sequence of the region. In a preferred embodiment, the modification does not create a new epitope or affect the MHC class II-binding properties of the region.
The peptide of the invention typically contains from 9 to 25 amino acids, and may contain 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids. It will be appreciated that the peptide of the invention may consist entirely of the region as defined above, or may comprise additional amino acids flanking the region upto a maximum of 25 amino acids, provided that the additional amino acids do not promote dimer formation. Additional amino acids which promote dimer formation may be assessed by the methods described in the “regions” section above.
Peptides longer than 25 amino acids are likely to possess sufficient tertiary structure to cross-link IgG or IgE on cell surfaces resulting in undesirable immune responses such as B cell activation or mast cell degranulation.
Peptide SynthesisThe peptides of the invention are derived in an intellectual sense from the polypeptide which comprises the region as defined above. This is done by making use of the amino acid sequence of the region and synthesising peptides based on the sequence. Peptides may be synthesised using methods well known in the art. Preferred methods include solid-phase peptide synthesis techniques and most preferably an automated or semiautomated peptide synthesizer. Typically, using such techniques, an α-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of a base such as diisopropyl-ethylamine. The α-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, and include t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).
The term “peptide” 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 pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al (1997) show that, at least for MHC class II and T helper cell responses, these pseudopeptides are useful. Retro-inverse peptides, which contain NH—CO bonds instead of CO—NH peptide bonds, are much more resistant to proteolysis.
Similarly, the 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. For example, the N-terminal amino group of the peptides 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. Other examples of 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 (—NH2→—NH(Me) or —N(Me)2).
Analogues of peptides according to the invention may also include peptide variants that increase or decrease the peptide's half-life in vivo. Examples of analogues capable of increasing the half-life of peptides used according to the invention include peptoid analogues of the peptides, D-amino acid derivatives of the peptides, and peptide-peptoid hybrids. A further embodiment of the variant 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.
CompositionsThe composition of the invention typically comprises:
-
- a) i) at least one peptide, wherein the peptide comprises the native sequence of a region as defined above; and
- ii) at least one agent which inhibits dimer formation;
- a) i) at least one peptide, wherein the peptide comprises the native sequence of a region as defined above; and
or
-
- b) i) at least one peptide, wherein the peptide comprises a region as defined above which has been engineered as defined above to reduce dimer formation; and optionally
- ii) at least one agent which inhibits dimer formation,
wherein a minimal proportion of the peptide is present in solution as a dimer.
- ii) at least one agent which inhibits dimer formation,
- b) i) at least one peptide, wherein the peptide comprises a region as defined above which has been engineered as defined above to reduce dimer formation; and optionally
Agents suitable for inhibiting dimer formation include agents suitable for reducing a disulfide bond, antioxidant agents or preservative agents. Suitable reducing agents include any trialkylphosphine compound, including tris(2-carboxyethyl)phosphine (TCEP), 2-Mercaptoethanol and dithiothreitol (DTT). Other suitable agents include thioglycerol, thioanisole, glutathione and cysteine. Particularly preferred compositions of the invention comprise 0.5% thioglycerol or 0.5% thioanisole.
The agent suitable for inhibiting dimer formation may be an agent which promotes cysteinylation of cysteine residues, such as cysteine, particularly cysteine hydrocholoride. The agent suitable for inhibiting dimer formation may be temporarily added to the composition and then removed. In one such embodiment, the agent is an agent which eliminates or reduces the presence of oxidising agents in a composition, since disulfide bond formation is dependent on the presence of oxidising agents. Preferred agents of this type are nitrogen, argon or other inert gases, which may be pulsed through the composition.
An example of a suitable composition of the invention comprises:
The above values are based on a typical 400 g batch comprising at least one peptide.
By a minimal proportion of peptide present in solution as a dimer it is meant that a maximum of 5%, 4%, 3%, 2% or 1% is present in solution as a dimer. It will be understood that the proportion of peptide present as a dimer in solution will be the proportion present as a dimer following a suitable period of time in solution. Suitable periods of time include ranges of time that a skilled practitioner might reasonably expect to keep a sequence in solution prior to use. For example, about 24 hours, about 48 hours, or about 72 hours. The proportion of a peptide present in a given form may be assessed by any suitable method as described in the “Regions” section above.
Where the epitope derives from an allergen, the compositions of the invention are typically capable of inducing a late phase response in an individual that is sensitised to the allergen. The term “late phase response” includes the meaning as set forth in Allergy and Allergic Diseases (1997) A. B. Kay (Ed.), Blackwell Science, pp 1113-1130. The late phase response may be any late phase response (LPR). Preferably, the compositions comprising an epitope derived from a protein allergen are capable of inducing a late asthmatic response (LAR) or a late rhinitic response, or a late phase skin response or a late phase ocular response. Whether or not a particular composition can give rise to a LPR can be determined using methods well known in the art; a particularly preferred method is that described in Cromwell O, Durham S R, Shaw R J, Mackay J and Kay A B. Provocation tests and measurements of mediators from mast cells and basophils in asthma and allergic rhinitis. In: Handbook of Experimental Immunology (4) Chapter 127, Editor: Weir D M, Blackwell Scientific Publications, 1986. Thus, preferably, the individual compositions of the invention are able to induce a LPR in an individual who has been sensitised to the protein allergen from which the epitope derives.
Whether or not an individual has been sensitised to the protein from which the epitope derives may be determined by well known procedures such as the detection of antibodies in the individual's blood or serum which are specific for the protein. Where the epitope derives from an allergen, suitable tests for sensitisation to the allergen include skin prick testing with solutions of protein extracts, induction of cutaneous LPRs, clinical history, allergen challenge and radioallergosorbent test (RAST) for measurement of protein specific IgE. Whether or not a particular individual is expected to benefit from treatment may be determined by the physician based, for example, on such tests or determinations.
Desensitising or tolerising an individual to the protein from which the epitope derives means inhibition or dampening of immunological tissue reactions induced by said protein in appropriately sensitised individuals. It has been shown that 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 protein. The desensitisation manifests itself as a reduction in response to a protein or protein-derived peptide, or preferably an elimination of such a response, on second and further administrations of the protein or protein-derived peptide. The 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 two weeks is preferred.
Although the compositions of the invention are able to induce a LPR in an individual who has been sensitised to the protein, it should be appreciated that when a composition is used to treat a patient it is preferable that a sufficiently low concentration of the composition is used such that no observable LPR will occur but the response will be sufficient to partially desensitise the T cells such that the next (preferably higher) dose may be given, and so on. In this way the dose is built up to give full desensitisation but often without ever inducing a LPR in the patient. Although, the composition or peptide is able to do so at a higher concentration than is administered.
The composition of the invention typically has a reduced ability to provoke an early phase response in an individual. By “reduced ability to provoke an early phase response”, it will be understood that the composition of the invention will result in a lower severity of early phase symptoms (such as basophil or mast cell degranulation) relative to a composition comprising a peptide comprising the same region as that in the composition of the invention, but without modification of its sequence to reduce dimer formation, and lacking an agent which reduces dimer formation. Accordingly, the composition of the invention will produce a lesser early phase response than an equivalent peptide predominantly present in dimeric form. The peptide is equivalent because it comprises the same MHC Class II-binding T cell epitope.
Alternatively or additionally, the composition of the invention typically has an improved ability to induce tolerance in an individual. By “improved ability to induce tolerance”, it will be understood that the composition of the invention will produce a greater level of desensitisation in an individual than a composition comprising a peptide comprising the same region as that in the composition of the invention, but without modification of its sequence to reduce dimer formation, and lacking an agent which reduces dimer formation. Accordingly, the composition of the invention will produce a greater level of desensitisation than an equivalent peptide predominantly present in dimeric form. The peptide is equivalent because it comprises the same MHC Class II-binding T cell epitope.
Desensitisation is as defined above, and its level may be characterised by any suitable means. For example, in allergic asthma, a smaller LAR produced in response to inhalation of the protein from which the epitope derives (or a protein-derived peptide) would indicate a greater level of desensitisation following treatment with the composition of the invention. The size of a LAR can be assessed by any suitable means in the art, for example, detection of the reduction in Forced Expired Volume (FEV) of an individual post-administration of protein. A greater reduction in FEV indicates a larger LAR. The composition of the invention preferably results in an LAR at least 10%, 20%, 30%, 40% or 50% smaller than a composition comprising an equivalent peptide predominantly present in dimeric form.
Alternatively, a greater level of desensitisation may be indicated by a greater reduction in the protein-specific production by T cells of inflammatory cytokines such as interferon gamma, interleukin 4 and interleukin 13. Cytokine production by T cells may be detected by any suitable method, for example an ELISA, ELISPOT assay or flow cytometric assay. Particularly preferred methods include Multiplex bead array assays as described in, for example de Jager et al; Clinical and Diagnostic Laboratory Immunology, 2003, Vol 10(1) p. 133-139. By “a greater reduction”, it is preferred that treatment with the composition of the invention will result in the production of preferably at least 10%, 20%, 30%, 40% or 50% less inflammatory cytokines than a composition comprising an equivalent peptide predominantly present in dimeric form.
Preferred compositions of the invention comprise at least one peptide comprising or consisting of the sequence corresponding to any one of SEQ ID NOS: 1 to 71 and optionally thioglycerol. Particularly preferred compositions comprise at least a first and a second peptide, wherein the first and second peptide each comprise or consist of a different sequence selected from the sequences of SEQ ID NO: 37 (MLA01), SEQ ID NO: 38 (MLA04), SEQ ID NO: 39 (MLA05), or SEQ ID NO: 40 (MLA12). For example, the first and second peptide may comprise or consist of the sequences of a) SEQ ID NOS: 37 (MLA01) and 38 (MLA04); b) SEQ ID NOS: 37 (MLA01) and 39 (MLA05); c) SEQ ID NOS: 37 (MLA01) and 40 (MLA12); d) SEQ ID NOS: 38 (MLA04) and 39 (MLA05); e) SEQ ID NOS: 38 (MLA04) and 40 (MLA12); or SEQ ID NOS: 39 (MLA05) and 40 (MLA12), respectively.
Polynucleotides, Vectors and CellsThe terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of 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. A polynucleotide of the invention may be provided in isolated or purified form. A nucleic acid sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence.
Polynucleotides of the invention 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).
The polynucleotide molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the peptide of the invention in vivo in a targeted subject. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors) which are suitable for use as reagents for nucleic acid immunization. Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a peptide of the invention.
The present invention thus includes expression vectors that comprise such polynucleotide sequences. Thus, the present invention provides a vector for use in preventing or treating allergy by tolerisation comprising one or more polynucleotide sequences which encode different polypeptides of the invention and optionally one or more further polynucleotide sequences which encode different polypeptides as defined herein.
Furthermore, it will be appreciated that the compositions and products of the invention may comprise a mixture of polypeptides and polynucleotides. Accordingly, the invention provides a composition or product as defined herein, wherein in place of any one of the polypeptide is a polynucleotide capable of expressing said polypeptide.
Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a peptide of the invention. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al.
Thus, a polypeptide of the invention may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given regulatory sequence, such as a promoter, operably linked to a nucleic acid sequence is capable of effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleic acid sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
A number of expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. The vectors of the invention may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. A “plasmid” is a vector in the form of an extrachromosomal genetic element. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example to allow in vivo expression of the polypeptide.
A “promoter” is a nucleotide sequence which initiates and regulates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.
A polynucleotide, expression cassette or vector according to the present invention may additionally comprise a signal peptide sequence. The signal peptide sequence is generally inserted in operable linkage with the promoter such that the signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding sequence also in operable linkage with the promoter.
Typically a signal peptide sequence encodes a peptide of 10 to 30 amino acids for example 15 to 20 amino acids. Often the amino acids are predominantly hydrophobic. In a typical situation, a signal peptide targets a growing polypeptide chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved off in the endoplasmic reticulum, allowing for secretion of the polypeptide via the Golgi apparatus. Thus, a peptide of the invention may be provided to an individual by expression from cells within the individual, and secretion from those cells.
Alternatively, polynucleotides of the invention may be expressed in a suitable manner to allow presentation of a peptide of the invention by an MHC class II molecule at the surface of an antigen presenting cell. For example, a polynucleotide, expression cassette or vector of the invention may be targeted to antigen presenting cells, or the expression of encoded peptide may be preferentially stimulated or induced in such cells.
Polynucleotides of interest may be used in vitro, ex vivo or in vivo in the production of a peptide of the invention. Such polynucleotides may be administered or used in the prevention or treatment of allergy to cats by tolerisation.
Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859 and 5,589,466. 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. For example, 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 re-introduced 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.”
The polypeptides, polynucleotides, vectors or cells of the invention may be present in a substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.
FormulationsThe peptides, polynucleotides, vectors and cells of the invention may be provided to an individual either singly or in combination. Each molecule or cell of the invention may be provided to an individual in an isolated, substantially isolated, purified or substantially purified form. For example, a peptide of the invention may be provided to an individual substantially free from the other peptides.
Whilst it may be possible for the peptides, polynucleotides or compositions according to the invention to be presented in raw form, it is preferable to present them as a pharmaceutical formulation. Thus, according to a further aspect of the invention, the present invention provides a pharmaceutical formulation for tolerising an individual to a protein from which a peptide of the invention derives, comprising a composition, vector or product according to the invention together with one or more pharmaceutically acceptable carriers or diluents and optionally one or more other therapeutic ingredients. The carrier (s) must be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation (in particular they must not promote dimer formation) and not deleterious to the recipient thereof. Typically, carriers for injection, and the final formulation, are sterile and pyrogen free.
For example, compositions containing one or more molecules or cells of the invention can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, antioxidants, chelating agents and the like, may be present in the excipient or vehicle. These 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 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. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution solution or a powder for reconstitution. 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 an aqueous solution (including 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.
Other parentally-administrable compositions 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. Compositions 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.
Alternatively, the peptides or polynucleotides 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 and genetically engineered polymers such as silk-elastin like polymers (Ghandehari and Cappello (1998) Pharm. Res. 15: 813-815).
Also, the peptides may be formulated at high concentrations >100 nmol/mL with dimethyl sulphoxide, polyethylene oxide, polyethylene glycol or other suitable excipients for use with implantable drug delivery devices.
The formulation of any of the peptides, polynucleotides or cells mentioned herein will depend upon factors such as the nature of the substance and the method of delivery. Any such substance 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), parenterally, subcutaneously, by inhalation, intravenously, intramuscularly, intrasternally, transdermally, intradermally, sublingually, instranasally, buccally or by infusion techniques. The substance may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular individual.
The compositions of formulations of the invention will comprise a suitable concentration of each peptide/polynucleotide/cell to be effective without causing adverse reaction. Typically, the concentration of each peptide in the composition will be in the range of 0.03 to 200 nmol/ml. More preferably in the range of 0.3 to 200 nmol/ml, 3 to 180 nmol/ml, 10 to 150 nmol/ml or 30 to 120 nmol/ml. The composition or formulations should have a purity of greater than 95% or 98% or a purity of at least 99%.
A composition may therefore be formulated which comprises a molecule and/or cell of the invention and also one or more other therapeutic molecules. A composition of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic compositions as part of a combined treatment.
Therapeutic Methods and Individual to be TreatedThe present invention relates to compositions comprising peptides that are capable of desensitising or tolerising human individuals to proteins from which the peptides of the invention derive. Such proteins are typically allergens or other antigens to which an immune response is undesirable. Examples of such antigens include antigens associated with autoimmune diseases, antigens associated with graft-versus-host disease or transplant rejection (herein referred to as alloimmune conditions) and antigens associated with maternal-foetal immune responses, for example Rhesus D Haemolytic Disease of the Newborn. The compositions of the invention are therefore useful in the prevention or treatment an allergic disease, an autoimmune disease, an alloimmune condition or a maternal-foetal immune response. The invention provides compositions, products, vectors and formulations for use in preventing or treating the above conditions. The invention also provides a method of preventing or treating a subject having the above conditions, comprising administering, either singly or in combination the polypeptides/polynucleotides/cells of the invention as described above.
The individual to be treated or provided with the composition or formulation of the invention is preferably human. It will be appreciated that the individual to be treated may be known to be sensitised to the particular allergen or antigen, at risk of being sensitised or suspected of being sensitised. The individual can be tested for sensitisation using techniques well known in the art and as described herein. Alternatively, the individual may have a family history of the conditions described above. It may not be necessary to test an individual for sensitisation to allergens because the individual may display symptoms of allergy when brought into proximity to a suitable allergen source. 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 source. Symptoms of allergy can include itchy eyes, runny nose, breathing difficulties, red itchy skin or rash. The individual to be treated 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. Preferably, the individual to be treated is from a population that has MHC allele frequencies within the range of frequencies that are representative of the Caucasian population. Reference population allele frequencies for 11 common DRB 1 allele families are shown in Table 1 (Data from HLA Facts Book, Parham and Barber).
Reference frequencies were obtained by analysis of multiple studies reporting frequencies and the figures shown are mean values. Preferably therefore, the individual to be treated is from a population that has equivalent MHC allele frequencies as the reference population for the alleles referred to Table 1 (such as for at least 1, 2, 3, 4, 5 or all of the alleles), for example within the ranges of those figures plus or minus 1, 2, 3, 5, 10, 15 or 20%.
Preferably the individual is from a population where the allele frequencies of the following DRB1 alleles is:
4—at least 9%
7—at least 10%
11—at least 8%.
The individual to be treated for allergic disease may have had allergy for at least 2 weeks, 1 month, 6 months, 1 year or 5 years. The individual may suffer from a rash, nasal congestion, nasal discharge and/or coughing caused by the allergy. The individual may or may not have been administered with other compositions/compounds which treat allergy.
The invention is particularly suitable for use with individuals who may need to receive multiple administrations of the compositions of the invention as described above. Peptides which are more prone to dimer formation than the peptides of the invention are more likely to induce an adverse response in an individual receiving multiple administrations. Since monomeric peptides are less immunogenic than dimeric peptides, the invention is also particularly suitable for administration to an individual who has or is at risk of a condition, wherein the condition is characterised by an adverse inflammatory reaction to a treatment comprising a peptide. An adverse inflammatory reaction to a treatment comprising a peptide may be diagnosed as a result of the onset of any of the symptoms of allergy as defined above following administration of a treatment comprising a peptide. An individual may be considered to be at risk of such a reaction for any suitable medical reason, for example, a family history of similar reactions, a personal medical history of multiple allergic responses, or strongly positive skin prick or skin patch responses to common allergens.
Allergens and AntigensSuitable allergens from which the region containing a MHC Class II-binding T cell epitope may derive can of course be obtained and/or produced using known methods. Classes of suitable allergens include, but are not limited to, pollens, animal dander (in particular cat dander), grasses, molds, dusts, antibiotics, stinging insect venoms, and a variety of environmental (including chemicals and metals), drug and food allergens. Common tree allergens include pollens from cottonwood, poplar, ash, birch, maple, oak, elm, hickory, and pecan trees; common plant allergens include those from mugwort, ragweed, English plantain, sorrel-dock and pigweed; plant contact allergens include those from poison oak, poison ivy and nettles; common grass allergens include rye grass, Timothy, Johnson, Bermuda, fescue and bluegrass allergens; common allergens can also be obtained from molds or fungi such as Candida, Alternaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor and thermophilic actinomycetes; epidermal allergens can be obtained from house or organic dusts (typically fungal in origin), from arthropods such as house mites (Dermatophagoides pteronyssinus), or from animal sources such as feathers, and dog dander; common food allergens include milk and cheese (diary), egg, wheat, nut (e.g., peanut), seafood (e.g., shellfish), pea, bean and gluten allergens; common environmental allergens include metals (nickel and gold), chemicals (formaldehyde, trinitrophenol and turpentine), Latex, rubber, fiber (cotton or wool), burlap, hair dye, cosmetic, detergent and perfume allergens; common drug allergens include local anesthetic and salicylate allergens; antibiotic allergens include penicillin, tetracycline and sulfonamide allergens; and common insect allergens include bee, wasp and ant venom, and cockroach calyx allergens. Particularly well characterized allergens include, but are not limited to, the major allergen produced by the domestic cat Felis catus (Fells domesticus) glycoprotein Fel d1, the major and cryptic epitopes of the Der p I allergen (Hoyne et al. (1994) Immunology 83190-195), bee venom phospholipase A2 (PLA) (Akdis et al. (1996) J. Clin. Invest. 98:1676-1683), birch pollen allergen Bet v 1 (Bauer et al. (1997) Clin. Exp. Immunol. 107:536-541), and the multi-epitopic recombinant grass allergen rKBG8.3 (Cao et al. (1997) Immunology 90:46-51). These and other suitable allergens are commercially available and/or can be readily prepared as extracts following known techniques.
Preferably, the allergen is selected from the list of allergen sequences and database accession numbers (NCBI Entrez accession numbers) below. NCBI is the National Center for Biotechnology information and is a division of the US National Institutes of Health. The NCBI web site, from which access to the database may be sought, is www.ncbi.nlm.nih.gov/. Allergen sequences and database accession numbers (NCBI Entrez accession numbers):
Additional mite allergen sequences (NCBI entrez accession):
1170095; 1359436; 2440053; 666007; 487661; 1545803; 84702; 84699; 625532; 404370; 1091577; 1460058; 7413; 9072; 387592.
Felis sequences (NCBI entrez accession):
539716; 539715; 423193; 423192; 423191; 423190; 1364213; 1364212; 395407; 163827; 163823; 163825; 1169665; 232086; 1169666.
Additional Hevea sequences (NCBI entrez accession):
3319923; 3319921; 3087805; 1493836; 1480457; 1223884; 3452147; 3451147; 1916805; 232267; 123335; 2501578; 3319662; 3288200; 1942537; 2392631; 2392630; 1421554; 1311006; 494093; 3183706; 3172534; 283243; 1170248; 1708278; 1706547; 464775; 2661042; 231586; 123337; 116359; 123062; 2213877; 542013; 2144920; 1070656; 2129914; 2129913; 2129912; 100135; 82026; 1076559; 82028; 82027; 282933; 280399; 100138; 1086972; 108697; 1086976; 1086978; 1086978; 1086976; 1086974; 1086972; 913758; 913757; 913756; 234388; 1092500; 228691; 1177405; 18839; 18837; 18835; 18833; 18831; 1209317; 1184668; 168217; 168215; 168213; 168211; 168209; 348137.
Additional Lolium sequences (NCBI entrez accession):
135480; 417103; 687261; 687259; 1771355; 2388662; 631955; 542131; 542130; 542129; 100636; 626029; 542132; 320616; 320615; 320614; 100638; 100634; 82450; 626028; 100639; 283345; 542133; 1771353; 1763163; 1040877; 1040875; 250525; 551047; 515377; 510911; 939932; 439950; 2718; 168316; 168314; 485371; 2388664; 2832717; 2828273; 548867.
Additional Parietaria sequences (NCBI entrez accession):
543659; 1836011; 1836010; 1311513; 1311512; 1311511; 1311510; 1311509; 240971.
Additional Phleum sequences (NCBI entrez accession):
458878; 548863; 2529314; 2529308; 2415702; 2415700; 2415698; 542168; 542167; 626037; 542169; 541814; 542171; 253337; 253336; 453976; 439960.
Additional vespula sequences (NCBI entrez accession):
549193; 549192; 549191; 549190; 5491104; 117414; 126761; 69576; 625255; 6271104; 627188; 627187; 482382; 112561; 627186; 627185; 1923233; 1047645; 1047647; 745570; 225764; 162551.
Additional tree allergen sequences (NCBI entrez accession number):
131919; 128193; 585564; 1942360; 2554672; 2392209; 2414158; 1321728; 1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3015520; 2935416; 464576; 1705843; 1168701; 1168710; 1168709; 1168708; 1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1842188; 2564228; 2564226; 2564224; 2564222; 2564220; 2051993; 18131041; 15368104; 534910; 534900; 5341048; 1340000; 1339998; 2149808; 66207; 2129477; 1076249; 1076247; 629480; 481805; 81443; 1361968; 1361967; 1361966; 1361965; 1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 629483; 629482; 629481; 541804; 320545; 81444; 541814; 629484; 474911; 452742; 1834387; 298737; 298736; 1584322; 1584321; 584320; 1542873; 1542871; 1542869; 1542867; 1542865; 1542863; 1542861; 1542859; 1542857; 1483232; 1483230; 1483228; 558561; 551640; 488605; 452746; 452744; 452740; 452738; 452736; 452734; 452732; 452730; 452728; 450885; 17938; 17927; 17925; 17921; 297538; 510951; 2104331; 2104329; 166953.
Additional dog allergen protein (NCBI entrez accession):
1731859
Additional cockroach sequences (NCBI Entrez accession numbers):
2580504; 1580797; 1580794; 1362590; 544619; 544618; 15315104; 1580792; 1166573; 1176397; 21047849.
2739154; 3719257; 3703107; 3687326; 3643813; 3087805; 1864024; 1493836; 1480457; 25910476; 25910474; 1575778; 763532; 746485; 163827; 163823; 3080761; 163825; 3608493; 3581965; 2253610; 2231297; 21047849; 3409499; 3409498; 3409497; 3409496; 3409495; 3409494; 3409493; 3409492; 3409491; 3409490; 34094104; 3409488; 3409487; 3409486; 3409485; 3409484; 3409483; 3409482; 3409481; 3409480; 3409479; 3409478; 3409477; 3409476; 3409475; 3409474; 3409473; 3409472; 3409471; 3409470; 3409469; 3409468; 3409467; 3409466; 3409465; 3409464; 3409463; 3409462; 3409461; 3409460; 3409459; 3409458; 3409457; 3409456; 3318885; 3396070; 3367732; 1916805; 3337403; 2851457; 2851456; 1351295; 549187; 136467; 1173367; 2499810; 2498582; 2498581; 1346478; 1171009; 126608; 114091; 2506771; 1706660; 1169665; 1169531; 232086; 4161048; 114922; 2497701; 1703232; 1703233; 1703233; 1703232; 3287877; 3122132; 3182907; 3121758; 3121756; 3121755; 3121746; 3121745; 3319925; 3319923; 3319921; 3319651; 33187104; 3318779; 3309647; 3309047; 3309045; 3309043; 3309041; 3309039; 3288200; 3288068; 2924494; 3256212; 3256210; 3243234; 3210053; 3210052; 3210051; 3210050; 3210049; 3210048; 3210047; 3210046; 3210045; 3210044; 3210043; 3210042; 3210041; 3210040; 3210039; 3210038; 3210037; 3210036; 3210035; 3210034; 3210033; 3210032; 3210031; 3210030; 3210029; 3210028; 3210027; 3210026; 3210025; 3210024; 3210023; 3210022; 3210021; 3210020; 3210019; 3210018; 3210017; 3210016; 3210015; 3210014; 3210013; 3210012; 3210011; 3210010; 3210009; 3210008; 3210007; 3210006; 3210005; 3210004; 3210003; 3210002; 3210001; 3210000; 3209999; 3201547; 2781152; 2392605; 2392604; 2781014; 1942360; 2554672; 2392209; 3114481; 3114480; 2981657; 3183706; 3152922; 3135503; 3135501; 3135499; 3135497; 2414158; 1321733; 1321731; 1321728; 1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3095075; 3062795; 3062793; 3062791; 2266625; 2266623; 2182106; 3044216; 2154736; 3021324; 3004467; 3005841; 3005839; 3004485; 3004473; 3004471; 3004469; 3004465; 2440053; 1805730; 2970629; 29591048; 2935527; 2935416; 809536; 730091; 585279; 584968; 2498195; 2833325; 2498604; 2498317; 2498299; 2493414; 2498586; 2498585; 2498576; 2497749; 2493446; 2493445; 1513216; 729944; 2498099; 548449; 465054; 465053; 465052; 548671; 548670; 548660; 548658; 548657; 2832430; 232084; 2500822; 2498118; 2498119; 2498119; 2498118; 1708296; 1708793; 416607; 416608; 416608; 416607; 2499791; 2498580; 2498579; 2498578; 2498577; 2497750; 1705483; 1703445; 1709542; 1709545; 17105104; 1352699; 1346568; 1346323; 1346322; 2507248; 11352240; 1352239; 1352237; 1352229; 1351935; 1350779; 1346806; 1346804; 1346803; 1170095; 1168701; 1352506; 1171011; 1171008; 1171005; 1171004; 1171002; 1171001; 1168710; 1168709; 1168708; 1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1168696; 1168391; 1168390; 1168348; 1173075; 1173074; 1173071; 1169290; 11610470; 1168402; 729764; 729320; 729979; 729970; 729315; 730050; 730049; 730048; 549194; 549193; 549192; 549191; 549190; 5491104; 549188; 549185; 549184; 549183; 549182; 549181; 549180; 549179; 464471; 585290; 416731; 1169666; 113478; 113479; 113477; 113476; 113475; 130975; 119656; 113562; 113561; 113560; 416610; 126387; 126386; 126385; 132270; 416611; 416612; 416612; 416611; 730035; 127205; 1352238; 125887; 549186; 137395; 730036; 133174; 114090; 131112; 126949; 129293; 124757; 129501; 416636; 2801531; 2796177; 2796175; 2677826; 2735118; 2735116; 2735114; 2735112; 2735110; 2735108; 2735106; 2735104; 2735102; 2735100; 2735098; 2735096; 2707295; 2154730; 2154728; 1684720; 2580504; 2465137; 2465135; 2465133; 2465131; 2465129; 2465127; 2564228; 2564226; 2564224; 2564222; 2564220; 2051993; 1313972; 1313970; 1313968; 1313966; 2443824; 2488684; 2488683; 2488682; 2488681; 2488680; 2488679; 2488678; 2326190; 2464905; 2415702; 2415700; 2415698; 2398759; 2398757; 2353266; 2338288; 1167836; 414703; 2276458; 1684718; 2293571; 1580797; 1580794; 2245508; 2245060; 1261972; 2190552; 1881574; 511953; 1532058; 1532056; 1532054; 1359436; 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320610; 280409; 320607; 320606; 539051; 539050; 539049; 539048; 322803; 280407; 100501; 100498; 100497; 100496; 1362137; 1362136; 1362135; 1362134; 1362133; 1362132; 1362131; 1362130; 1362129; 1362128; 100478; 21291041; 1076531; 1362049; 1076486; 2129817; 2129816; 2129815; 2129814; 2129813; 2129812; 2129805; 2129804; 2129802; 2129801; 2129800; 2129799; 479902; 479901; 2129477; 1076247; 629480; 1076242; 1076241; 541803; 541802; 280372; 280371; 1361968; 1361967; 1361966; 1361965; 1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 2119763; 543622; 541804; 478825; 478824; 478823; 421788; 320545; 81444; 626037; 626028; 539056; 483123; 481398; 481397; 100733; 100732; 100639; 625532; 1083651; 322674; 322673; 81719; 81718; 2118430; 2118429; 2118428; 2118427; 419801; 419800; 419799; 419798; 282991; 100691; 322995; 322994; 101824; 626077; 414553; 398830; 1311457; 1916292; 1911819; 1911818; 1911659; 1911582; 467629; 467627; 467619; 467617; 915347; 1871507; 1322185; 1322183; 1047645; 1047647; 1850544; 1850542; 1850540; 2810417; 452742; 1842045; 1839305; 1836011; 1836010; 1829900; 18291049; 18291048; 18291047; 18291046; 18291045; 18291044; 1825459; 18010487; 159653; 1773369; 1769849; 1769847; 608690; 1040877; 1040875; 1438761; 1311513; 1311512; 1311511; 1311510; 1311509; 13116104; 1246120; 1246119; 1246118; 1246117; 1246116; 1478293; 1478292; 1311642; 1174278; 1174276; 1086972; 1086974; 1086976; 1086978; 1086978; 1086976; 1086974; 1086972; 999009; 999356; 999355; 994866; 994865; 913758; 913757; 913756; 913285; 913283; 926885; 807138; 632782; 601807; 546852; 633938; 544619; 544618; 453094; 451275; 451274; 407610; 407609; 404371; 409328; 299551; 299550; 264742; 261407; 255657; 250902; 250525; 1613674; 1613673; 1613672; 1613671; 1613670; 1613304; 1613303; 1613302; 1613240; 1613239; 1613238; 1612181; 1612180; 1612179; 1612178; 1612177; 1612176; 1612175; 1612174; 1612173; 1612172; 1612171; 1612170; 1612169; 1612168; 1612167; 1612166; 1612165; 1612164; 1612163; 1612162; 1612161; 1612160; 1612159; 1612158; 1612157; 1612156; 1612155; 1612154; 1612153; 1612152; 1612151; 1612150; 1612149; 1612148; 1612147; 1612146; 1612145; 1612144; 1612143; 1612142; 1612141; 1612140; 1612139; 1093120; 447712; 447711; 447710; 1587177; 158542; 1582223; 1582222; 15315104; 1580792; 886215; 15451047; 15451045; 15451043; 15451041; 15458104; 1545887; 1545885; 1545883; 1545881; 1545879; 1545877; 1545875; 166486; 1498496; 1460058; 972513; 1009442; 1009440; 1009438; 1009436; 1009434; 7413; 1421808; 551228; 452606; 32905; 1377859; 1364213; 1364212; 395407; 22690; 22688; 22686; 22684; 488605; 17680; 1052817; 1008445; 1008443; 992612; 706811; 886683; 747852; 939932; 19003; 1247377; 1247375; 1247373; 862307; 312284; 999462; 999460; 999458; 587450; 763064; 886209; 1176397; 1173557; 902012; 997915; 997914; 997913; 997912; 997911; 997910; 99790; 997908; 997907; 997906; 997905; 997904; 997903; 997902; 997901; 997900; 9971049; 9971048; 9971047; 9971046; 9971045; 9971044; 9971043; 9971042; 910984; 910983; 910982; 910981; 511604; 169631; 169629; 169627; 168316; 168314; 607633; 555616; 293902; 485371; 455288; 166447; 166445; 166443; 166435; 162551; 160780; 552080; 156719; 156715; 515957; 515956; 515955; 515954; 515953; 459163; 166953; 386678; 169865.
Particularly preferred T cell epitopes are derived from the allergens: cat dander protein Fel d1; House dust mite proteins Der P1, Der P2 and Der P7; Ragweed protein amb a 1.1, a 1.2, a1.3 or a1.4; Rye grass proteins lol p1 and lol p5; Timothy grass proteins phl p1 and phl p5; Bermuda grass protein Cyn d 5; Alternaria alternate proteins Alt a 1, Alt a 2 and Enolase (Alt a 6); Birch protein Bet v1 and P14; German Cockroach proteins Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5 and Bla g 6; Mugwort protein Art v 1; Russian thistle protein Sal k 1 and Sal k 2; peanut Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, plant profilins or lipid transfer proteins or a human leukocyte antigen.
Suitable autoimmune antigens from which the MHC Class II-binding T cell epitope may derive can of course be obtained and/or produced using known methods. Suitable autoimmune antigens include the major antigens in the following autoimmune diseases: Acute disseminated encephalomyelitis (ADEM); Addison's disease; Ankylosing spondylitis; Antiphospholipid antibody syndrome (APS); Aplastic anemia; Autoimmune hepatitis; Autoimmune Oophoritis; Coeliac disease; Crohn's disease; Diabetes mellitus type 1; Gestational pemphigoid; Goodpasture's syndrome; Graves' disease; Guillain-Barré syndrome (GBS); Hashimoto's disease; Idiopathic thrombocytopenic purpura; Kawasaki's Disease; Lupus erythematosus; Multiple sclerosis; Myasthenia gravis; Narcolepsy, Opsoclonus myoclonus syndrome (OMS); Optic neuritis; Ord's thyroiditis; Pemphigus; Pernicious anaemia; Polyarthritis in dogs; Primary biliary cirrhosis; Rheumatoid arthritis; Reiter's syndrome; Sjögren's syndrome; Takayasu's arteritis; Temporal arteritis (also known as “giant cell arteritis”); Warm autoimmune hemolytic anemia; Wegener's granulomatosis.
Other preferred eptiopes may be derived from antigens involved with maternal-foetal immunes responses, for example Rhesus D antigens involved in Rhesus D Haemolytic Disease of the Newborn.
Other preferred epitopes may be derived from antigens involved in graft-versus-host disease or transplant rejection (alloimmune responses), for example from MHC Class I molecules (otherwise referred to as human leukocyte antigens—HLA), preferably from the α3 domain and/or transmembrane domain of MHC Class I molecules, most preferably from the human MHC Class I molecule HLA-A2.
The epitopes may be of proteins which are administered to the individual, for example for therapy. Such proteins may act as neoantigens in the individual, such as for example in the situation where the individual does not express the protein. The therapeutic protein may be factor VIII, salcatonin or human growth hormone.
The following Examples illustrate the invention:
Example 1 Peptides Derived from Human Leukocyte AntigensThe peptides in Table 2 derive from HLA-A2 and were identified by in silico analysis as containing MHC class II-binding T cell epitopes. Native sequences from HLA-A2 are in rows with a shaded background. Peptides marked with * were engineered to reduce dimer formation. Altered residues are shown in bold and underlined. =2-aminobutyric acid. The binding affinity of each peptide for different MHC class II molecules was then assessed in vitro by ELISA, as was the ability of the peptides to stimulate specific T cells.
Peptide TRA30 (HAVSDHEATLRCWAL—SEQ ID NO: 1) corresponds to amino acids 192-206 of HLA-A2 protein and is derived from the α3 domain of the HLA-A2 molecule. This peptide has a molecular weight of 1708. Peptide TRA31 (HPISDHEATLRCWAL—SEQ ID NO: 4) is an analogue of TRA30 and is also derived from the α3 domain of the HLA-A2 molecule, except that the alanine residue is replaced with a proline residue, and the valine residue is replaced with an isoleucine residue. TRA32 (HPVSDHEATLRCWAL—SEQ ID NO: 7) is another analogue of TRA30 and is also derived from the α3 domain of the HLA-A2 molecule, except that the alanine residue is replaced with a proline residue. Peptide TRA39 (RCWALSFYPAEITLT—SEQ ID NO: 10) corresponds to amino acids 202-216 of HLA-A2 protein, and is derived from the α3 domain of the HLA-A2 molecule. This peptide has a molecular weight of 1770. Peptide TRA 40 (RCWALGFYPAEITLT—SEQ ID NO: 12) is an analogue of TRA39, and is derived from the α3 domain of the HLA-A2 molecule, except that the serine residue at position 207 is replaced with a glycine residue.
All eight engineered peptides in Table 2 were engineered by the replacement of the cysteine residue with either serine or 2-aminobutyric acid (as shown) to reduce dimer formation and improve solubility. The following table illustrates the success of this strategy in that TRA33 and 36 have superior solubility to TRA 30, and TRA42 has superior solubility to TRA40.
Furthermore, some of the peptides above have been tested to determine whether modified peptides were more or less able to activate T cells than the original peptides. In particular, the peptides shown in the table below were tested against T cells from two subjects. The two subjects were renal transplant patients who were >1 yr post transplant and unselected for renal function. Subject 1 had a medium HLA peptide-specific T cell Elispot response whilst subject 2 had a very low Elispot response (see table below—original peptides are shaded grey).
The assay was performed as follows: Mononuclear cells are prepared from peripheral blood (PBMCs) of patients by ficoll gradient (30 mins). The PBMCs are incubated with peptides, positive control or negative control (medium only) in an Interferon gamma Elispot plate (48 hrs incubation). Following incubation, the Elispot plate is washed (30 mins) and the Anti-interferon gamma antibody-enzyme conjugate is added to Elispot plate (1.5 hr incubation). The Elispot plate is washed (30 mins) and the substrate is added to stain the Elispots (20 mins incubation). The plate is then read. The number of spots equates to the number of activated T cells.
As shown, in the subject (subject 1) with good responses, these were maintained when testing with modified peptides versus original peptides. Similarly, for the subject who did not respond to the original peptide (subject 2), modifying the peptides has not changed this. Thus, modification does not affect the ability of the peptides to activate T cells and in particular does not created a new false epitope. That is, engineering the peptides does not diminish their ability to induce an immune response.
The peptides in Table 3 derive from major allergens from House Dust Mites and were identified by in silico analysis as containing MHC class II-binding T cell epitopes. Native sequences from House Dust Mite allergen proteins are in rows with a shaded background. Peptides marked with * were engineered to reduce dimer formation. Altered residues are shown in bold and underlined. =2-aminobutyric acid. Binding affinity for each original peptide for different MHC class II molecules was assessed by in vitro binding studies.
Peptides HDM02, HDM03, HDM06, HDM100, 101, 102 and 203 derive from the major dust mite allergen Der p1. Peptides HDM19 and HDM26 derive from the major dust mite allergen Der p2. All the engineered peptides in Table 3 were engineered by the replacement of the cysteine residue with either serine or 2-aminobutyric acid (as shown) to reduce dimer formation.
The suitability of several of the above engineered peptides for use in tolerisation to treat or prevent house dust mite allergy is demonstrated below. The following Table presents results from a cytokine release assay performed on PBMCs taken from a population of house dust mite allergic individuals (N=number of individuals in population). A positive response is considered to be production of at least 100 pg/ml of cytokine. As shown, the number of individuals in the population who produce the cytokines IFN-γ and IL-13 in response to the peptides indicated is not significantly altered by the engineering process. Thus, engineering the peptides does not diminish their ability to induce an immune response.
Cytokine secretion profiles from PBMC's were analysed in response to the peptide stimulation using the peptides indicated. Supernatants from the cytokine release assay were tested for the presence of 2 cytokines, IFN-γ and IL-13, using either an ELISA assay or a multiplex bead array assay.
A typical cytokine release assay requires 40×106 PBMC's per subject. In more detail, 250 μl of a 200 μg/ml solution of the appropriate antigen or peptide concentration is distributed into the appropriate wells of 48 well plates. Plates are the incubated in a humidified 5% CO2 incubator at 37° C. for a maximum of 4 hours. 250 μl of a 5×106 cell/ml PBMC suspension is then added to each well and the plates returned to the incubator for 5 days. Following stimulation, samples of culture supernatant are harvested for testing by ELISA or multiplex bead assay according to standard protocols.
The peptides below derive from the major allergen in Ragweed pollen (Amb a 1, NCBI Acc. No. AAA32669) and were identified by in silico analysis as containing MHC class II-binding T cell epitopes. Native sequences from ragweed allergen proteins are in rows with a shaded background. Peptides marked with * were engineered to reduce dimer formation. Altered residues are shown in bold and underlined. Binding affinity for each original peptide for different MHC class II molecules was assessed by in vitro binding studies.
Using methods equivalent to those in Example 2, these peptides were tested for the ability to induce cytokine production in PBMCs taken from a population of ragweed allergic individuals. The levels of IFN-gamma produced by each subject are shown in
An equivalent substitution could be made with 2 amino-butyric acid to give RGW02c: GSSQIWIDHSLSKS (SEQ ID NO.72).
The suitability of several of the above engineered peptides for use in tolerisation to treat or prevent ragweed allergy is demonstrated below. The following Table presents results from a cytokine release assay performed on PBMCs taken from seven ragweed allergic individuals (A-G). As shown, the level of production of IL-10 by the modified peptide (RGW02) is not significantly different to the level produced by the original peptide (RGW02A) Thus, engineering the peptide does not diminish its ability to induce an immune response.
Cytokine secretion profiles from PBMC's were analysed in response to the peptide stimulation using the peptide indicated. Supernatants from the cytokine release assay were tested for the presence of IL-10, using either an ELISA assay or a multiplex bead array assay.
A typical cytokine release assay requires 40×106 PBMC's per subject. In more detail, 250 μl of a 200 μg/ml solution of the appropriate antigen or peptide concentration is distributed into the appropriate wells of 48 well plates. Plates are the incubated in a humidified 5% CO2 incubator at 37° C. for a maximum of 4 hours. 250 μl of a 5×106 cell/ml PBMC suspension is then added to each well and the plates returned to the incubator for 5 days. Following stimulation, samples of culture supernatant are harvested for testing by ELISA or multiplex bead assay according to standard protocols.
The peptides in Table 4 derive from the major cat allergen Fel d1, identified by in vitro analysis as containing MHC class II-binding T cell epitopes. Each of the five peptides contains a single cysteine residue, the side-chain of which contains a thiol functional group. Although free thiols can exist in the free state, they are readily oxidised to form intermolecular disulphide bridges or cystine residues. Oxidation of the cysteine residues present in the peptides will result in the formation of dimers. These dimers may arise due to crosslinking of two peptides with the same sequence in individual formulations, or different peptides within a mixture.
While individual peptides have a maximum chain length of 17 amino acids, dimerization will result in larger molecules that could trigger mast cell degranulation with a concomitant release of histamine. Obviously this is undesirable, but the presence of cysteine residues in some of the selected peptides may result in this effect being observed.
Consequently, the primary focus of this Example was to assess the ability of each agent or mixture of agents, to reduce or inhibit the formation of peptide dimers arising through the oxidation of free thiols to form intermolecular disulphide bridges. The output of the Example is the identification of agents which may be included in a formulation or composition of each of the individual peptides to reduce dimer formation.
These sequences may also be engineered to replace cysteine residues as described above. Thus:
The engineered peptides are not tested further in this Example.
The peptides used in this study have a minimum purity of >90%. The effectiveness of each additive to reduce dimer formation was assessed by size exclusion chromatography (SEC) and RP-HPLC. In SEC, molecules are separated based upon their apparent molecular weight. Smaller molecules can distribute into a greater proportion of the column matrix and are retained for longer than analytes of higher molecular weight. Dimers will elute from the column with an apparent molecular weight approximately twice that of the corresponding monomers. Separation by RP-HPLC separates species based on differences in their hydrophobicities. The amount of dimer formed is determined as a percentage of the total peak area ratio (% PAR) for the chromatogram.
Basic FormulationsStudies were conducted using a universal matrix into which each of the potential agents were added to the appropriate concentration. The universal matrix was prepared in deionised water and contained
5 mM hydrochloric acid (HCl)
140 mM sodium chloride (NaCl)
The 5 mM HCl was utilised to provide a low pH environment, i.e. ca. pH 2.3. At low pH the free thiol groups will be fully protonated and as such are much less susceptible to oxidation than at pH>6. The low pH provides an environment that promotes the solubility of each of the peptides. At pH 2.3 all the peptides should exhibit cationic properties, i.e. be positively charged, and therefore should be soluble to some extent. HCl has been used at concentration of up to 10% v/v in intravenous injections.
NaCl was included at 140 mM to produce a matrix with an ionic strength roughly equivalent to the physiological environment, i.e. isotonic. Since the peptides will be administered intradermally during Clinical studies it is important that the formulations used are close to isotonic. It is expected that similar effects would be observed in low tonicity matrices.
Agents TestedThe agents added to the universal matrix are shown in Table 5 together with the concentrations at which they were used.
The level of dimer formation in the presence of each agent is shown in Table 6. Agents which successfully reduced dimer formation are highlighted in bold.
The data generated by size exclusion chromatography on samples prepared as above and stored for up to one week identified two agents, 1-Thioglycerol and Cysteine hydrochloride, as being effective at preventing peptide dimer formation. In addition, a mixture of agents, i.e. EDTA, BHA and 1-Thioglycerol (Mix 2), also appeared to prevent dimer formation. Of the remaining agents ascorbic acid and DL-Methionine appear to retard dimer formation compared to the control matrix, i.e. 5 mM HCl and 140 mM NaCl, but the presence of the other additives resulted in increased dimer formation.
Evaluation of dimer content was continued for the peptide mixtures prepared in the Control matrix and the matrices containing 1-Thioglycerol, L-Cysteine hydrochloride and Mix 2 at two week and five week timepoints under the conditions as shown in Table 6.
Following the generation of data from the preliminary screening a further piece of work was undertaken to evaluate the ability of matrices containing Cysteine hydrochloride and 1-thioglycerol to inhibit the propensity of individual cysteine containing peptides and mixed pairs of these peptides to dimerize compared to the matrix alone. For each excipient mixtures of all the possible binary peptide combinations were prepared. Samples were analysed by RP-HPLC immediately and after storage at 25° C./60% RH for one week. The amounts of dimer formed are presented in Table 7. The amount of dimer formed is determined as a percentage of the total peak area ratio (% PAR) for the chromatogram in each case.
The effectiveness of Cysteine hydrochloride in preventing dimerization is considered to proceed through the formation of cysteinylated peptides.
All of the peptides in tables 8, 9 and 10 derive from proteins associated with alloimmune diseases as indicated, and were previously identified by in silico or in vitro analysis as containing MHC class II-binding T cell epitopes. Native sequences from the proteins are in rows with a shaded background. Peptides marked with * were engineered to reduce dimer formation. Altered residues are shown in bold and underlined.
Derived from Platelet Glycoprotein IIIa
Derived from Rhesus Blood Group D Antigen
Derived from Platelet Glycoprotein IIIa
Claims
1. A composition comprising:
- i) at least one peptide of 9 to 25 amino acids in length wherein the peptide comprises a region comprising at least one T cell epitope; and
- ii) at least one agent which inhibits peptide dimer formation which is thioglycerol or thioanisole;
- wherein a minimal proportion of the peptide of the composition is present in solution as a dimer.
2. A composition according to claim 1, wherein the proportion of peptide as defined in i) that is present as a dimer in solution in the absence of the agent is at least 0.5%; and/or wherein the epitope is an MEW Class II-binding T cell epitope
3. A composition according to claim 2, wherein the proportion of peptide present as a dimer in solution is measured after the peptide has been in solution for at least 72 hours at about 25° C. and about 60% relative humidity.
4. A composition according to claim 1, wherein less than 5% of the peptide is present in dimeric form in solution.
5. A composition according to claim 1, wherein the peptide has an improved ability to induce tolerance in an individual compared to the dimer form of the peptide.
6. A composition according to claim 1, wherein the native sequence of the region comprises at least one cysteine residue.
7. A composition according to claim 1, wherein the native sequence of the protein from which the region derives comprises approximately 33% cysteine residues; and/or wherein the native sequence of the region comprises one, two, three or more cysteine residues up to a maximum of 25% of the total number of amino acid residues in the peptide.
8. A composition according to claim 1, wherein the peptide does not comprise an epitope capable of cross-linking IgG expressed on the cell surface of B cells or IgE expressed on the surface of mast cells or basophils and/or wherein the region consists entirely of the minimal sequence of the T cell epitope.
9. A composition according to claim 1, wherein the epitope derives from:
- i) an allergen selected from: a plant allergen (particularly a grass allergen), animal dander allergens, a mold or fungal allergen, a dust allergen, an antibiotic or other drug, a stinging insect venom, an environmental allergen or a food allergen; or
- ii) an antigen selected from the major antigens associated with Acute disseminated encephalomyelitis (ADEM); Addison's disease; Ankylosing spondylitis; Antiphospholipid antibody syndrome (APS); Aplastic anemia; Autoimmune hepatitis; Autoimmune Oophoritis; Coeliac disease; Crohn's disease; Diabetes mellitus type 1; Gestational pemphigoid; Goodpasture's syndrome; Graves' disease; Guillain-Barré syndrome (GBS); Hashimoto's disease; Idiopathic thrombocytopenic purpura; Kawasaki's Disease; Lupus erythematosus; Multiple sclerosis; Myasthenia gravis; Narcolepsy, Opsoclonus myoclonus syndrome (OMS); Optic neuritis; Ord's thyroiditis; Pemphigus; Pernicious anaemia; Polyarthritis in dogs; Primary biliary cirrhosis; Rheumatoid arthritis; Reiter's syndrome; Sjögren's syndrome; Takayasu's arteritis; Temporal arteritis (also known as “giant cell arteritis”); Warm autoimmune hemolytic anemia; or Wegener's granulomatosis
10. A composition according to claim 1, wherein the epitope derives from: cat dander protein Fel d1; House dust mite proteins Der P1, Der P2 and Der P7; Ragweed protein amb a 1.1, a 1.2, a1.3 or a1.4; Rye grass proteins lol p1 and lol p5; Timothy grass proteins phl p1 and phl p5; Bermuda grass protein Cyn d 5; Alternaria alternate proteins Alt a 1, Alt a 2 and Enolase (Alt a 6); Birch protein Bet v1 and P14; German Cockroach proteins Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5 and Bla g 6; Mugwort protein Art v 1; Russian thistle protein Sal k 1 and Sal k 2; peanut Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, plant profilins or lipid transfer proteins or a human leukocyte antigen.
11. A composition according to claim 1 for use in treating or preventing a disease by tolerisation of an individual to the protein from which the T cell epitope derives.
12. A composition according to claim 1 for use in treating or preventing an allergic disease, an autoimmune disease, an alloimmune response or a maternal-foetal immune response by tolerisation, or for use in tolerising an individual to a neoantigen or to a protein which is being provided to the individual in therapy.
13. A composition according to claim 12, wherein the allergic disease or autoimmune disease comprises an immune response to an allergen or antigen as defined in i) or ii) below: or the alloimmune response is involved in transplant rejection or graft-versus-host disease, or the maternal-foetal immune response is Rhesus D Haemolytic Disease of the Newborn.
- i) an allergen selected from: a plant allergen (particularly a grass allergen), animal dander allergens, a mold or fungal allergen, a dust allergen, an antibiotic or other drug, a stinging insect venom, an environmental allergen or a food allergen; or
- ii) an antigen selected from the major antigens associated with Acute disseminated encephalomyelitis (ADEM); Addison's disease; Ankylosing spondylitis; Antiphospholipid antibody syndrome (APS); Aplastic anemia; Autoimmune hepatitis; Autoimmune Oophoritis; Coeliac disease; Crohn's disease; Diabetes mellitus type 1; Gestational pemphigoid; Goodpasture's syndrome; Graves' disease; Guillain-Barré syndrome (GBS); Hashimoto's disease; Idiopathic thrombocytopenic purpura; Kawasaki's Disease; Lupus erythematosus; Multiple sclerosis; Myasthenia gravis; Narcolepsy, Opsoclonus myoclonus syndrome (OMS); Optic neuritis; Ord's thyroiditis; Pemphigus; Pernicious anaemia; Polyarthritis in dogs; Primary biliary cirrhosis; Rheumatoid arthritis; Reiter's syndrome; Sjögren's syndrome; Takayasu's arteritis; Temporal arteritis (also known as “giant cell arteritis”); Warm autoimmune hemolytic anemia; or Wegener's granulomatosis
14. A composition according to claim 11 wherein the individual to be treated is from a population where the allele frequencies of the following DRB1 alleles is:
- 4—at least 9%
- 7—at least 10%
- 11—at least 8%.
15. A composition according to claim 11 wherein the individual to be treated has or is at risk of a condition, wherein the condition is an adverse inflammatory reaction to a treatment comprising a peptide.
16. A composition as defined in claim 1 for use in an in vitro method of diagnosing the presence or absence in a subject of a T-cell immune response to the protein from which the epitope derives, the method comprising:
- i) contacting the composition with T cells in a sample taken from the subject, under conditions which allow the peptide and the T cells to interact;
- ii) determining whether or not any of the T cells are stimulated; and
- thereby determining whether or not a T-cell immune response is present or absent.
17. A composition according to claim 15, wherein the T cells are present in a population of PBMCs isolated from a blood or serum sample taken from the subject and/or wherein step (ii) comprises measuring the production of a cytokine by the T cells.
18. A composition according to claim 17, wherein the production of a cytokine is detected by an ELISPOT or multiplex bead array assay
19. A composition according to claim 17, wherein the cytokine is interferon-gamma.
20. A composition according to claim 1, wherein the at least one peptide comprises or consists of the sequence corresponding to any one of SEQ ID NOS: 1 to 72.
21. A composition according to claim 1, comprising at least a first and a second peptide, wherein the first and second peptide each comprise or consist of a different sequence selected from the sequences of SEQ ID NO: 37 (MLA01), SEQ ID NO: 38 (MLA04), SEQ ID NO: 39 (MLA05), or SEQ ID NO: 40 (MLA12).
22. A composition according to claim 21, wherein the first and second peptides comprise or consist of the sequences of:
- a) SEQ ID NOS: 37 (MLA01) and 38 (MLA04);
- b) SEQ ID NOS: 37 (MLA01) and 39 (MLA05);
- c) SEQ ID NOS: 37 (MLA01) and 40 (MLA12);
- d) SEQ ID NOS: 38 (MLA04) and 39 (MLA05);
- e) SEQ ID NOS: 38 (MLA04) and 40 (MLA12); or
- f) SEQ ID NOS: 39 (MLA05) and 40 (MLA12), respectively.
23. A composition according to claim 20, wherein the agent is thioglycerol.
24. An antibody which binds to the peptide of the composition according to claim 1.
25. An antibody according to claim 24 which binds to the peptide when the peptide is associated with an MHC Class II molecule.
Type: Application
Filed: Aug 23, 2013
Publication Date: Dec 19, 2013
Applicant: CIRCASSIA LIMITED (OXFORD)
Inventors: RODERICK PETER HAFNER (OXFORD), PAUL LAIDLER (OXFORD)
Application Number: 13/974,603
International Classification: A61K 39/36 (20060101); C07K 16/16 (20060101); C07K 16/18 (20060101); A61K 39/00 (20060101); A61K 39/35 (20060101);
