Title of Invention


Abstract A peptide which is capable of binding to an MHC class I or II molecule without further processing, and which is selected from the following myelin basic protein peptides: YKSAHKGFKGVDAQG (134-148), KSAHKGFKGVDAQGT (135-149), SAHKGFKGVDAQGTL (136-150), AHKGFKGVDAQGTLS (137-151), HKGFKGVDAQGTLSK (138-152), GFKGVDAQGTLSKIF (140-154).
The present invention relates to peptides from myelin basic protein. In particular, the invention relates to peptides which comprise a portion of the region 131-158 of myelin basic protein and their use in the treatment and/or prevention of a disease.
In an adaptive immune response, T lymphocytes are capable of recognising internal epitopes of a protein antigen. Antigen presenting cells (APC) take up protein antigens and degrade them into short peptide fragments. A peptide may bind to a major histocompatability complex (MHC) class I or II molecule inside the cell and be carried to the cell surface. When presented at the cell surface in conjunction with an MHC molecule, the peptide may be recognised by a T cell (via the T cell receptor (TCR)), in which case the peptide is a T cell epitope.
T cell epitopes play a central role in the adaptive immune response to any antigen, whether self or foreign. The central role played by T cell epitopes in hypersensitivity diseases (which include allergy, autoimmune diseases and transplant rejection) has been demonstrated through the use of experimental models. It is possible to induce inflammatory or allergic diseases by injection of synthetic peptides (based on the structure of T cell epitopes) in combination with adjuvant.
By contrast, it has been shown to be possible to induce immunological tolerance towards particular peptide epitopes by administration of peptide epitopes in soluble form. Administration of soluble peptide antigens has been demonstrated as an effective means of inhibiting disease in experimental autoimmune encephalomyelitis (EAE - a model for multiple sclerosis (MS)) (Metzler and Wraith (1993) Int. Immunol. 5:1159-1165; Liu and Wraith (1995) Int. Immunol. 7:1255-1263; Anderton and Wraith (1998) Eur. J. Immunol. 28:1251-1261); and experimental models of arthritis, diabetes, and uveoretinitis (reviewed in Anderton and Wraith (1998) as above). This has also been demonstrated as a means of treating an ongoing disease in EAE (Anderton and Wraith (1998) as above).
The use of tolerogenic peptides to treat or prevent disease has attracted considerable attention. One reason for this is that it has been shown that certain tolerogenic epitopes can down-regulate responses of T cells for distinct antigens within the same tissue. This phenomenon, known as "bystander suppression" means that it should be possible to induce tolerance to more than one epitope (preferably all epitopes) within a given antigen, and to more than one antigen for a given disease, using a particular tolerogenic peptide (Anderton and Wraith (1998) as above). This would obviate the need to identify all of the pathogenic antigens within a particular disease.
Peptides are also a favourable option for therapy because of their relatively low cost and the fact that peptide analogues can be produced with altered immunological properties. Peptides may thus be modified to alter their interactions with either MHC or TCR.
One possible problem with this approach is that it has been shown that not all peptides which act as T cell epitopes are capable of inducing tolerance. The myelin basic protein (MBP) peptide 89-101 is an immunodominant antigen after immunisation and is also a very effective immunogen both in terms of priming for T cell reactivity and induction of EAE. However, this peptide has been shown to be ineffective at inducing tolerance when administered in solution (Anderton and Wraith (1998), as above).
A number of explanations for the observed hierarchy in the ability of T cell epitopes to induce tolerance have been proposed (reviewed in Anderton and Wraith (1998) as above). In particular, it has been proposed that there is a correlation between the affinity of the peptide for the MHC and tolerogenicity (Liu and Wraith (1995) as above), but this does not tally with some of the observations. For example, MBP[89-101], which is not tolerogenic, binds to I-A with relatively high affinity. It is thus not straightforward to predict which peptides will induce tolerance.
The present inventors have shown that if a peptide epitope is of an appropriate size to be presented by immature APC without antigen processing, it can induce immunological tolerance (International patent application number PCT/GBOl/03702). The observation
that some T cell epitopes are tolerogenic and others are incapable of inducing tolerance can therefore be explained by the fact that some epitopes require further processing before they are capable of being presented by an MHC molecule. These epitopes which require further processing do not induce tolerance when administered in a soluble form, despite their capacity to induce disease when injected in combination with adjuvant.
The epitopes which do not require further processing are capable of inducing tolerance, and have been termed "apitopes" (Antigen Processing Independent epiTOPES) by the inventors.
The present inventors have examined the region 131-158 of MBP, and found a number of peptides which can be presented by fixed antigen presenting cells to T-cells
These peptides are defined as apitopes, because they are capable of binding to MHC class I or II without further processing.
In a first aspect, therefore, the present invention provides an apitope which comprises a portion of the region 131-158 of myelin basic protein.
The present inventors have also identified two minimal epitopes in this region which are recognised by particular T-cell clones. The peptide may comprise one or both of these epitopes which are MBP 142-152 and 140-148.
In a preferred embodiment the peptide is selected from the following myelin basic protein peptides: 134-148,135-149,136-150,137-151,138-152,139-153,140-154.
In a second aspect, the present invention provides a pharmaceutical composition comprising one or more peptide according to the first aspect of the application.
In a third aspect, the present invention provides a method for treating and/or preventing a disease in a subject in need of same which comprises the step of administering a peptide according to the first aspect of the invention to the subject.
The present invention also provides the use of a peptide according to the first aspect of the invention in the manufacture of a medicament for use in the treatment and/or prevention of a disease.
The peptides of the present invention are useful in the prevention and/or treatment of multiple sclerosis.
Figure 1 shows the response of T cell clone MS60:D2 to presentation of nested MBP peptides in the region 131 -158 by APC.
Figure 2 shows the response of T cell clone N5 to presentation of MBP peptide 140-154 by APC.
Figure 3 shows the response of T cell clone N5 to presentation of nested MBP peptides in the region 136-157 by APC.
In a first aspect, the present invention relates to a peptide.
The term "peptide" is used in the normal sense to mean a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the a-amino
and carboxyl groups of adjacent amino acids The term includes modified peptides and synthetic peptide analogues.
A peptide of the present invention may be any length that is capable of binding to an MHC class I or II molecule without further processing.
Peptides that bind to MHC class I molecules are typically 7 to 13, more usually 8 to 10 amino acids in length. The binding of the peptide is stabilised at its two ends by contacts between atoms in the main chain of the peptide and invariant sites in the peptide-binding groove of all MHC class I molecules. There are invariant sites at both ends of the groove which bind the amino and carboxy tennini of the peptide. Variations is peptide length are accommodated by a kinking in the peptide backbone, often at proline or glycine residues that allow the required flexibility.
Peptides which bind to MHC class II molecules are typically between 8 and 20 amino acids in length, more usually between 10 and 17 amino acids in length, and can be much longer. These peptides lie in an extended conformation along the MHC II peptide-binding groove which (unlike the MHC class I peptide-binding groove) is open at both ends. The peptide is held in place mainly by main-chain atom contacts with conserved residues that line the peptide-binding groove.
The peptide of the present invention may be made using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example, peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
The peptide may alternatively be made by recombinant means, or by cleavage from a longer polypeptide. For example, the peptide may be obtained by cleavage from myelin
basic protein. The composition of a peptide may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure).
The peptide of the invention is derivable from region 131-158 of MBP. Preferably the peptide is derivable from a fragment of the antigen which arises by natural processing of the antigen by an APC.
For practical purposes, there are various other characteristics which the peptide should show. For example, the peptide should be soluble at a concentration which permits its use in vivo. Preferably the peptide should be soluble at concentrations of up to 0.5 mg/ml, more preferably the peptide should be soluble at concentrations of up to 1 mg/ml, most preferably the peptide should be soluble at concentrations of up to 5 mg/ml.
For intranasal administration the maximum volume of dose which can be taken up using current procedures is approximately 200µ1 per nostril. If the peptide is soluble at 1 mg/ml, a double dose to each nostril enables 800ug to be given to the patient. It is unusual to give more that 5mg in any individual dose.
It is also important that the peptide is sufficiently stable in vivo to be therapeutically useful. The present inventors have found that in vivo, 30 minutes after administration the total amount of a test peptide drops to about 50%, 4 hours after administration the amount drops to about 30%, but that after 5 days the peptide is still detectable (at about 5%). The half-life of the peptide in vivo should be at least 10 minutes, preferably at least 30 minutes, more preferably at least 4 hours, most preferably at least 24 hours.
The present inventors have found that following intranasal administration, the amount of peptide in the draining lymph node peaks at about 4 hrs following administration, however peptide is still detectable (at levels of about 5% maximum) after 5 days. Preferably the peptide is sufficiently stable to be present at a therapeutically active concentration in the draining lymph node for long enough to exert a therapeutic effect.
The peptide should also demonstrate good bioavailability in vivo. The peptide should maintain a conformation in vivo which enables it to bind to an MHC molecule at the cell surface without due hindrance.
Myelin Basic Protein (MBP)
MBP is an antigen from myelin, which has been previously characterised and sequenced (Roth et al (1987) J. Neurosci. Res. 17(4) 321-328; Accession number M30516).
The peptides of the present invention are derivable from MBP region 131-158.
Preferably the peptide is or comprises one or more of the minimal T-cell epitopes in the region, for example the minimal epitope MBP142-152 or 140-148.
Preferably the peptide is selected from the following myelin basic protein peptides:
(Table removed)
Antigen Processing Independent Epitopes (APITOPES)
The peptide of the present invention is capable of binding to an MHC class I or II molecule (in the peptide-binding groove) without further processing. Such peptides are known herein as "apitopes" (Antigen Processing Independent epiTOPES).
Cell surface presentation of peptides derived from a given antigen is not random and tends to be dominated by a small number of frequently occurring epitopes. The dominance of a particular peptide will depend on many factors, such as relative affinity for binding the MHC molecule, spatio-temporal point of generation within the APC and resistance to degradation. The epitope hierarchy for an antigen can change with progression of an immune response, which has important implications for self-tolerance and autoimmunity. Immunodominant determinant regions are likely to be good tolerogens. Hence, in a preferred embodiment, the apitope of the present invention is based on a dominant epitope.
However, after a primary immune response to the immunodominant peptides, epitope "spreading" may occur to sub-dominant determinants (Lehmann et al (1992) Nature 358:155-157). Presentation of sub-dominant epitopes may be important in triggering autoimmunity. The apitope of the present invention may, therefore be based on a subdominant epitope.
For any given antigen, cryptic epitopes may also exist. Cryptic epitopes are those which can stimulate a T cell response when administered as a peptide but which fail to produce such a response when administered as a whole antigen. It may be that during processing of the antigen into peptides in the APC the cryptic epitope is destroyed.
A cryptic epitope may act as an apitope in vitro, in that it may be capable of binding to an MHC molecule without further processing, and inducing anergy in a T cell which recognises the cryptic epitope. However, such an apitope would be unlikely to be therapeutically useful because it should be incapable of tolerising T cells which recognise a naturally processed epitope of the antigen.
Epitopes for an antigen may be identified by measuring the T cell response to overlapping peptides spanning the entire antigen (see below) when presented by APC. Such studies usually result in "nested sets" of peptides, and the minimal epitope for a particular T cell line/clone can be assessed by measuring the response to truncated peptides.
It cannot be assumed that a minimal epitope of an antigen will behave as an apitope. It may well be that amino acids flanking the minimal epitope will be required for optimal binding to the MHC. The apitope should be designed to cover the possibility that there may be subtle differences between the minimal epitopes of different T cell clones.
It should be emphasised mat it may not be possible to identify an apitope for all epitopes. There is clear evidence that some epitopes bind MHC in a way that is dependent on MHC-loading in endosomes and hence require processing (Viner et al (1995) Proc. Natl. Acad. Sci. 92:2214-2218). This is another reason why one cannot assume that each minimal epitope will inevitably behave as an apitope.
Identification of peptides containing T cell epitopes
There are a number of methods known in the art to identify the T cell epitopes within a given antigen.
Naturally processed epitopes may be identified by mass spectrophotometnc analysis of peptides eluted from antigen-loaded APC. These are. APC that have either been encouraged to take up antigen, or have been forced to produce the protein intracellularly by transformation with the appropriate gene. Typically APC are incubated with protein either in solution or suitably targeted to the APC cell surface. After incubation at 37°C the cells are lysed in detergent and the class II protein purified by, for example affinity chromatography. Treatment of the purified MHC with a suitable chemical medium (for example, acid conditions) results in the elution of peptides from the MHC. This pool of peptides is separated and the profile compared with peptide from control APC treated in the same way. The peaks unique to the protein expressing/fed cells are analysed (for example by mass spectrometry) and the peptide fragments identified. This procedure usually generates information about the range of peptides (usually found in "nested sets") generated from a particular antigen by antigen processing.
Another method for identifying epitopes is to screen a synthetic library of peptides which overlap and span the length of the antigen in an in vitro assay. For example, peptides
which are 15 amino acids in length and which overlap by 5 or 10 amino acids may be used. The peptides are tested in an antigen presentation system which comprises antigen presenting cells and T cells. For example, the antigen presentation system may be a murine splenocyte preparation, a preparation of human cells from tonsil or PBMC. Alternatively, the antigen presentation system may comprise a particular T cell line/clone and/or a particular antigen presenting cell type.
T cell activation may be measured via T cell proliferation (for example using H-thymidine incorporation) or cytokine production. Activation of THl-type CD4+ T cells can, for example be detected via IFNy production which may be detected by standard techniques, such as an ELISPOT assay.
Overlapping peptide studies usually indicate the area of the antigen in which an epitope is located. The minimal epitope for a particular T cell can then be assessed by measuring the response to truncated peptides. For example if a response is obtained to the peptide comprising residues 1-15 in the overlapping library, sets which are truncated at both ends (i.e. 1-14, 1-13, 1-12 etc. and 2-15, 3-15, 4-15 etc.) can be used to identify the minimal epitope.
Antigen Processing Independent Presentation Systems (APIPS)
Once an epitope has been identified, the next step is to investigate whether it also behaves as an apitope.
An apitope must be presented to T cells without the need for antigen processing. Having identified peptides containing T cell epitopes, apitopes may be identified using a processing free system. Truncated peptides and peptide analogues may be tested for activation using an antigen processing independent presentation system (APIPS).
Examples of APIPS include:
a) fixed APC (with or without antibodies to CD28);
b) Lipid membranes containing Class I or II MHC molecules (with or without antibodies to CD28); and
c) purified natural or recombinant MHC in plate-bound form (with or without antibodies to CD28).
It is known to use fixed APC to investigate T cell responses, for example in studies to investigate the minimal epitope within a polypeptide, by measuring the response to truncated peptides (Fairchild et al (1996) Int Immunol. 8:1035-1043). APC may be fixed using, for example formaldehyde (usually paraformaldehyde) or glutaraldehyde.
Lipid membranes (which may be planar membranes or liposomes) may be prepared using artificial lipids or may be plasma membrane/microsomal fractions from APC.
In use, the APIPS may be applied to the wells of a tissue culture plate. Peptide antigens are then added and binding of the peptide to the MHC portion of the APIPS is detected by addition of selected T cell lines or clones. Activation of the T cell line or clone may be measured by any of the methods known in the art, for example via 3H-thymidine incorporation or cytokine secretion.
The peptides of the present invention should be capable of inducing tolerance to MBP as they are apitopes for this antigen.
As used herein, the term "tolerogenic" means capable of inducing tolerance.
Tolerance is the failure to respond to an antigen. Tolerance to self antigens is an essential feature of the immune system, when this is lost, autoimmune disease can result. The adaptive immune system must maintain the capacity to respond to an enormous variety of infectious agents while avoiding autoimmune attack of the self antigens contained within its own tissues. This is controlled to a large extent by the sensitivity of immature T lymphocytes to apoptotic cell death in the thymus (central tolerance). However, not all
self antigens are detected in the thymus, so death of self-reactive thymocytes remains incomplete. There are thus also mechanisms by which tolerance may be acquired by mature self-reactive T lymphocytes in the peripheral tissues (peripheral tolerance). A review of the mechanisms of central and peripheral tolerance is given in Anderton et al (1999) {Immunological Reviews 169:123-137).
Tolerance may result from or be characterised by the induction of anergy in at least a portion of CD4+ T cells. In order to activate a T cell, a peptide must associate with a "professional" APC capable of delivering two signals to T cells. The first signal (signal 1) . is delivered by the MHC-peptide complex on the cell surface of the APC and is received by the T cell via the TCR. The second signal (signal 2) is delivered by costimulatory molecules on the surface of the APC, such as CD80 and CD86, and received by CD28 on the surface of the T cell. It is thought that when a T cell receives signal 1 in the absence of signal 2, it is not activated and, in fact, becomes anergic. Anergic T cells are refractory to subsequent antigenic challenge, and may be capable of suppressing other immune responses. Anergic T cells are thought to be involved in mediating T cell tolerance.
Without wishing to be bound by theory, the present inventors predict that peptides which require processing before they can be presented in conjunction with MHC molecules do not induce tolerance because they have to be handled by mature antigen presenting cells. Mature antigen presenting cells (such as macrophages, B cells and dendritic cells) are capable of antigen processing, but also of delivering both signals 1 and 2 to a T cell, leading to T cell activation. Apitopes, on the other hand, will be able to bind class II MHC on immature APC. Thus they will be presented to T cells without costimulation, leading to T cell anergy and tolerance.
Of course, apitopes are also capable of binding to MHC molecules at the cell surface of mature APC. However, the immune system contains a greater abundance of immature than mature APC (it has been suggested that less than 10% of dendritic cells are activated, Summers et al. (2001) Am. J. Pathol. 159: 285-295). The default position to an apitope will therefore be anergy/tolerance, rather than activation.
it has been shown that, when tolerance is induced by peptide inhalation, the capacity of antigen-specific CD4+ T cells to proliferate is reduced. Also, the production of IL-2, IFN-y and IL-4 production by these cells is down-regulated, but production of IL-10 is increased. Neutralisation of IL-10 in mice in a state of peptide-induced tolerance has been shown to restore completely susceptibility to disease. It has been proposed that a population of regulatory cells persist in the tolerant state which produce IL-10 and mediate immune regulation (Burkhart et al (1999) Int. Immunol. 11:1625-1634).
The induction of tolerance can therefore be monitored by various techniques including:
(a) reduced susceptibility to contract the disease for which the peptide is a target epitope in vivo;
(b) the induction of anergy in CD4+ T cells (which can be detected by subsequent challenge with antigen in vitro);
(c) changes in the CD4+ T cell population, including
(i) reduction in proliferation;
(ii) down-regulation in the production of IL-2, BFN-y and IL-4; and (iii) increase in the production of IL-10.
Target diseases
The peptide of the invention may be used in the treatment and/or prevention of a disease.
The peptides of the present invention are particularly useful in the treatment and/or prevention of multiple sclerosis (MS). Multiple sclerosis (MS) is a chronic inflammatory disease characterised by multiple demyelinating lesions disseminated throughout the CNS white matter and occurring at various sites and times (McFarlin and McFarland, 1982 New England J. Medicine 307:1183-1188 and 1246-1251). MS is thought to be mediated by autoreactive T cells.
MBP is immunogenic and MBP-specific T lymphocytes have encephalitogenic activity in animals (Segal et al., 1994 J. Neuroimmunol. 51:7-19; Voskuhl et al., 1993 J. Neuroimmunol 42:187-192; Zamvil etal, 1985 Nature 317:355-8).
Pliarmaceutical composition
In a second aspect, the present invention relates to a pharmaceutical composition comprising one or more peptide(s) of the first aspect of the invention. The composition may also comprise one or more apitopes from another region of MBP or from a different antigen.
The present inventors predict that, despite "bystander suppression" it may be necessary to target a number of different T cell clones in order to induce tolerance effectively. Hence a plurality of peptides may be administered to an individual in order to prevent or treat a disease.
The pharmaceutical composition may, for example comprise between 1 and 50 apitopes, preferably between 1 and 15 apitopes. If there is more than one apitope, preferably the apitopes are either all able to bind to MHC class I, or all able to bind MHC class II, without further processing.
Where there are two or more apitopes, the pharmaceutical composition may be in the form of a kit, in which some or each of the apitopes are provided separately for simultaneous, separate or sequential administration.
Alternatively (or in addition) if the pharmaceutical composition (or any part thereof) is to be administered in multiple doses, each dose may be packaged separately.
The pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of the or each apitope and optionally a pharmaceutically acceptable carrier, diluent or excipient.
Also, in the pharmaceutical compositions of the present invention, the or each apitope may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilising agent(s).
The peptide may be administered in soluble form in the absence of adjuvant.
Preferably the peptide is administered by a mucosal route.
Studies have shown that peptide, when given in soluble form intraperitoneally (i.p.), intravenously (i.v.) or intranasally (i.n.) or orally can induce T cell tolerance (Anderton and Wraith (1998) as above; Liu and Wraith (1995) as above; Metzler and Wraith (1999) Immunology 97:257-263).
Preferably the peptide is administered intranasally.
Studies in mice have demonstrated that the duration of peptide administration required to induce tolerance depends on the precursor frequency of T cells in the recipient (Burkhart et al (1999) as above). In many experimental studies, it has been shown that repeated doses of peptide are required to induce tolerance (Burkhart et al (1999) as above). The exact dose and number of doses of peptide will therefore depend on the individual, however, in a preferred embodiment a plurality of doses is administered.
If a plurality of peptides is administered simultaneously, they may be in the form of a "cocktail" which is suitable for administration in single or multiple doses. Alternatively it may be preferably to give multiple doses but vary the relative concentrations of the peptides between doses.
In a preferred embodiment a "dose escalation" protocol may be followed, where a plurality of doses is given to the patient in ascending concentrations. Such an approach has been used, for example, for phospholipase A2 peptides in immunotherapeutic applications against bee venom allergy (Muller et al (1998) J. Allergy Clin Immunol. 101:747-754 and Akdis et al (1998) J. Clin. Invest. 102:98-106).
The following examples serve to illustrate the present invention, but should not be construed as a limitation thereof. The invention particularly relates to the specific embodiments described in these examples
EXAMPLE 1 - Identification of apitopes within MBP region 134-154
Materials and Methods
Human MBP is prepared from brain white matter as described by Deibler et al. (Deibler et ai, 1972 Preparative Biochemistry 2:139), and its purity assessed by SDS-PAGE. MBP and Mycobacterium tuberculosis purified protein derivative (PPD) (UK Central Veterinary Laboratory, Surrey) are used in proliferative assays at previously determined optimal concentrations; the optimum concentration for each antigen is 20 ug/ml. A panel of 15-mer overlapping peptides spanning MBP region 131-158 are synthesized using standard F-moc chemistry on an Abimed AMS 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany). Each peptide is displaced by 1 aa and overlapped by 14 aa.
Tissue culture medium
RPMI-1640 medium supplemented with 20mM HEPES (Sigma, Poole, UK), penicillin (l00U/ml), streptomycin sulphate (100 mg/ml), and 4 mM L-glutamine (all from Life Technologies, Paisley, Scotland), is used as the tissue culture medium. Medium without serum is used for washing lymphoid cells and TCL. For all culture conditions and assays, medium is supplemented with 10 % heat inactivated autologous plasma.
Generation of T cell clones
MBP-specific T cell lines (TCL) are generated from 8 MS patients and 2 healthy control donors. PBMC from each subject are separated as described above and plated out at lxl06 cells/ml in 6-well plates in the presence of MBP (50 ug/ml); a portion of PBMC from each subject is regularly frozen and stored for subsequent restimulations. Seven days later the
cells are ted with fresh medium containing 2% IL-2 (Lymphocult-HT; Biotest LTD., Birmingham, UK), and on day 12 of culture all cells are restirnulated with antigen, EL-2 and irradiated (2500 Rad) autologous PBMC as a source of antigen presenting cells (APC), at a cell ratio of IT cell:5 APC. Cells are expanded in IL-2 every 3-4 days, and on day 14 are restirnulated with antigen, IL-2 and PBMC, as described above. On the day of the first restimulation cells are examined for specific proliferation to MBP. Briefly, 2x10 T cells and lxl 05 irradiated autologous PBMC are cultured in triplicate, in 96-well round-bottom plates, in the presence of MBP. Cells are cultured for 2 days and pulsed with ( H)-Thymidine at 0.4 µCi/well during the last 18 hours of the culture. Cells are then harvested as described above, and a TCL is considered to be MBP-specific with a Scpm >1000 and a . SI>3.
Following 3 restimulation/expansion cycles TCL are cloned using PEA (Sigma, Poole, Dorset, UK)) in the presence of autologous irradiated PBMC as APC. T cells are plated under, limiting dilution conditions at 0.1 cell/well, 0.3 cell/well and 1 cell/well and cultured in Terasaki plates (Nunc International, Costar) with lx104 irradiated PBMC, 5µg/ml PHA, and 2% IL-2. After 10-12 days, growth-positive wells are expanded onto 96-well round-bottom plates, using lxl05 irradiated PBMC, 5µg/ml PHA and IL-2. Three days later wells are fed with fresh medium containing IL-2, and on day 7 the clones are expanded onto 48-well plates using 5xl05 irradiated PBMC, PHA and IL-2; at this point clones are tested in proliferation assays for specific responses to MBP. MBP-specific clones are expanded a week later onto 24-well plates, using lxl06 irradiated PBMC with PHA or Dynabeads (Dynal, UK) and IL-2. The clones are maintained in 24-well plates using a 7-10 day restimulation/expansion cycle.
Proliferation assay
Live or p-formaldehyde fixed Mgar (HLA-DR2 +ve) cells were incubated with peptides in serum or in serum alone, together with T cells. The T cell proliferative response was measured by 3H-thymidine uptake as follows. Triplicate aliquots of 100 u.1 of each culture were grown in a 96-well round bottom microtitre plate for 48 hours, then pulsed with 0.4 µCi [3H]-Thymidine (Amersham International, Amersham, UK). After 20 hours cells are harvested onto glass fibre mats (LKB-Wallac, Turku, Finland) using a Mach 111 harvester
96 (Tomtec, Orange, New Jersey, USA). [3H]- Thymidine incorporation is determined using a Microbeta liquid scintillation counter (LKB-Wallac). Test wells containing antigen are considered positive when the 5cpm >1000 and the Stimulation Index (SI)3, where SI = CPM antigen containing culture/CPM culture without antigen.
The response of T cell clone MS60:D2 to presentation of nested MBP peptides in the region 131-158 is shown in Figure 1. Peptides 134-148, 135-149, 136-150, 137-151, 138-152,139-153 and 140-154 are defined as apitopes as they could be presented by fixed APC to T cells without further processing.
The response of T cell clone N5 to presentation of peptide 140-154 is shown in Figure 2. This further confirms that peptide 140-154 can be presented by fixed APC without further processing.
EXAMPLE 2 - Identification of the minimum epitope recognised by T cell clones N5 and MS60:D2
Live Mgar cells were incubated with overlapping peptides from MBP region 136-157 in serum or serum alone, together with N5 T cells. T cell proliferation was measured by 3H-thymiding uptake.
The results are shows in Figure 3. The minimal epitope recognised by T cell clone N5 is MBP 142-152.
A similar experiment was performed for T cell clone MS60:D2 (Fig. 1, unfixed Mgar). The minimal epitope recognised by this T cell clone is MBP 140-148.
various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or biology or related fields are intended to be covered by the present invention. All publications mentioned in the above specification are herein incorporated by reference.

1. A peptide which is capable of binding to an MHC class I or II molecule without further
processing, and which is selected from the following myelin basic protein peptides:
2. A peptide as claimed in claim 1 which is myelin basic protein peptide GFKGVDAQGTLSKIF (140-154).







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2044-delnp-2004-description (complete).pdf
























Patent Number 249728
Indian Patent Application Number 2044/DELNP/2004
PG Journal Number 45/2011
Publication Date 11-Nov-2011
Grant Date 04-Nov-2011
Date of Filing 15-Jul-2004
# Inventor's Name Inventor's Address
PCT International Classification Number C07K7/00
PCT International Application Number PCT/GB03/00399
PCT International Filing date 2003-01-30
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0202399-2 2002-02-01 U.K.