WO2009095646A2

WO2009095646A2 - Peptides from factor viii

Info

Publication number: WO2009095646A2
Application number: PCT/GB2009/000187
Authority: WO
Inventors: Roderick Peter Hafner
Original assignee: Circassia Limited
Priority date: 2008-01-28
Filing date: 2009-01-23
Publication date: 2009-08-06
Also published as: WO2009095646A3; GB0801513D0

Abstract

The present invention relates to compositions comprising peptides for preventing or treating the development of inhibitors in patients with haemophilia, in particular certain peptides and / or peptide fragments derived from factor VIII.

Description

PEPTIDES FROM FACTOR VHI Field of the Invention

The present invention relates to compositions for preventing or treating the development of inhibitors in patients with haemophilia.

Background of the Invention

T-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 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 recognised.

Recognition of external antigens by the immune system of an organism, such as man, can in some cases be undesirable. For example, Haemophilia is an hereditary blood clotting disorder. Modern treatment uses clotting concentrates such as factor VTII for Haemophilia A and factor IX for Haemophilia B. However some people do not respond as expected to treatment because they develop inhibitors (or antibodies) to the clotting concentrates. A therapeutic treatment that would decrease or eliminate the unwanted immune response to clotting factors, without altering the immune reactivity to other foreign antigens or triggering an immune response itself would be of great benefit to haemophiliac individuals with inhibitors.

It is estimated that up to one third of people with severe or moderately severe haemophilia A (less than 5% clotting factor) may develop an inhibitor at some time in their lives. Among people with haemophilia B₅ inhibitors seem to affect approximately 3 to 5% of people with severe or moderately severe haemophilia. Inhibitor development is less common among people with mild haemophilia, but has been known to occur. A therapeutic or preventative treatment would therefore be of great benefit to humans that have or are at risk of developing clotting factor inhibitors. Summary of the Invention

The present inventors have discovered that certain peptide fragments derived from factor VIII are useful in desensitising individuals to this protein.

The peptides of the invention were selected as MHC class II-binding T cell epitopes through use of in silico analysis to identify peptide-MHC interactions and MHC class II binding assays.

A difficulty associated with approaches to desensitisation based on peptide immunisation lies in how to select an appropriate size and region of the antigen as the basis for the peptide to be used for immunisation. The size of the peptide of choice is crucial. If the peptide is too small, the vaccine would not be effective in inducing an immunological response. If the peptides are too large, or if the whole protein is introduced into an individual, there is the risk of inducing greater inhibitor titres, or other adverse reactions such as anaphylaxis, which may be fatal.

An advantage of the invention is the ability of the peptides to broadly target Major Histocompatibility Complex (MHC) molecules. T cell receptors (TCRs) are highly variable in their specificity. Variability is generated, as with antibody molecules, through gene recombination events within the cell. TCRs recognise antigen in the form of short peptides bound to molecules encoded by the genes of the Major Histocompatibility Complex (MHC). These gene products are the same molecules that give rise to "tissue types" used in transplantation and are also referred to as Human Leukocyte Antigen molecules (HLAs) which terms may be used interchangeably. Individual MHC molecules possess peptide binding grooves which, due to their shape and charge are only capable of binding a limited group of peptides. The peptides bound by one MHC molecule may not necessarily be bound by other MHC molecules.

When a protein molecule such as an antigen or antigen is taken up by antigen presenting cells such as B lymphocytes, dendritic cells, monocytes and macrophages, the molecule is enzymatically degraded within the cell. The process of degradation gives rise to peptide fragments of the molecule which, if they are of the appropriate size, charge and shape, may then bind within the peptide binding groove of certain MHC molecules and be subsequently displayed upon the surface of antigen presenting cells. If the peptide/MHC complexes are present upon the antigen presenting cell surface in sufficient numbers they may then activate T cells which bear the appropriate peptide/MHC-specific T cell receptors.

Due to the polymorphic nature of the MHC, individuals in an outbred population such as man will express different combinations of MHC molecules on their cell surfaces. Since different MHC molecules can bind different peptides from the same molecule based on the size, charge and shape of the peptide, different individuals will display a different repertoire of peptides bound to their MHC molecules. Identification of universal MHC-binding peptide epitopes in an outbred population such as man is more difficult than in inbred animals (such as certain strains of laboratory mice). On the basis of differential MHC expression between individuals and the inherent differences in peptide binding and presentation which this brings, it is unlikely that a single peptide can be identified which will be of use for desensitisation therapy in man. The peptides of the invention, however, provide a broad coverage of efficacy over the human population by targeting multiple different MHC molecules. A vaccine formulated with a peptide of the invention would therefore have broad utility.

Accordingly, the present invention provides a composition for use in preventing or treating inhibitors of Factor VIE by tolerisation comprising at least one polypeptide selected from SEQ ID NOS: 1 to 47 or variant thereof. Typically the composition may comprise up to seven different polypeptides selected from SEQ ID NOS: 1 to 47, or a variant thereof. The invention further provides a composition for use in preventing or inhibitors of Factor VIII by tolerisation comprising (i) at least one polypeptide selected from SEQ ID NOS: 1 to 47; or

(ii) a polypeptide of length 9 to 30 amino acids that comprises a region consisting of:

- any of the sequences of (i), or

- a sequence which has at least 65% homology to any of the sequences of (i) which sequence is a functional variant of any of the sequences of

(i), or (iii) a polypeptide of length 9 to 30 amino acids that comprises a region consisting of a sequence that represents either: a fragment of any of the sequences of (i), or a homologue of a fragment of any of the sequences of (i), which sequence is a functional variant of any of the sequences of (i) and has a length of at least 9 amino acids, and wherein said homologue has at least 65% homology to any 9 contiguous amino acids in any of the sequences of (i).

Description of the sequences mentioned herein SEQ ID NOS: 1 to 47 provide the polypeptide sequences of the invention as set out in Table 1.

Detailed description of the invention

The invention concerns peptides which can be used in tolerisation. Such peptides may comprise, consist of, or consist essentially of the sequences shown in any of SEQ ID NOS: 1 to 47. Variants of these specific peptides may also be used.

The variants may comprise, consist of, or consist essentially of sequences which are fragments of either any of SEQ ID NOS: 1 to 47 or homologues of any of SEQ ID

NOS: 1 to 47. In one embodiment the invention relates to a composition for use in preventing or treating inhibitors of Factor VIII comprising at least one polypeptide or variant thereof selected from SEQ ID NOS: 1 to 47.

The invention also provides products and formulations comprising the polypeptides of the invention and compositions, products and vectors comprising polynucleotides capable of expressing the polypeptides of the invention for use in preventing or treating inhibitors of Factor VIII by tolerisation. Such tolerisation will typically be to an epitope (for example a MHC class II-binding T cell epitope) present in any of SEQ ID NOS: 1 to 47.

Peptide fragments of Factor VIII

The present inventors have identified the regions in the Factor VIII protein which comprise MHC Class II-binding T cell epitopes. Based on this information, peptides derived from the relevant regions are suitable for preventing or treating inhibitors of Factor VIH by tolerisation to that protein. The terms "peptide" and "polypeptide" are used interchangeably herein. Factor VIII is also referred to herein as "the antigen".

Table 1 sets out the sequences of the peptides of the invention. The composition of the invention typically comprises at least one polypeptide or variant thereof (for example a functional variant) selected from SEQ ID NOS: 1 to 47. Optionally, the composition may comprise further polypeptides up to a total of seven unique polypeptides. The composition may therefore comprise one, two, three, four, five, six or seven unique polypeptides as provided in any of SEQ ID NOS: 1 to 47. The further polypeptides relate to (i.e. are typically homologues and/or fragments of) the other sequences, i.e. SEQ ID NOS: 1 to 47, that are not amongst the polypeptides already selected. The further peptides are typically functional variants of one of the peptides of SEQ ED NOS: 1 to 47. The further polypeptides may be identical to any of SEQ ID NOS: 1 to 47. However, the optional further polypeptides do not need to be 100% identical to any of SEQ ID NOS: 1 to 47. They are preferably at least 65% identical to at least 9 (for example at least 10, 11₃ 12 or 13) or more contiguous amino acids in any of SEQ ID NOS: 1 to 47, not already selected amongst the at least one polypeptide selected from SEQ ID NOS: 1 to 47. These contiguous amino acids may comprise a MHC class II epitope, for example which binds to any of the MHC molecules mentioned herein.

In other words, the invention provides a composition for use in the prevention or treatment of inhibitors of Factor VIII by tolerisation comprising i) at least one polypeptide selected from SEQ ED NOS: 1 to 47;

And optionally, the composition may comprise (ii) further polypeptides up to a total of seven unique polypeptides, wherein the further polypeptides: a) comprise a sequence having at least 65% sequence identity to at least 9 or more contiguous amino acids in any of SEQ ID NOS: 1 to 47 above not selected in (i); and b) are 9 to 30 amino acids in length. The invention also provides a product containing i) at least one polypeptide selected from SEQ ID NOS: 1 to 47; And optionally, the product may comprise (ii) further polypeptides up to a total of seven unique polypeptides, wherein the further polypeptides: a) comprise a sequence having at least 65% sequence identity to at least

9 or more contiguous amino acids in any of SEQ ID NOS: 1 to 47 above not selected in (i); and b) are 9 to 30 amino acids in length. wherein each different polypeptide is for simultaneous, separate or sequential use in the prevention or treatment of inhibitors of Factor VIQ by tolerisation.

In more detail therefore, the invention provides a product containing:

(a) A polypeptide selected from any one of SEQ ED NOS: 1 to 47; and optionally

(b) A polypetide:

(i) comprising sequence having at least 65% sequence identity to at least 9 or more contiguous amino acids in any of SEQ ID NOS: 1 to 47 not selected in a) above; and (ii) 9 to 30 amino acids in length; and optionally

(c) A polypeptide as defined in b), but additionally not selected in a) or b); and optionally (d) A polypeptide as defined in b), but additionally not selected in a) to c) above; and optionally

(e) A polypeptide as defined in b), but additionally not selected in a) to d) above; and optionally

(f) A polypeptide as defined in b), but additionally not selected in a) to e) above; and optionally

(g) A polypeptide as defined in b), but additionally not selected in a) to f) above; and optionally; for simultaneous, separate or sequential use in the prevention or treatment of inhibitors of Factor VIII by tolerisation. The composition or products of the invention may therefore comprise variants of any of SEQ ID NOS: 1 to 47. The variant typically comprises 1 , 2, 3 or more of the MHC class II epitopes present in the corresponding peptide of SEQ ED NOS: 1 to 47.

Functional variants are mentioned herein. Such a variant may be able to tolerise an individual to a class II MHC epitope present in the corresponding peptide of SEQ ID NOS: 1 to 47, and thus it will typically comprise sequence that binds to the same MHC class II molecule and/or is recognised by a T cell which recognises the corresponding epitope in the polypeptide of SEQ ID NOS: 1 to 47.

Variants of SEQ ID NOS: 1 to 47 may be fragments derived by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Fragments may also be generated by one or more internal deletions, provided that the core 9 amino acids that makes up the T cell epitope is not substantially disrupted.

For example, a variant of SEQ ID NO: 1 may comprise a fragment of SEQ ID NO: 1, i.e. a shorter sequence. This may include a deletion of one, two, three or four amino acids from the N-terminal end of SEQ ID NO: 1 or from the C-terminal end of SEQ ID NO: 1. Such deletions may be made from both ends of SEQ ID NO: 1. A variant of SEQ ID NO: 1 may include additional amino acids (for example from the sequence of the parent protein from which the peptide derives) extending beyond the end(s) of SEQ ED NO: 1. A variant may include a combination of the deletions and additions discussed above. For example, amino acids may be deleted from one end of SEQ ID NO: 1 , but additional amino acids from the full length parent protein sequence may be added at the other end of SEQ ID NO: 1. The same discussion of variants above also applies to SEQ ID NOS: 2 to 47.

A variant peptide may include one or more amino acid substitutions from the amino acid sequence of any of SEQ ID NOS: 1 to 47 or a fragment thereof. A variant peptide may comprise sequence having at least 65% sequence identity to at least 9 or more contiguous amino acids in any of SEQ ID NOS: 1 to 47. More preferably a suitable variant may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid identity to at least 9 contiguous amino acids of any of SEQ ID NOS: 1 to 47. This level of amino acid identity may be seen at any section of the peptide, although it is preferably the core region. The level of amino acid identity is over at least 9 contiguous amino acids but it maybe at least 10, 11₅ 12, 13, 14, 15 or at least 16 or 17 amino acids, depending on the size of the peptides of comparison. Accordingly, any of the above-specified levels of identity may be across the entire length of sequence. In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10; Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue- specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized. A variant peptide may comprise 1, 2, 3, 4, 5 or more, or up to 10 amino acid substitutions from any of SEQ ID NOS: 1 to 47. Substitution variants preferably involve the replacement of one or more amino acids with the same number of ammo acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:

Further variants include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be modified, e.g. labelled, providing the function of the peptide is not significantly adversely affected.

Where the peptide has a sequence that varies from the sequence of any of SEQ ID NOS: 1 to 47 or a fragment thereof, the substitutions may occur across the full length of the sequence, within the sequence of any of SEQ ID NOS: 1 to 47 or outside the sequence of any of SEQ ID NOS: 1 to 47. For example, the variations described herein, such as additions, deletions, substitutions and modifications, may occur within the sequence of any of SEQ ID NOS: 1 to 47. A variant peptide may comprise or consist essentially of the amino acid sequence of any of SEQ ID NOS: 1 to 47 in which one, two, three, four or more amino acid substitutions have been made. A variant peptide may comprise a fragment of the parent protein that is larger than any of SEQ ID NOS: 1 to 47. In this embodiment, the variations described herein, such as substitutions and modifications, may occur within and/or outside the sequence of any of SEQ ID NOS: 1 to 47.

The variant peptides of the invention are 9 to 30 amino acids in length inclusive. Preferably, they may be from 9 to 20 or more preferably 13 to 17 amino acids in length. The peptides may be the same length as the peptide sequences in any one of SEQ ID NOS: 1 to 47.

The peptides may be chemically derived from the polypeptide antigen, for example by proteolytic cleavage or can be derived in an intellectual sense from the polypeptide antigen, for example by making use of the amino acid sequence of the polypeptide antigen and synthesising peptides based on the sequence. Peptides may be synthesised using methods well known in the art. 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. The peptides provided by the present invention may be derived from splice variants of the parent proteins encoded by mRNA generated by alternative splicing of the primary transcripts encoding the parent protein chains. The peptides may also be derived from amino acid mutants, glycosylation variants and other covalent derivatives of the parent proteins which retain at least an MHC-binding property of the antigen. Exemplary derivatives include molecules wherein the peptides of the invention are covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Further included are naturally occurring variants of the parent proteins found in different mites. Such a variant may be encoded by an allelic variant or represent an alternative splicing variant.

Variants as described above may be prepared during synthesis of the peptide or by post- production modification, or when the peptide is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.

In accordance with the invention, the further peptides that the composition may comprise are preferably functional variants of any of SEQ E⁾ NOS: 1 to 47. That is, the peptides are preferably capable of inducing an immune response. This may be tested by the ability of the peptide to induce T cell proliferation in a sample of T cells. Methods of testing the induction of T cell proliferation are well known in the art and one such method is exemplified in Example 1. Preferably the one or more further peptides are capable of causing T cell proliferation in at least 20 % of samples of T cells, wherein each sample is obtained from different individuals in the population with inhibitors of Factor VIH The compositions of the invention are preferably capable of inducing T cell proliferation in 30 % or more samples of T cells obtained from of a panel of individuals in the population with inhibitors of Factor VIII. More preferably, the compositions are capable of inducing T cell proliferation in 35% or more, 40 % or more, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, or 90 % or more of samples obtained from sensitized individuals in a panel. The number of individuals in a panel of individuals in the population with inhibitors of Factor VHI may be any number greater than one, for example at least 2, 3, 5, 10, 15, 20, 30, 50, 80, or at least 100 individuals. It is preferred if the peptides cause T cell proliferation, but do not lead to the release of histamine from enriched basophils or mast cell preparations from a sensitised individual. There may be some histamine release, but preferably the composition does not cause significant amounts of histamine to be released. Significant histamine release may be considered to be the release of 20% or more of the total available leukocyte histamine when a sample of leukocytes from an individual is stimulated with a composition in vitro. A normal individual typically has an approximate leukocyte histamine content of 150ng/10⁷ cells. Suitable variants capable of binding to TCRs may be derived empirically or selected according to known criteria. Within a single peptide there are certain residues which contribute to binding within the MHC antigen binding groove and other residues which interact with hypervariable regions of the T cell receptor (Allen et al (1987) Nature 327: 713-5). Within the residues contributing to T cell receptor interaction, a hierarchy has been demonstrated which pertains to dependency of T cell activation upon substitution of a given peptide residue. Using peptides which have had one or more T cell receptor contact residues substituted with a different amino acid, several groups have demonstrated profound effects upon the process of T cell activation. Evavold & Allen (1991) Nature 252: 1308-10) demonstrated the dissociation of T cell proliferation and cytokine production. In this in vitro model, a T cell clone specific for residues 64-76 of haemoglobin (in the context of I-E^k), was challenged with a peptide analogue in which a conservative substitution of aspartic acid for glutamic acid had been made. This substitution did not significantly interfere with the capacity of the analogue to bind to I-E^k.

Following in vitro challenge of a T cell clone with this analogue, no proliferation was detected although EL-4 secretion was maintained, as was the capacity of the clone to help B cell responses. In a subsequent study the same group demonstrated the separation of T cell-mediated cytolysis from cytokine production. In this instance, the former remained unaltered while the latter was impaired. The efficacy of altered peptide ligands in vivo was initially demonstrated in a murine model of EAE (experimental allergic encephalomyelitis) by McDevitt and colleagues (Smilek et al (1991) Proc Natl Acad Sci USA 88 : 9633-9637). In this model EAE is induced by immunisation with the encephalitogenic peptide AcI-11 of MBP (myelin basic protein). Substitution at position four (lysine) with an alanine residue generated a peptide which bound well to its restricting element (Aα^uAβ^u), but which was non- immunogenic in the susceptible PL/JxSJLFl strain and which, furthermore prevented the onset of EAE when administered either before or after immunisation with the encephalitogenic peptide. Thus, residues can be identified in peptides which affect the ability of the peptides to induce various functions of T-cells. Advantageously, peptides may be designed to favour T-cell proliferation and induction of desensitisation. Metzler and Wraith have demonstrated improved tolerogenic capacity of peptides in which substitutions increasing peptide-MHC affinity have been made (Metzler & Wraith(1993) Int Immunol ~ : 1159-65). That an altered peptide ligand can cause long-term and profound anergy in cloned T cells was demonstrated by Sloan-Lancaster et al (1993) Nature 363: 156-9.

Preferably, the individual peptides of the invention are able to desensitise in an individual who has been sensitised to Factor VIII. Whether or not an individual has been sensitised to Factor VlH may be determined by well known procedures such as the Bethesda inhibitor assay. Whether or not a particular individual is expected to benefit from treatment may be determined by the physician based, for example, on such tests.

Desensitising or tolerising an individual to the antigen means inhibition or dampening of antibody responses induced by Factor VIII 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 antigen. The desensitisation manifests itself as a reduction in response to antigen or antigen-derived peptide, or preferably an elimination of such a response, on second and further administrations of the antigen or antigen-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.

It will be understood that the peptides of the invention comprise a T cell epitope that consists of a core number of amino acids (typically 9) which are the minimal essential sequence required for MHC class II binding. However, the peptides may also comprise additional residues flanking the core amino acids. The peptides may therefore comprise a region containing a T cell epitope, in which some residues may be modified without affecting the function of the epitope. Accordingly, functional variants of the peptides as defined above include peptides which are altered to improve their solubility relative to the native sequence of the peptides. Improved solubility is advantageous for the tolerisation of subjects to antigens from which the peptides of the invention derive, since administration of poorly soluble agents to subjects causes undesirable, non-tolerising inflammatory responses. The solubility of the peptides may be improved by altering the residues which flank the region containing a T cell epitope. A peptide of the invention may be engineered to be more soluble such that it comprises: i) N terminal to the residues of the peptide which flank a T cell epitope: one to six contiguous amino acids corresponding to the two to six contiguous amino acids immediately N terminal to said residues in the sequence of the protein from which the peptide derives; and/or ii) C terminal to the residues of the peptide which flank a T cell epitope: one to six contiguous amino acids corresponding to the one to six contiguous amino acids immediately C terminal to the said residues in the sequence of the protein from which the peptide derives; or iii)both N and C terminal to the residues of the peptide which flank a T cell epitope, at least one amino acid selected from arginine, lysine, histidine, glutamate and aspartate.

Optionally, the peptides may additionally be engineered to be more soluble such that: i) any cysteine residues in the native sequence of the peptide are replaced with serine or 2-aminobutyric acid; and /or ii) any hydrophobic residues in the upto three amino acids at the N or C terminus of the native sequence of the peptide, which are not comprised in a T cell epitope, are deleted; and/or iii) any two consecutive amino acids comprising the sequence Asp-Gly in the upto four amino acids at the N or C terminus of the native sequence of the peptide, which are not comprised in a T cell epitope, are deleted.

Nucleic acids and vectors

The individual peptides that make up the compositions and products of the invention may be administered directly, or may be administered indirectly by expression from an encoding sequence. For example, a polynucleotide may be provided that encodes a peptide of the invention, such as any of the peptides described above. A peptide of the invention may thus be produced from or delivered in the form of a polynucleotide which encodes, and is capable of expressing, it. Any reference herein to the use, delivery or administration of a peptide of the invention is intended to include the indirect use, delivery or administration of such a peptide via expression from a polynucleotide that encodes it.

Accordingly, the invention provides a composition for use in preventing or treating inhibitors of Factor VUI by tolerisation comprising at least one polynucleotide sequence which when expressed cause the production of a composition for use in preventing or treating inhibitors of Factor VIII by tolerisation comprising at least one polypeptide or variant thereof selected from SEQ ID NOS: 1 to 47.

The invention also provides a product containing upto seven polynucleotides, wherein a each polynucleotide encodes a different polypeptide selected from SEQ ID NOS: 1 to 47; and wherein each different polypeptide is for simultaneous, separate of sequential use in the prevention or treatment of inhibitors of Factor VITI in a human. The 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

(rnRNA), 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 inhibitors of Factor VIH 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. The vector may comprise 2, 3 , 4, 5, 6 or 7 polynucleotide sequences which encode different polypeptides of the invention.

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 resistence 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. La 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.

In some embodiments, the polynucleotide, expression cassette or vector will encode an adjuvant, or an adjuvant wall otherwise be provided. As used herein, the term "adjuvant" refers to any material or composition capable of specifically or non- specifically altering, enhancing, directing, redirecting, potentiating or initiating an antigen-specific immune response.

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 by tolerisation.

Methods for gene delivery are known in the art. See, e.g., U.S. Patent 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.

Antigen presenting cells (APCs)

The invention encompasses the use in vitro of a method of producing a population of APCs that present the peptides of the invention on their surface, that may be subsequently used in therapy. Such a method may be carried out ex vivo on a sample of cells that have been obtained from a patient. The APCs produced in this way therefore form a pharmaceutical agent that can be used in the treatment or prevention of inhibitors of Factor VIII by tolerisation. The cells should be accepted by the immune system of the individual because they derive from that individual. Delivery of cells that have been produced in this way to the individual from whom they were originally obtained, thus forms a therapeutic embodiment of the invention.

Formulations and compositions

The 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 use in preventing or treating inhibitors of Factor VHI by tolerisation 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 and not deleterious to the recipient thereof. Typically, carriers for injection, and the final formulation, are sterile and pyrogen free.

Formulation of a composition comprising the peptide, polynucleotides or cells of the invention can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan.

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 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., NJ. 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. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides. 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.

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%.

In one aspect of the invention an adjuvant may be used in combination with the polypeptide/polynucleotides/cells of the invention. The adjuvant is preferably administered in an amount which is sufficient to augment the effect of the polypeptide/polynucleotides/cells of the invention or vice versa. The adjuvant or other therapeutic agent may be an agent that potentiates the effects of the molecule of the invention. For example, the other agent may be an immunomodulatory molecule or an adjuvant which enhances the response to the peptide or cell of the invention. In one embodiment, therefore, the peptides, polynucleotides, cells or compositions of the invention are used for therapy in combination with one or more other therapeutic agents. The agents may be administered separately, simultaneously or sequentially. They may be administered in the same or different compositions. Accordingly, in a method of the invention, the subject may also be treated with a further therapeutic agent.

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. Non-limiting examples of adjuvants include vitamin D, and rapamycin.

Therapeutic methods and individual to be treated

The present invention relates to peptides, polynucleotides, vectors and cells that are capable of desensitising or tolerising human individuals to the antigen described above and are therefore useful in the prevention or treatment of inhibitors of Factor VIH. The invention provides compositions, products, vectors and formulations for use in preventing or treating inhibitors of Factor VHI by tolerisation. The invention also provides a method of tolerising or desensitizing an individual who has or is at risk of developing inhibitors of Factor VIE 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. The individual is preferably a patient with haemophilia, at risk of haemophilia, or suspected of having haemophilia. The haemophilia typically involves a deficiency or defect in clotting Factor VIII. The haemophilia is typically haemophilia A. Haemophilia is a hereditary genetic disorder that impair the body's ability to control blood clotting, or coagulation. The main risk factor for haemophilia is therefore a family history of the disorder.

The individual may have inhibitors of Factor VIE, be at risk of developing inhibitors of Factor VHI, or be suspected of having inhibitors to Factor VlTf. The individual may be diagnosed as having inhibitors of Factor VIII using any suitable assay. Typically the assay is the Bethesda inhibitor assay, for which the amount of antibody is measured and is reported as a Bethesda unit or titre. A titre of greater than about 5 indicates the presence of clinically significant levels of inhibitors of Factor VlH and the subject is likely to show symptoms of not responding to Factor Viπ therapy. The patient to be treated using the polypeptides/polynucleotides/cells of the invention may typically have a Bethesda titre of greater than 5.

A Bethesda titre of less than 5 indicates that the levels of inhibitor are unlikely to have an effect on Factor VIII therapy. Titres of this level may indicate that the inhibitors are transient and will disappear in time. However, they may also indicate that a subject is at risk of developing higher levels in the future. There are a number of inherited factors that can also indicate an increased risk of developing inhibitors: having haemophilia A or severe haemophilia A; a family history of inhibitors; the presence of a mutation in intron 22 of the Factor VIII gene; large deletions in the Factor VIII gene; specific HLA (human leucocyte antigen) alleles which increase susceptibility and race (the risk is higher among people of African- American or Hispanic origin). There are also certain environmental factors that may increase the risk of inhibitor development: exposure - the majority of individuals develop inhibitors within their first 20 exposures to factor V-H treatment; change of Factor VIII product administered (it is considered good clinical practice not to change products unless there is a good reason); administration of sub-cutaneous infusions of factor VTII; exposure to large amounts of factor VIE.

It may not be necessary to test an individual for inhibitors to Factor VIII because the individual may display symptoms of such inhibitors. For example, a previously effective does of Factor VHI becomes insufficient to prevent bleeding. The individual to be treated may be of any age. However, preferably, the individual may be in the age group of 1 to 90, 1 to 60, 1 to 40, 1 to 30, 1 to 18, 1 to 10 years or more preferably less than about 4 years of age.

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 DRBl allele families are shown in the table below (Data from HLA Facts Book, Parham and Barber).

DRBl allele frequencies

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 above (such as for at least 1, 2, 3, A, 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 DRBl alleles is: 4 - at least 9% 7 - at least 10% 11 - at least 8%. The individual may have had inhibitors of Factor VIII for at least 2 weeks, 1 month, 6 months, 1 year or 5 years. The individual may suffer from symptoms associated with Factor VIII inhibitors. The individual may or may not have been administered with other compositions/compounds which treat inhibitors of Factor viπ.

Delivery methods

Once formulated the compositions of the invention can be delivered to a subject in vivo using a variety of known routes and techniques. For example, a composition can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, sublingual administration, and active or passive transdermal delivery techniques.

Where a peptide of the invention is to be administered, it is preferred to administer the peptide to a site in the body where it will have the ability to contact suitable antigen presenting cells, and where it, or they, will have the opportunity to contact T cells of the individual. Where an APC is to be administered, it is preferred to administer the APC to a site in the body where it will have the ability to contact, and activate, suitable T cells of the individual.

Delivery regimes

Administration of the peptides/polynucleotides/cells (such as the composition containing a plurality of peptides) may be by any suitable method as described above. Suitable amounts of the peptide may be determined empirically, but typically are in the range given below. A single administration of each peptide may be sufficient to have a beneficial effect for the patient, but it will be appreciated that it may be beneficial if the peptide is administered more than once, in which case typical administration regimes may be, for example, once or twice a week for 2-4 weeks every 6 months, or once a day for a week every four to six months. As will be appreciated, each peptide or polynucleotide, or combination of peptides and/or polynucleotides may be administered to a patient singly or in combination. Dosages for administration will depend upon a number of factors including the nature of the composition, the route of administration and the schedule and timing of the administration regime. Suitable doses of a molecule of the invention may be in the order of up to 15μg, up to 20μg, up to 25μg, up to 30μg, up to 50μg, up to lOOμg, up to 500 μg or more per administration. Suitable doses may be less than 15μg, but at least Ing, or at least 2ng, or at least 5ng, or at least 50ng, or least 1 OOng, or at least 500ng, or at least lμg, or at least lOμg. For some molecules of the invention, the dose used may be higher, for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or higher. Such doses may be provided in a liquid formulation, at a concentration suitable to allow an appropriate volume for administration by the selected route.

Kits

The invention also relates to a combination of components described herein suitable for use in a treatment of the invention which are packaged in the form of a kit in a container. Such kits may comprise a series of components to allow for a treatment of the invention. For example, a kit may comprise one or more different peptides, polynucleotides and/or cells of the invention, or one or more peptides, polynucleotides or cells of the invention and one or more additional therapeutic agents suitable for simultaneous administration, or for sequential or separate administration. The kit may optionally contain other suitable reagent(s) or instructions and the like. The invention is illustrated by the following Examples:

Example 1 MHC Class II binding search

The aim of this study is to identify a distinct panel of peptides with strong affinities for the seven most common human MΗC Class II ΗLA-DRB1* allotypes (covering in total around 63% of the allotypes found in the average Caucasian population). In order to identify binding peptides in Factor VDI, the inventors employed an in silico approach known as "peptide threading" using the commercially available EpiMatrix algorithm (EpiVax Inc.). This is a bioinformatic analysis of peptides from a sequence for the potential to be accommodated within the binding groove of MΗC class π HL A-DR molecules. EpiMatrix is a matrix-based algorithm that ranks 10 amino acid long segments, overlapping by 9 amino acids, from any polypeptide sequence by estimated probability of binding to each of the selected MHC molecules. (De Groot et al., AIDS Research and Human Retroviruses 13:539-41 (1997)). The procedure for developing matrix motifs was published by Schafer et al, 16 Vaccine 1998 (1998). In this Example, binding potential for HLA DRl, DR2, DR3, DR4, DR7, DR8, DRIl, DRl 3 and DRl 5 is assessed. Putative MHC ligands are selected by scoring each 10-mer frame in a protein sequence. This score is derived by comparing the sequence of the 10-mer to the matrix of 10 amino acid sequences known to bind to each MHC allele. Retrospective studies have demonstrated that EpiMatrix accurately predicts published MHC ligands (Jesdale et al., in Vaccines '97 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1997)). Successful Prediction of peptides which bind to multiple MHC molecules has also been confirmed.

Estimated probability of binding to a selected MHC molecule is calculated by EpiMatrix as follows. The peptides are scored by estimating the relative promotion or inhibition of binding for each amino acid, compared to known MHC binders for a given MHC allele. This information is summed across the peptide and a summary score (EMX score) is assigned to the entire peptide. After comparing the EMX score to the scores of known MHC ligands, EpiMatrix arrives at an "estimated binding probability" (abbreviated as EBP, but not strictly a probability). The EBP describes the proportion of peptides with EpiMatrix scores as high or higher that will bind to a given MHC molecule. EBPs range from 100% (highly likely to bind) to less than 1% (very unlikely to bind). EpiMatrix analyses were performed on the entire sequence of the known isoform of Ara h 1 (NCBI accession no: AAA52484). This analysis identified core peptides (and their flanking sequences) derived from the above sequences which are predicted to have good MHC class-II binding. These sequences are shown below in Table 1. In Table 1 : "Residues in sequence" gives the location of the peptide within the sequences that were analysed. The core peptide (middle amino acids in bold) defines the actual binding sequence that was identified during the analysis. The stabilizing flanks (N-terminal and C-terminal, not bold) were included for use with the core sequence and are typically required to aid manufacture of the peptides. "Number of hits" refers to the number of high predicted binding affinities for all MHC types tested within the sequence. The "EpiMatrix Cluster Score" is derived from the number of hits normalized for the length of the cluster. Cluster Score is thus the excess or shortfall in predicted aggregate MHC binding properties relative to a random peptide standard. A score above 10 is considered to indicate broad MHC binding properties.

Example 2 In vitro binding analysis

The peptides identified as being potential MHC Class II-binding are pre-screened for solubility in an aqueous, acidic milieu and the peptides are tested in an in vitro MHC Class π binding assay.

Methods

The assay employed is a competitive MHC class II binding assay, wherein each peptide is analysed for its ability to displace a known control binder from each of the human MHC class II allotypes investigated. The allotypes and control peptides used in this study are typically those shown below:

Control peptides used in the in vitro binding assays

Each of the peptides from Table 1 are analysed in the competition assay and screened for relative binding compared to the control peptides. Due to the nature of the competitive assay the data for each peptide is determined as a ratio of its own IC50 to that of the control peptide. Thus, a peptide that has an IC50 value that is parity to the control peptide has an identical binding affinity, while peptides with a ratio less than one have a higher affinity and those with a ratio greater than one have a lower affinity.

Solubility in aqueous solution is an essential criterion for a peptide to be an effective therapeutic agent. Therefore, as a consequence of the solubility screen very hydrophobic peptides with a high frequency of large hydrophobic amino acid residues in multiple binding registers will be eliminated. This is a characteristic of promiscuous HLA-DRBl * binders. Peptides which bind to one or more of the MHC Class II allotypes are identified. It is expected that such peptides would have the ability to bind similar allotypes that have not been tested through the homology of MHC structures.

Example 3

The following methods are applied to the same peptides as in Example 2.

Cell proliferation assay The cell proliferation assay is performed on PBMCs (14OxIO⁶ cells required for all parameters to be tested). Proliferation is measured by the incorporation of the radiolabeled compound 3H-thymidine. In more detail, lOOμl of the appropriate antigen or peptide concentration is distributed into the appropriate wells of 96 well plates. The plates are then placed into a humidified 5% CO2 incubator set at 37oC for a maximum of 4 hours. PBMCs isolated as described above are prepared to a concentration of 2x10⁶ cells/ml in complete medium at room temperature. 1 OOμl of cell solution is then distributed into each of the wells of the 96 well plates containing antigen/peptide. The plates are then incubated for 6 to 8 days. The cultures are pulsed with tritiated thymidine solution by adding lOμl of tritiated thymidine stock solution (1.85MBq/ml in serum-free RPMI medium) to each well. The plates are then returned to the incubator for between 8 and 16 hours. Cultures are then harvested using a Canberra Packard FilterMate 196 cell harvester. Dried filter mats are counted using an appropriate beta scintillation counter.

Counts from wells containing peptide are compared statistically to wells containing media alone (12 wells per group). The non-parametric Mann- Whitney test is used. The same statistical test is used for all subjects. A statistically significant difference between media only wells and peptide-stimulated wells is considered a positive stimulation of PBMCs by the peptide.

Cytokine release assay Cytokine secretion profiles from PBMCs are analysed in response to the peptide stimulation. Supernatants from the cytokine release assay are tested for the presence of 3 cytokines, IFN-γ, IL-IO and EL-13, using ELISA assays. The cytokine release assay requires 4OxIO⁶ PBMCs per subject. In more detail, 250μl of a 200μg/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 37⁰C for a maximum of 4 hours. 250μl of a 5x10⁶ 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 into 3 aliquots and frozen until the ELISA assays can be performed. One aliquot is tested for the presence of one cytokine (therefore all 3 aliquots are required to test for the 3 cytokines). The cytokine levels in the samples are determined by interpolation from standard curves also generated in the assay.

Claims

1. A composition for use in preventing or treating inhibitors of Factor VHI by tolerisation comprising

(i) at least one polypeptide selected from SEQ ID NOs: 1 to 47; or (iϊ) a polypeptide of length 9 to 30 amino acids that comprises a region consisting of: any of the sequences of (i), or

- a sequence which has at least 65% homology to any of the sequences of (i) which sequence is capable of tolerising an individual to any of the sequences of (i), or (iii) a polypeptide of length 9 to 30 amino acids that comprises a region consisting of a sequence that represents either: a fragment of any of the sequences of (i), or a homologue of a fragment of any of the sequences of (i), which sequence is capable of tolerising an individual to any of the sequences of (i) and has a length of at least 9 amino acids, and wherein said homologue has at least 65% homology to any 9 contiguous amino acids in any of the sequences of (i).

2. The composition according to claim 1, wherein the composition comprises a polypeptide selected from l(i) or a variant thereof defined in claim l(ii) or l(iii), and/or at least one further polypeptide up to a total of seven different polypeptides, wherein the further polypeptides:

(a) comprise a sequence having at least 65% sequence identity to at least 9 or more contiguous amino acids in any of SEQ H) NO: 1 to 47 above not selected in claim l(i); and

(b) are 9 to 30 amino acids in length.

3. The composition according to any one of the preceding claims, comprising at least one said polypeptide which is 9 to 20 or 13 to 17 amino acids in length and/or wherein said polypeptide has at least 70% sequence identity to any of SEQ ID NOS: 1 to 47.

4. The composition according to any one of the preceding claims, wherein one or more of the polypeptides have one or more modifications selected from the following:

(i) N terminal acetylation;

(ii) C terminal amidation;

(iii) one or more hydrogen on the side chain amines of

Arginine and/or Lysine replaced with a methylene group; (iv) glycosylation; and

(v) phosphorylation.

5. The composition according to any one of the preceding claims wherein at least one of the peptides has been engineered to be soluble such that it comprises: i) N terminal to the residues of the peptide which flank a T cell epitope: one to six contiguous amino acids corresponding to the two to six contiguous amino acids immediately N terminal to said residues in the sequence of the protein from which the peptide derives; and/or ii) C terminal to the residues of the peptide which flank a T cell epitope: one to six contiguous amino acids corresponding to the one to six contiguous amino acids immediately C terminal to the said residues in the sequence of the protein from which the peptide derives; or iii) both N and C terminal to the residues of the peptide which flank a T cell epitope, at least one amino acid selected from arginine, lysine, histidine, glutamate and aspartate, wherein the polypeptide has a solubility of at least 3.5mg/ml and the T cell epitope has a solubility of less than 3.5mg/ml.

6. The composition according to any one of claims 1 to 5 wherein at least one of the peptides has been engineered to be soluble such that additionally: i) any cysteine residues in the native sequence of the peptide are replaced with serine or 2-aminobutyric acid; and /or ii) any hydrophobic residues in the upto three amino acids at the N or C terminus of the native sequence of the peptide, which are not comprised in a T cell epitope, are deleted; and/or iii) any two consecutive amino acids comprising the sequence

Asp-Gly in the upto four amino acids at the N or C terminus of the native sequence of the peptide, which are not comprised in a T cell epitope, are deleted.

7. The composition according to any one of the preceding claims wherein each polypeptide has a concentration in the range of 0.03 to 200 nrnol/ml, 0.3 to 200 nmol/ml or 30 to 120 nmol/mL

8. A composition for use in preventing or treating inhibitors of Factor VIII by tolerisation comprising at least one polynucleotide sequence which when expressed cause the production of a composition as defined in any one of claims 1 to 6.

9. The composition according to claim 8, wherein each polynucleotide sequence capable of expressing a different polypeptide is present in the same or different polynucleotide vectors.

10. A vector for use in preventing or treating inhibitors of Factor VIII by tolerisation comprising at least one polynucleotide sequence, which encodes a polypeptide as defined in claim 1 and optionally one or more further polynucleotide sequences which encode different polypeptides as defined in claim 3.

11. A vector for use in preventing or treating inhibitors of Factor VIII by tolerisation comprising upto seven different polynucleotide sequences, wherein each polynucleotide encodes a different polypeptide selected from SEQ ID NOS: 1 to 47 or a variant thereof as defined in claim l(ii) or l(iii).

12. A product containing a polypeptide selected from SEQ ED NOS: 1 to 47, and additionally comprising at least one further different polypeptide selected from SEQ ID NOS: 1 to 47, wherein each different polypeptide is for simultaneous, separate or sequential use in preventing or treating inhibitors of Factor VlH by tolerisation.

13. A product containing at least one polynucleotide which encodes a polypeptide as defined in claim 1, and additionally comprising at least one further polynucleotide sequence which encodes a different polypeptide as defined in claim 1, wherein each different polypeptide is for simultaneous, separate of sequential use in the prevention or treatment of inhibitors of Factor VDI in a human.

14. A pharmaceutical formulation for use in preventing or treating inhibitors of Factor VIII by tolerisation comprising a composition according to any one of claims 1 to 6; a vector according to any one of claims 10 or 11; or a product according to any one of claims 12 or 13; and a pharmaceutically acceptable carrier or diluent.

15. The composition, vector or product according to claim 14, formulated for oral administration, nasal administration, subcutaneous administration, sublingual administration, intradermal administration, buccal administration or for administration by inhalation or by injection.

16. An in vitro method of determining whether T cells recognize a polypeptide as defined in claim 1 comprising contacting said T cells with said polypeptide and detecting whether said T cells are stimulated by said polypeptide.

17. A method according to claim 16 which is carried out to determine whether an individual has, or is at risk of having, an inhibitors of Factor VIH.