Patent Application
20110142866
The present invention relates to linear and cyclic peptide analogues of myelin antigens and conjugates thereof. The peptides and conjugates of the invention are useful candidates for the immunotherapy of Multiple Sclerosis (MS).
Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and is characterized by a dysregulated T cell response. An association between MHC class II alleles and disease has been observed in MS patients, in particular HLA-DR1, HLA-DR2 and HLA-DR4 (Valli et al., J. Clin. Invest. 1993).
Current peptide therapies of multiple sclerosis include treatment with interferons (Interferon beta-1α and Interferon beta-1β) and glatiramer acetate (copolymer-1) which is a mixture of synthetic polypeptides comprised of the major aminoacids Glu, Gln, Lys, Arg of MBP. These immunomodulators have been approved by the FDA for patients with relapsing-remitting MS. Interferons given by subcutaneous or intramuscular injection reduce the frequency, severity and duration of exacerbation but their impact on preventing disability over the long-term is not yet established. Side effects are also common and include reactions at the injection site, fever, myalgia and flu-like syndrome. So far the reported benefits from the use of interferons and copolymer are marginal and there is therefore a need for improved therapeutics. Another approach under clinical investigation for autoimmune suppression is the oral administration of autoantigens. Orally administered antigens suppress autoimmunity in animal models, including EAE, collagen and adjuvant-induced arthritis, uveitis and diabetes in the non-obese diabetic mouse. Low doses of oral antigen induce antigen-specific regulatory T-cells which act by releasing inhibitory cytokines such as transforming growth factor-beta, IL-4, and IL-10 at the target organ. Thus, one can suppress inflammation at a target organ by orally administering an antigen derived from the side of inflammation, even if it is not the target of the autoimmune response. Initial human trials of orally administered antigen have shown positive findings in patients with MS and Rheumatoid Arthritis. A double-blind, placebo-controlled, phase III multi-centre trial of oral myelin in relapsing-remitting multiple sclerosis patients is in progress, as are phase II clinical trials investigating the oral administration of type II collagen in rheumatoid arthritis, S-antigen in uveitis and insulin in type I diabetes. This promising method is advantageous because it allows oral administration in contrast to previous methods using interferons and copolymer-1. However, issues related to the peptide nature and cost of administered substance renders the non-peptide mimetic approach, even in its infancy, an attractive goal to pursue.
In the immunotherapeutic approach towards the development of therapeutic vaccines for MS, the assumption is that MBP epitopes or their analogues can actively inhibit or prevent disease through the activation of antigen-specific regulatory T cells, or antibodies related to myelin sheath destruction. The myelin sheath is constituted from the proteins: MBP, Proteolipid Protein (PLP), Myelin Oligodentrocyte Glycoprotein (MOG) and heat-shock protein which are implicated in MS. Thus, epitopes of these myelin sheath proteins are targets for immunotherapeutic techniques. In areas of inflammation in MS, antibodies against the minor protein MOG have been demonstrated. MOG antibodies are related to significant myelin disruption, probably by coating the myelin so that macrophages can engulf and destroy coated sections of myelin, blocking nerve impulses temporarily or permanently. Thus, it is known that antibodies play a role in MS, and cooperate with antigen presenting cells in myelin destruction. Blocking the effects of these MOG antibodies with secondary antibodies or non-peptide mimetics may be an important avenue for future therapy.
Another direction in the immunotherapy of autoimmune diseases is the use of Multiple-Antigen Peptide (MAP) systems introduced by Tam [Tam et al., 1990]. This system represents a novel approach to anti-peptide antibody production. It is built on a resin which bears a core of radial branching lysine dendrites on which a number of copies of a given peptide antigen can be synthesized. Lysine derivatives have been used for the solid phase synthesis of lysine cores suitable for the assembly of antigenic peptides. These peptides have found applications in raising antibodies and in the preparation of synthetic vaccines. On a lysine core several different epitopes of a protein or of different proteins can be assembled to create the required antigenic synthetic protein. Following assembly of the peptide on the MAP core, the peptide is deprotected and cleaved from the support using standard techniques, yielding a highly immunogenic macromolecular structure without the need for conjugation to a carrier protein. The MAP approach has been shown to yield higher antibody titers than using monomeric peptide-conjugates. Alternatively, the two epitopes can be synthesized on alternate branches of the lysine core, using Boc and Fmoc chemistry. T-cell and B-cell epitopes can also be combined sequentially within a single linear sequence.
Another challenging strategy in the immunotherapy of MS or other autoimmune diseases is the use of peptide poly-lysine analogues conjugated to reduced mannan via KLH linker in its oxidized or reduced form to develop Th1/Th2 responses followed by release of appropriate cytokines [Apostolopoulos, et al., 1995, Apostolopoulos, et al., 1996, Apostolopoulos, et al., 1998, Lofthouse, et al., 1997]. The aim of this approach is the development of an immunotherapeutic approach for prevention or control of disease. Presently, antigen peptides from MBP, PLP and MOG proteins conjugated to mannan in its oxidized or reduced form are under investigation. In order to study their effect on cellular proliferation, antibody production and cytokines secretion assays were determined in Lewis rats, mouse models and in human peripheral blood T-cells. These studies include the development of recently rationally designed constrained cyclic antagonist peptide analogues based on MBP epitope 87-99 which suppresses the development of clinical E.A.E., CNS inflammation and demyelination. The use of oxidized or reduced mannan to develop a Th1 or Th2 response (and the appropriate cytokines) to myelin peptides expressed in MS constitutes a novel strategy for the treatment of disease.
Although the pathology of MS remains unclear, there is evidence that T cells recognizing encephalitogenic epitopes of myelin, such as Myelin Basic Protein (MBP), play a pathogenic role in the induction of MS. Studies have shown that T cell responses in patients are associated with the recognition of the 81-105 region of MBP (QDENPVVHFFKNIVTPRTPPPSQGK; SEQ ID NO: 1) (Valli et al., J. Clin. Invest. 1993), and with highest affinity and binding to HLA-DR2 for the peptide epitope MBP83-99 (ENPVVHFFKNIVTPRTP; SEQ ID NO: 2). T cell recognition of this region of MBP has also been shown in healthy individuals, but at relatively low precursor frequencies. The binding of MBP83-99 to HLA-DR2 is via hydrophobic V87 and F90 residues, whilst, H88, F89, and K91 are TCR contact residues.
The pathogenic role of autoimmune T cells recognizing encephalitogenic epitopes of MBP has also been noted in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Similar clinical and histopathological features to MS can be induced in susceptible mouse strains by immunization of myelin components. EAE is mediated by CD4+ T cells of the Th1 phenotype (IFNγ). Similar to MS, EAE susceptibility is dependent on the MHC background of the mouse and different peptides are immunogenic and induce EAE in different mouse strains. Myelin oligodendrocyte glycoprotein (MOG) residues 35-55 induce chronic EAE in C57BL/6 mice, MBP residues 74-85 induce acute EAE in Lewis rats and proteolipid protein (PLP) residues 139-153 induce acute EAE in SJL/J mice (Zamvil et al., Annu Rev Immunol. 1990). The SJL/J mouse strain (H-2s haplotype), is commonly used for EAE, since numerous histopathological, clinical and immunological features resemble that of human MS, more similarly compared to other mouse or rat strains (Sommer et al., J Neuroimmunol, 1997). The SJL/J mouse does not express the H2-E-α chain, thus, H2-As (I-As) is the only functional MHC class II molecule in the SJL/J mouse. In the SJL/J mouse strain residues from the encephalitogenic epitope MBP81-100 have been shown to bind with high affinity. In fact the minimum epitope required for binding is MBP83-99 (Kalbus et al., Eur J. Immunol. 2001). Thus, the MBP83-99 peptide may act as a lead peptide for the design of altered peptide ligands and peptide analogues, which could be used to alter T cell responses in the SJL/J mouse model and may aid in new therapeutic approaches in autoimmune diseases.
The present invention seeks to provide altered peptide ligands and conjugates thereof which have potential therapeutic applications in the treatment of autoimmune disorders, and in particular, multiple sclerosis.
A first aspect of the invention relates to a peptide comprising the amino acid sequence of formula (I),
wherein at least one of K91 and P96 is substituted by a natural or unnatural amino acid, wherein said peptide is in linear or cyclic form.
A second aspect of the invention relates to a peptide comprising the amino acid sequence of formula (Ia),
wherein each of K91 and P96 is substituted by a natural or unnatural amino acid.
A third aspect of the invention relates to a peptide comprising the amino acid sequence of formula (Ib),
wherein at least one of K91 and P96 is substituted by an amino acid selected from R, E, F and Y.
A fourth aspect of the invention relates to a cyclic peptide comprising the amino acid sequence of formula (Ic),
or a variant thereof wherein one or two amino acids are substituted by a natural or unnatural amino acid.
Compared to linear peptides, cyclic peptides have been considered to have greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectively, and improved pharmacodynamic properties.
A fifth aspect of the invention relates to a peptide comprising the amino acid sequence of formula (IIa),
wherein K91 is the amino acid A and P96 is substituted by the amino acid A, wherein said peptide is in linear form.
A sixth aspect of the invention relates to a peptide comprising the amino acid sequence of formula (IIb),
wherein K91 is substituted by the amino acid A and P96 is substituted by the amino acid A, wherein said peptide is in cyclic form.
A seventh aspect of the invention relates to a conjugate which comprises a peptide as defined above and mannan.
Advantageously, biological assays have demonstrated that specific altered peptide ligands of the epitope MBP83-99 (E83NPVVHFFK91NIVTP96RTP; SEQ ID NO: 2) with modifications at positions 91 and 96, and which are conjugated to reduced mannan:
An eighth aspect of the invention relate to a peptide or conjugate as described above for use in medicine.
A ninth aspect of the invention relates to the use of a peptide or a conjugate as described above in the preparation of a medicament for treating an immune disorder.
A tenth aspect of the invention relate to a pharmaceutical composition comprising a peptide or a conjugate as defined above, admixed with a pharmaceutically acceptable diluent, excipient or carrier.
An eleventh aspect of the invention relates to the use of a peptide or a conjugate as described above in an assay for elucidating agents capable of regulating experimental autoimmune encephalomyelitis (EAE) or regulating multiple sclerosis.
A twelfth aspect of the invention relates to a process for preparing a conjugate as described above, said process comprising the steps of:
(i) reacting a peptide as defined above with a Keyhole Limpit Hemocyanin (KLH);
(ii) reacting the product formed in step (i) with oxidized mannan; and
(iii) reducing the product formed in step (ii) to form a reduced mannan conjugate.
Another aspect of the invention relates to a method of treatment comprising inversely modulating the Th1 and Th2 responses of lymphocytes in a subject so as to increase the Th2 response and decrease the Th1 response respectively above and below the levels prevailing without said treatment, said method comprising administering to the subject a peptide or a conjugate as described above.
Another aspect of the invention relates to a method of treatment comprising administering to a subject a peptide or a conjugate as described above, wherein said treatment enhances/induces the Th2-type response in a subject, as compared to the Th2-type response prior to peptide/conjugate administration and reduces the Th1-type response as compared to the Th1-type response prior to peptide/conjugate administration.
Another aspect of the invention relates to a method of diverting the immune response in a subject from a Th1 response to a Th2 response, said method comprising administering to the subject a peptide or a conjugate as described above.
A further aspect of the invention relates to a method of selectively reducing the Th1 immune response in a subject relative to the Th2 immune response, said method comprising administering to the subject a peptide or a conjugate as described above.
A further aspect of the invention relates to a method of selectively increasing the Th2 immune response in a subject relative to the Th1 immune response, said method comprising administering to the subject a peptide or a conjugate as described above.
Yet another aspect of the invention relates to a method of inducing a Th2-specific immune response in a subject, said method comprising administering to the subject a peptide or a conjugate as described above.
A further aspect of the invention relates to the use of a peptide or a conjugate as described above for treating a disorder associated with an imbalance in lymphokine expression.
A further aspect of the invention relates to the use of a peptide or a conjugate as described above in the preparation of a medicament for treating a disorder associated with an imbalance in lymphokine expression.
Another aspect of the invention relates to a method of treating a disorder associated with an imbalance in lymphokine expression in a subject, said method comprising administering to the subject a peptide or a conjugate as described above.
Another aspect of the invention relates to a vaccine composition comprising a peptide or a conjugate as described above and a suitable adjuvant.
Similarly, a further aspect relates to the use of a peptide or a conjugate as described above in the preparation of a vaccine composition.
The present invention relates to variants of the MBP peptide, and more specifically to variants of particular fragments of the MBP peptide, namely the MBP83-99 peptide and the MBP87-99 peptide.
As used herein, the term “variant” includes any variation wherein; (a) one or more amino acid residues are replaced by a naturally or non-naturally occurring amino acid residue (b) the order of two or more amino acid residues is reversed, (c) both (a) and (b) are present together, (d) a spacer group is present between any two amino acid residues, (e) one or more amino acid residues are in peptoid form, (f) the (N—C—C) backbone of one or more amino acid residues of the peptide has been modified, or any of (a)-(f) in combination. Preferably, the variants arise from one of (a), (b) or (c). More preferably, one or two, amino acids residues are substituted by one or more other amino acid residues. Even more preferably, one amino acid residue is substituted by another amino acid residue. Preferably, the substitution is homologous.
In one embodiment, one or more amino acids are substituted by the corresponding D-amino acids.
Homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 0), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine, a more detailed list of which appears below. Within each peptide more than one amino acid residue may be modified at a time.
As used herein, amino acids are classified according to the following classes;
basic; H, K, R
acidic; D, E
non-polar; A, F, G, I, L, M, P, V, W
polar; C, N, Q, S, T, Y,
(using the internationally accepted single letter amino acid notation)
and homologous and non-homologous substitution is defined using these classes. Thus, homologous substitution is used to refer to substitution from within the same class, whereas non-homologous substitution refers to substitution from a different class or by an unnatural amino acid.
Suitable spacer groups that may be inserted between any two amino acid residues of the peptide include alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, type (e), involving the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134. Type (f) modification may occur by methods such as those described in International Application PCT/GB99/01855.
Where there is variation within the general formulae, it is preferable for amino acid variation, preferably of type (a) or (b), to occur independently at any position. As mentioned above more than one homologous or non-homologous substitution may occur simultaneously. Further variation may occur by virtue of reversing the sequence of a number of amino acid residues within a sequence.
In one embodiment the replacement amino acid residue is selected from the residues of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
The replacement amino acid residue may additionally be selected from unnatural amino acids. Non-natural amino acid derivatives that may be used in the context of the present invention include alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above, to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.
The peptide of the present invention may comprise amino acids in the L or D form, i.e. one or more residues, preferably all the residues may be in the L or D form.
One aspect of the invention relates to a peptide comprising the amino acid sequence of formula (I),
wherein at least one of K91 and P96 is substituted by a natural or unnatural amino acid.
As mentioned above, one embodiment of the invention relates to a peptide comprising the amino acid sequence of formula (Ia),
wherein each of K91 and P96 is substituted by a natural or unnatural amino acid.
In one preferred embodiment, the peptide consists of the amino acid sequence of formula (Ia), wherein each of K91 and P96 is substituted by a natural or unnatural amino acid.
In one preferred embodiment, K91 is substituted by a natural amino acid.
In one preferred embodiment, K91 is substituted by an amino acid selected from A, R, E, F and Y.
In a more preferred embodiment, K91 is substituted by an amino acid selected from A, E and Y.
In one highly preferred embodiment, K91 is substituted by the amino acid Y.
In one preferred embodiment, P96 is substituted by a natural amino acid.
In one particularly preferred embodiment, P96 is substituted by the amino acid A.
In one especially preferred embodiment, K91 is substituted by an amino acid selected from A, R, E, F and Y and P96 is substituted by the amino acid A.
In one preferred embodiment, K91 is substituted by the amino acid R and P96 is substituted by the amino acid A.
In another preferred embodiment, K91 is substituted by the amino acid A and P96 is substituted by the amino acid A.
In one particularly preferred embodiment, the peptide of the invention is selected from the following sequences:
[A91,A96]MBP83-99 is particularly preferred.
In another embodiment, the peptide of the invention comprises the amino acid sequence of formula (Ib),
wherein at least one of K91 and P96 is substituted by an amino acid selected from R, E, F and Y.
In one preferred embodiment, the peptide consists of the amino acid sequence of formula (Ib), wherein at least one of K91 and P96 is substituted by an amino acid selected from R, E, F and Y.
In one highly preferred embodiment, K91 is substituted by an amino acid selected from R, E, F and Y.
More preferably still, the peptide of formula (Ib) is selected from the following:
Peptide [Y91]MBP83-99 is particularly preferred, linear or cyclic.
Preferably, the peptide of formula (Ia) or (Ib) is a linear peptide
Preferably, the peptide of formula (Ia) or (Ib) is selected from the above-mentioned sequences, each of which is attached to mannan, preferably reduced mannan.
Based on the minimum binding peptide MBP83-99 to I-As, the applicants have designed and synthesized analogues by mutating the residue at position K91 with alanine (A), glutamic acid (E), arginine (R), phenylalanine (F) and tyrosine (Y), in order to determine if immune responses can be altered from Th1 to Th2. K91 was mutated as this is one of the principal TCR contact residues when bound to HLA-DR2 and has been predicted to also be a TCR contact residue when bound to H2 I-As (Kalbus et al, Eur J. Immunol. 2001).
In one preferred embodiment, the peptide of the invention is in cyclic form. Compared to linear peptides, cyclic peptides have been considered to have greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectively, and improved pharmacodynamic properties.
Preferably, the peptide consists of the amino acid sequence of the invention cyclised head to tail.
One aspect of the invention relates to a cyclic peptide comprising the amino acid sequence of formula (Ic),
or a variant thereof wherein one or two amino acids are substituted by a natural or unnatural amino acid.
Preferably, the peptide consists of the amino acid sequence of formula (Ic) cyclised head to tail.
Preferably, at least one of K91 and P96 is substituted by a natural or unnatural amino acid.
In one preferred embodiment, one or two amino acids are substituted by an amino acid selected from A, R, E, F, S and Y.
In one preferred embodiment, K91 is substituted by an amino acid selected from A, R, E, F and Y.
In one preferred embodiment, P96 is substituted by a natural amino acid.
More preferably, P96 is substituted by the amino acid A.
In one particularly preferred embodiment, K91 is substituted by an amino acid selected from A, R, F, S and Y and P96 is substituted by the amino acid A.
More preferably still, the K91 residue is substituted by the amino acid A. Thus, in one particularly preferred embodiment, the cyclic analogue is cyclo(83-99)[A91]MBP83-99, with alanine substitution at position 91 and head-tail cyclization between residues 83-99.
In one particularly preferred embodiment, the cyclic peptide is selected from the following SEQ ID NOS: 13-23:
In the above nomenclature, (83-99) denotes the site of cyclisation. For example, amino acid residue 83 is joined to residue 99; i.e. the peptide is cyclised “head-to-tail”. Cyclo(83-99)[Y91]MBP83-99 is particularly preferred.
The cyclization of MBP83-99 peptide and analogues was pursued as a result of conformation studies in linear MBP87-99 indicating a cyclic structure. In particular, design of the cyclic analogues of Human MBP epitope 83-99 was based on Nuclear Magnetic Resonance and Molecular Dynamics. These studies revealed a head to tail intramolecular proximity (ROE connectivity between ζNHLys91-nNHArg97) indicating a pseudocyclic conformation for the immunogenic peptide MBP87-99. Similarly NMR studies of [Arg91, Ala96]MBP87-99 (antagonist-altered peptide ligand) revealed a head to tail intramolecular interaction (ROE connectivity between αVal87-βThr98, αVal87-γThr98).
Cyclization of amino acid sequences results in increased metabolic stability, potency, receptor selectivity and bioavailability all of them reflecting a better pharmacological profile [Scott, et al., 1999, Oligino, et al., 1997]. In particular, cyclic peptides have been used in several cases as synthetic immunogens [Bruggle, et al., 1999], potent vaccine for diabetes [Berezhkovskiy, et al., 1999], antigens for Herpes Simplex Virus [Mezo, et al., 1999], transmembrane ion channels [Chaloin et al., 1999], inhibitors of HIV-1 Tat-TAR interactions in human cells [Tamilarasu et al., 2000], of α-amylase, pancreatic tripsin and as protein stabilizer [Iwai, et al., 1999].
Furthermore, advantages of cyclic analogues over their linear counterparts include:
To date, this is the first time that cyclic analogues of MBP epitopes have been synthesized and shown to prevent the development of EAE.
In one particularly preferred embodiment of the invention, the peptide is of formula A, in which N to C termini (or head-to-tail) cyclo(83-99)MBP83-99 analogues are optionally substituted at positions 91 and/or 96, which are the TCR contact sites.
The MBP72-85 peptide (25 μg) induced an acute monophasic disease with a peak clinical score at day 13 after the initial injection, followed by complete recovery in all animals by day 18. Co-injection of [Arg91, Ala96]MBP87-99 (500 μg) with the potent agonist peptide MBP72-85 (25 μg) completely prevented the development of EAE. Further modification and cyclization resulted in two cyclic antagonist peptides. The Lys side chain and C-terminus amide-linked cyclic analogue, cyclo(91-99)[Ala96] MBP87-99 (Val-His-Phe-Phe-Lys91-Asn-Ile-Val-Thr-Ala96-Arg-Thr-Pro99; SEQ ID NO: 25) had low inhibitory activity in the EAE system while the amide-linked cyclic analogue, cyclo(87-99)[Arg91, Ala96] MBP87-99 was a strong inhibitor of EAE when co-administered with MBP72-85. Co-injection of any cyclic analogue (500 μg) with MBP72-85 (25 μg) did not inhibit EAE activity. However all cyclic analogues except cyclo(1-5)Phe1-Ala-Arg-Gln-Acp5 (SEQ ID NO: 26) resulted in delay of onset of EAE (from day 13 to day 15) but not its severity.
Linear MOG35-55 injection in C57BL/6 mice produces a chronic disease with a single relapse at the beginning of the disease (acute phase) followed by an incomplete remission which leads to chronic phase with a residual disease. This chronic EAE is produced by injection of 600 μg of MOG35-55, administered in 2 doses of 300 μg each and 2 days apart. Co-injection of the cyclic MBP83-99 peptide at disease induction at 1:1 ratio seems to offer a mild preventive benefit which is not always constant and depends on the statistical sample size and variance. On the other hand, histopathology reveals that cyclic MBP83-99 is reducing some of the basic histopathological effects that are responsible for the clinical picture of the animals.
A further aspect of the invention relates to a peptide comprising the amino acid sequence of formula (IIa),
wherein K91 is substituted by the amino acid A and P96 is substituted by the amino acid A, wherein said peptide is in linear form.
In one highly preferred embodiment, the peptide of formula (IIa) consists of the sequence VHFFANIVTARTP (SEQ ID NO: 7).
Another aspect of the invention relates to a peptide comprising the amino acid sequence of formula (IIb),
wherein K91 is substituted by the amino acid A and P96 is substituted by the amino acid A, wherein said peptide is in cyclic form.
In one highly preferred embodiment, the peptide of formula (IIb) consists of the sequence VHFFANIVTARTP (SEQ ID NO: 8) cyclised head to tail, i.e. cyclization between residues 87-99.
Another aspect of the invention relates to a conjugate which comprises a peptide as described above and mannan. The mannan (oxidized or reduced) may be attached at any suitable site on the peptide. The conjugate may contain more than one mannan residue and more than one peptide according to the invention (each of which may be the same or different).
Preferably, the mannan is reduced mannan.
Thus, in one preferred embodiment, the invention relates to MBP83-99 or linear or cyclic analogues thereof conjugated to reduced mannan.
One preferred embodiment of the invention relates to a conjugate comprising mannan and a peptide comprising the amino acid sequence of formula (I),
or a variant thereof wherein at least one of K91 and P96 is substituted by a natural or unnatural amino acid.
Mannan has been used as a successful carrier to target peptides to the macrophage/dendritic cell mannose receptor. Upon binding, MHC class I or MHC class II presentation of peptides is generated, stimulating either CTL/Ab or Th1/Th2 immune responses. Preliminary results suggest that conjugations of reduced mannan to cyclic antagonist/agonist peptides are more potent than cyclic analogues alone. Further investigations are being done to measure cytokines and T cells after immunization of oxidized/reduced mannan conjugates to cyclic MBP analogues.
Th1 cytokines released after therapeutic administration are associated with exacerbation of MS. However, Th2 cytokines (such as IL-4 and IL-10) have anti-inflammatory properties and down-regulate Th1 responses. Mannan has been investigated extensively for its ability to generate responses in several model systems. Its adjuvant function has been shown to stem from its ability to target the mannose receptor on antigen presenting cells. Mice immunized with mannan-MUC1 protein are protected against a MUC1 expressing tumor challenge, as well as reversing established tumors in mice. Similar results were observed in MUC1 transgenic mice. Either a Th1 response (IL-2, IFN-γ, IL-12, TNF-α and IgG2a antibodies) or Th2 response (no IFN-γ or IL-12, but significant amounts of IL-4, IL-10 and TGF-β and IgG1 antibodies), depending on the mode of conjugation, and oxidized/reduced mannan respectively. Other cytokines, IL-5, IL-6, IL-13, IL-15, and IL-18 have also been measured with either oxidized or reduced mannan immunogens. In addition to Th1/Th2 type responses to MUC1 in mice, similar responses have been demonstrated in humans and monkeys with MUC1 protein and to an Anaplasma marginale MSP-1 peptide in cows. The use of reduced mannan to further divert immune responses to Th2 when conjugated to MBP peptides constitutes a novel strategy for immunotherapy of the disease.
The present applicants have demonstrated that mutated MBP83-99 peptide analogues conjugated to reduced mannan diverts immune responses from Th1 to Th2 in SJL/J mice and generate antibodies which do not cross react with native MBP protein. [A91]MBP83-99, [E91]MBP83-99 and [Y91]MBP83-99 peptides gave the best cytokine and antibody reactivity profile with [Y91]MBP83-99 being the most promising as a therapeutic peptide against MS.
As used herein, to “divert immune responses from Th1 to Th2” means an increased production of at least one of the Th2-type cytokines, including, without limitation, IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 and/or a decreased production of at least one of the Th1-type cytokines, including, without limitation, IL-2, TNFα and IFNγ, as compared to the levels of these cytokines prior to peptide/conjugate administration, i.e. in the absence of the treatment.
In one especially preferred embodiment, the invention relates to the peptide cyclo(83-99)[A91]MBP83-99 conjugated to reduced mannan.
In another highly preferred embodiment, the invention relates to the peptide [Y91]MBP83-99 or [A91,A96]MBP83-99 conjugated to reduced mannan.
In one preferred embodiment, the peptide is linked to the reduced mannan via a linker group.
Preferably, the linker group is a Keyhole Limpit Hemocyanin (KLH).
Keyhole Limpet Hemocyanin (KLH) is a copper-containing protein isolated from the mollusck, Megathura crenulata. It is a potent immunoactivator commonly used as a carrier in the conjugation of peptides for antibody production, and CD4+ helper responses. It is composed of high and low molecular weight monomeric forms. KLH is used as a linker between mannan and peptide. KLH-peptide is conjugated to KLH via glutaraldehyde. Conjugation occurs via Schiff base formation between free amino groups of KLH and oxidized mannan. The oxidized mannan-KLH-peptide complex is then reduced with sodium borohydride to form reduced mannan-KLH-peptide complex. In addition, KLH provides CD4+ help to further initiate immune responses. Reduced mannan, is able to divert immune responses from Th1 to Th2.
Preferably, mannan is oxidized to a polyaldehyde by treating with sodium periodate. Ethanedithiol is then added to stop oxidation. The mixture is passed through a PD-10 column pre-calibrated with carbonate buffer and the oxidized mannan fraction is collected. For the conjugation of peptide analogues to oxidized mannan, each peptide pre-conjugated to KLH using glutaraldehyde is allowed to react with oxidized mannan overnight at room temperature. Oxidized mannan contains aldehydes and Schiff bases (
A further aspect of the invention relates to a process for preparing a conjugate, said process comprising the steps of:
(i) reacting a peptide as defined above with a Keyhole Limpit Hemocyanin (KLH);
(ii) reacting the product formed in step (i) with oxidized mannan; and
(iii) reducing the product formed in step (ii) to form a reduced mannan conjugate.
There is gathering evidence that analogues of disease-associated epitopes can be conjugated to oxidized or reduced mannan via KLH linker and actively generate antigen specific regulatory CD4+/CD8+ T cells and Th1/Th2 cytokines. The ability to alter the cytokine secretion of autoreactive T cell lines using peptides or peptide mimetic treatment even in longstanding autoimmune disease indicates that cytokine therapy might have therapeutic benefits by switching the function of myelin autoreactive T cells which are non-pathogenic. Oxidized or reduced mannan conjugates of myelin antigens are promising to induce appropriate cytokine secretion.
In one particularly preferred embodiment of the invention, the conjugate is of formula B, wherein a linear MBP83-99 peptide is optionally substituted at positions 91 and/or 96 and conjugated to mannan in its oxidized or reduced forms.
In another particularly preferred embodiment of the invention, the conjugate is of formula C, wherein a cyclo(83-99)MBP83-99 peptide is optionally substituted at positions 91 and/or 96 and conjugated to mannan in its reduced form.
In one particularly preferred embodiment of the invention, the conjugate is of formula D, in which a cyclo(83-99)MBP83-99 peptide is optionally substituted at positions 91 and/or 96 and conjugated to mannan in its oxidized form.
Another aspect of the invention relates to a peptide or conjugate as described above for use in medicine.
Yet another aspect relates to the use of a peptide or conjugate of the invention in the preparation of a medicament for treating an immune disorder.
In one preferred embodiment, the immune disorder is an autoimmune disease.
In one particularly preferred embodiment, the disorder is multiple sclerosis (MS).
Multiple Sclerosis (MS) is an autoimmune demyelinating disease mediated primarily by CD4+ T cells of the Th1 subset. Candidate autoantigens include constituents of the myelin sheath—Myelin Basic Protein (MBP), Proteolipid Protein (PLP) and Myelin Oligodendrocyte Glycoprotein (MOG). Modern approaches toward the therapeutic management of MS involve the design and use of peptide analogues of disease-associated myelin epitopes to induce peripheral T-cell tolerance. The present invention provides a number of peptide ligands altered by mutating the principal TCR contact residues based on MBP83-99 epitope. Experiments by the applicant have demonstrated that mutated MBP83-99 peptide analogues conjugated to reduced mannan unexpectedly divert immune responses from Th1 to Th2 in SJL/J mice and generate antibodies which do not cross react with native MBP protein. [A91]MBP83-99, [E91]MBP83-99 and [Y91]MBP83-99 peptides gave the best cytokine and antibody reactivity profile with [Y91]MBP83-99 being the most promising as a therapeutic peptide against MS.
Furthermore, cyclic MBP83-99 analogues suppressed chronic EAE induced by the linear MOG35-55 and also immunization with linear MBP83-99-KLH-reduced mannan conjugates prevented EAE induction.
In another preferred embodiment, the disorder is experimental autoimmune encephalomyelitis (EAE).
Structure-activity studies based on the human MBP83-99 [Vergeli, et al 1996, Brocke, et al 1996] epitope (Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro; SEQ ID NO: 27) resulted in linear and cyclic analogues namely [Arg91,Ala96]MBP87-99 (Val-His-Phe-Phe-Arg1-Asn-Ile-Val-Thr-Ala96-Arg-Thr-Pro; SEQ ID NO: 7) and cyclo(87-99)[Arg91,Ala96]MBP87-99 (Val87-His-Phe-Phe-Arg91-Asn-Ile-Val-Thr-Ala96-Arg-Thr-Pro99; SEQ ID NO: 8) which completely prevented the induction of EAE when co-injected to Lewis rats together with encephalitogenic agonist MBP72-85. Blockade of MBP72-85 induced EAE by the previous unrelated peptides could indicate that the mechanism of inhibition is not due to binding competition but rather due to the delivery of a negative signal by the antagonist which overcomes the agonist response possibly through the activation of antigen specific regulatory T cells.
A further aspect of the invention relates to a method of treating an immune disorder, said method comprising administering to a subject a peptide or conjugate as defined above.
Preferably, the immune disorder is an autoimmune disease. More preferably, the disorder is multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE).
Yet another aspect of the invention relates to a method of immunizing a subject against an immune disorder, said method comprising administering to a subject a peptide or a conjugate as defined above.
Preferably, the peptide or conjugate is administered in an amount sufficient to cause suppression of MOG35-55-induced chronic experimental autoimmune encephalomyelitis (EAE).
Another aspect of the invention relates to a method of treatment comprising inversely modulating the Th1 and Th2 responses of lymphocytes in a subject so as to increase the Th2 response and decrease the Th1 response respectively above and below the levels prevailing without said treatment, said method comprising administering to the subject a peptide or a conjugate as described above.
Immunological assays to measure specific T cell responses to antigens can be carried out by ELISpot analysis to detect the secretion of specific cytokines such as IFN-γ, IL-4 or IL-10. Further details of these assays may be found in the accompanying Examples section.
A further aspect of the invention relates to a method of treatment comprising administering to a subject a peptide or a conjugate as described above, wherein said treatment enhances/induces the Th2-type response in a subject, as compared to the Th2-type response prior to peptide/conjugate administration (i.e. in the absence of treatment) and reduces the Th1-type response as compared to the Th1-type response prior to peptide/conjugate administration.
Another aspect of the invention relates to a method of diverting the immune response in a subject from a Th1 response to a Th2 response, said method comprising administering to the subject a peptide or a conjugate as described above.
A further aspect of the invention relates to a method of selectively inhibiting the Th1 immune response over the Th2 immune response in a subject, said method comprising administering to the subject a peptide or a conjugate as described above.
A further aspect of the invention relates to a method of selectively increasing the Th2 immune response in a subject relative to the Th1 immune response, said method comprising administering to the subject a peptide or a conjugate as described above.
Another aspect of the invention relates to a method of selectively reducing the Th1 response of lymphocytes in a subject relative to the Th2 response of lymphocytes, said method comprising administering to the subject a peptide or a conjugate as described above.
Yet another aspect relates to a method of inducing a Th2-specific immune response in a subject, said method comprising administering to the subject a peptide or a conjugate as described above.
Another aspect of the invention relates to a method of reducing the level of IFNγ in a subject, said method comprising administering to the subject a peptide or a conjugate as described above.
Another aspect relates to the use of a peptide or a conjugate as described above for treating a disorder associated with an imbalance in lymphokine expression.
The lymphokines are a group of polypeptides belonging to the family of cytokines, i.e. hormone-like molecules that can affect various cell functions and enable communication between different cells. Recent developments have helped to clarify the role of lymphokines in the immune response. Lymphokine production by helper CD4 (and also in CD8+) T cells frequently fall into one of two phenotypes, Th1 and Th2, in both murine and human systems (Romagnani. 1991, Immunol Today 12: 256-257, Mosmann. 1989, Annu Rev Immunol, 7: 145 173). Th1 cells produce interleukin 2 (IL-2), tumor necrosis factor (TNFα) and interferon gamma (IFNγ) and they are responsible primarily for cell-mediated immunity such as delayed type hypersensitivity. Th2 cells produce interleukins, IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 and are primarily involved in providing optimal help for humoral immune responses such as IgE and IgG4 antibody isotype switching (Mosmann, 1989, Annu Rev Immunol, 7: 145-173). Strongly polarized Th1 and Th2 responses not only play different roles in protection, they can promote different immunopathological reactions.
Another aspect relates to the use of a peptide or a conjugate as described above in the preparation of a medicament for treating a disorder associated with an imbalance in lymphokine expression.
Preferably, the peptide or conjugate is administered in an amount sufficient to inversely modulate the Th1 and Th2 responses of lymphocytes in the subject so as to increase the Th2 response and decrease the Th1 response respectively above and below the levels prevailing without said treatment.
Yet another aspect relates to a method of treating a disorder associated with an imbalance in lymphokine expression in a subject, said method comprising administering to the subject a peptide or a conjugate as described above.
In one preferred embodiment, the peptide or conjugate is administered in an amount sufficient to enhance/induce the Th2-type response in a subject, as compared to the Th2-type response prior to peptide/conjugate administration (i.e. in the absence of treatment) and reduce the Th1-type response as compared to the Th1-type response prior to peptide/conjugate administration.
In another preferred embodiment, the peptide or conjugate is administered in an amount sufficient to inversely modulate the Th1 and Th2 responses of lymphocytes in the subject so as to increase the Th2 response and decrease the Th1 response respectively above and below the levels prevailing without said treatment.
In one highly preferred embodiment, the disorder associated with an imbalance in lymphokine expression is an inflammatory autoimmune disease with a Th1 immune profile.
Another aspect relates to a pharmaceutical composition comprising a peptide or a conjugate according to the invention, admixed with a pharmaceutically acceptable diluent, excipient or carrier.
Even though the peptide/conjugate of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
The peptides/conjugates of the invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
Pharmaceutically acceptable salts of the peptides/conjugates of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers and tautomers of the peptides/conjugates. The man skilled in the art will recognise compounds that possess an optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
Some of the peptides/conjugates of the invention may exist as stereoisomers and/or geometric isomers—e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the peptides/conjugates or pharmaceutically acceptable salts thereof. An isotopic variation is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the peptides/conjugates and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F and 36Cl, respectively. Certain isotopic variations of the peptides/conjugates and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the peptides/conjugates of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
The present invention also includes the use of solvate forms of the peptides/conjugates of the present invention. The terms used in the claims encompass these forms.
The invention furthermore relates to the peptides/conjugates of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
The invention further includes the peptides/conjugates of the present invention in prodrug form. Such prodrugs are generally peptides/conjugates wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
In a particularly preferred embodiment, one or more peptides/conjugates of the invention are administered in combination with one or more other therapeutically active agents, for example, existing drugs available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other agents.
Combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to administer most drugs at their maximum tolerated doses with minimum time intervals between such doses. The major advantages of combining chemotherapeutic drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance in early tumor cells which would have been otherwise responsive to initial chemotherapy with a single agent.
Another aspect relates to the use of a peptide or a conjugate according to the invention, in an assay for elucidating agents capable of regulating experimental autoimmune encephalomyelitis (EAE) or regulating multiple sclerosis.
Another aspect of the invention relates to a vaccine composition comprising a peptide or a conjugate as described above. Preferably, the vaccine composition further comprises one or more adjuvants. The term “adjuvant” as used herein includes an agent having the ability to enhance the immune response of a vertebrate subject's immune system to an antigen or antigenic determinant.
The preparation of vaccines which contain an immunogenic polypeptide as active ingredient will be familiar to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
Further examples of adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.).
Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used.
The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al2O3 basis). Conveniently, the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.
The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic agent resulting from administration of this agent in vaccines which are also comprised of the various adjuvants.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
Another aspect of the invention relates to the use of a peptide or a conjugate as described above in the preparation of a vaccine composition.
The present invention is further described by way of the following non-limiting examples, and with reference to the figures below wherein:
Peptides (Table 1) were prepared on 2-chlorotrityl chloride resin (CTLR-C1) using Fmoc/tBu methodology. The cyclization was achieved with TBTU/HOAt and 2,4,6-collidine as base, as previously described. Preparative HPLC for peptide analogues were performed using a Lichrosorb RP-18 reversed phase semipreparative column with 7 μm packing material. The peptides were >95% pure as analysed by mass spectrometry.
Cyclization of linear MBP83-99 protected analogue was achieved using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine in DMF solution, allowing fast reactions and high yield cyclization products. Recent results indicate that HOAt, a 4-nitrogen containing variant, is a very effective coupling additive, more efficient than HOBt for solution or solid phase synthesis leading in high yield products.
Cyclo(83-99)[Arg91, Ala96] MBP83-99 (Glu-Asn-Pro-Val-Val87-His-Phe-Phe-Arg91-Asn-Ile-Val-Thr-Ala96-Arg-Thr-Pro99) (SEQ ID NO: 20)
Cyclo(91-99)[Ala96]MBP83-99 (Glu-Asn-Pro-Val-Val87-His-Phe-Phe-Lys91-Asn-Ile-Val-Thr-Ala96-Arg-Thr-Pro99) (SEQ ID NO: 25)
Human MBP cyclic analogues were prepared on 2-chlorotrityl chloride resin using Fmoc/tBu methodology. The peptide synthesis was achieved using DIC/HOBt in DMF and the Nα—NH2 of amino acids was protected with the Fmoc group. The side chain of peptides was protected as following: Trt for His, Pbf for Arg, tBu for Ser, Thr, Asp, Glu, Boc for Lys, as regarding the cyclic analogue cyclo(91-99)[Ala96]MBP83-99 (by Nε-NH2 of Lys and C-terminous) Mtt protected group was used, because it can easily be removed using mixture HFIP(1,1,1,3,3,3 hexafluoro-2-propanol)/DCM (2/8), which cleaves peptides from the resin. Otherwise, the side chain of Lys of the cyclo(87-99)[Arg91, Ala96]MBP87-99 was protected with Boc group. The final protected linear peptides on resin were dried in vacuo and then treated with the splitting mixture DCM/HFIP (8/2) for 7 h at room temperature to release the peptide from the resin and deprotect Lys from Mtt of the cyclo(91-99)[Ala96]MBP83-99. Each one of the linear protected peptide was dissolved in DMF and collidine/HOAt was added. This mixture was added dropwise in a solution of TBTU in DMF for 8 hours. The cyclization was determined by TLC and analytical reversed phase HPLC(RP-HPLC). The solvent was removed under reduced pressure affording a light yellow oily residue. The cyclic protected peptide (purity ≧90%) was precipitated from H2O and dried in vacuo for 16 h. The cyclic protected peptide was treated with 65% TFA in DCM and 3% ethanodithiol as scavanger for 4 hours at room temperature. The resulting solution was concentrated to a small volume and the final free peptide was precipitated as a light yellow amorphous solid added diethylether (purity ≧80%). Peptide purity was assessed by analytical HPLC reruns, thin layer chromatography (TLC) and mass spectrometry (ESIMS) (
MBP83-99 peptide analogues (Table 1) were conjugated to keyhole limpet hemocyanin (KLH) via glutaraldehyde (
Female 6-8 week old SJL/J mice used in all experiments, were purchased from Walter and Eliza Hall Institute (VIC Australia) and housed at the Biological Research Laboratory at Burnet Institute at Austin, Heidelberg, Australia. SJL/J mice were immunized twice on days 0, 14 intradermally (base of tail) with 50 μg MBP83-99 peptide analogue-KLH-reduced mannan.
ELISpot analyses specific T cell responses to antigens by detecting the secretion of specific cytokines. Spleen cells from immunized SJL/J mice were isolated 14 days after immunization and assessed by ELISpot for IFN-γ, IL-4 or IL-10 secretion by T cells. IFN-γ ELISpot assay was performed on MultiScreen-IP Filter Plate (MAIP S4510) with hydrophobic PVDF filters (Millipore, UK) while IL-4 and IL-10 ELISpot was performed on MultiScreen-HA Filter Plate (MAHA S4510) with mixed cellulose esters filters (Millipore, UK). MAIP S4510 plates were pre-wet with 50 μl of 70% ethanol, washed 5 times with 200 μl sterile PBS and coated with 70 μl of 5 μg/ml anti-IFN-γ capture antibody, AN18 (Mabtech, Australia) in PBS and left to incubate at 4° C. O/N. While 70 μl of 5 μg/ml anti-IL-4 and anti-IL-10 capture antibody (Mabtech, Australia) was added directly to MAHA S4510 plates and incubated at 4° C. O/N without 70% ethanol treatment. Following 5 washes with PBS, plates were blocked by adding 200 μl of culture media (supplemented with 2.5% FCS) and incubated for 2 hours at 37° C. The blocking media was discarded and 10 μg/ml recall peptides (MBP83-99 peptide analogues not conjugated to KLH-reduced mannan) were added into each defined well. Con A (1.0 μg/ml; IL-4 and IFN-γ) or LPS (1.0 μg/ml; IL-10) were used as an internal positive controls and no peptide was used as negative control. Triplicate wells were set up for each condition. 5×105 spleen cells in 100 μl culture media were seeded into each well and incubated for 18 hours for IFN-γ, 24 hours for IL-4 and 48 hours for IL-10 at 37° C. Plates were washed 5 times PBS/0.05% Tween 20 and 5 times PBS and incubated for 2 hours at RT with anti-murine IFN-γ, IL-4 or IL-10 monoclonal antibody-biotin. Plates were washed and streptavidin-ALP was added at 1.0 μg/ml and incubated for 2.0 hours at RT. Spots of activity were detected using a colorimetric AP kit (Biorad, Hercules, Calif. USA) and counted using an AID ELISpot plate reader (Autoimmun Diagnostika GmbH, Germany). Data are presented as mean spot forming units (sfu) per 0.5×106 cells +/−standard error of the mean (SEM).
Spleen cells from immunized SJL/J mice were isolated 14 days after immunization and assessed by T cell proliferation assay. 1×105 spleen cells in 100 μl culture media were seeded into 96 U-bottom plates and incubated for 1-6 days at 37° C. in the presence of recall peptide (10 μg/ml), ConA (positive control) or no peptide (negative control). Proliferation was assessed by adding 1 μCi of [3H]-thymidine per well to one plate per time point (days 1-6). Cells were incubated for a further 6 h before harvesting onto glass fiber filters. [3H] uptake was measured using a β-scintillation counter (Top Count Gamma Counter, Packard, USA).
Blood was collected and sera isolated from mice prior to and 2 weeks after immunization. MBP83-99 peptides conjugated to BSA were coated onto polyvinyl chloride (PVC) microtiter plates at 10 μg/ml or with 2.5 μg/ml native MBP bovine protein in 0.2 M NaHCO3 buffer, pH 9.6, overnight at 4′C and non-specific binding was blocked with 2% BSA for 1.0 hour at room temperature. After washing (0.05% Tween 20/PBS), serial dilutions of sera were added and incubated for a further 2.0 hours at room temperature. The plates were washed and bound antibody was detected using HRP-conjugated sheep anti-mouse antibody (1:1000 in PBS) (Amersham, UK) and developed using 2,2″-azino-di(3-ethylbenzthiazoline) sulphonate (ABTS) (Sigma, UK). Absorption at 405 nm was recorded using an ELISA microplate reader.
Inbred Lewis rats bred and maintained in the animal facility of the Hellenic Pasteur Institute were used in all experiments. Female rats (220 g) were immunized with linear MBP72-85 (30 μg) (n=10, as positive control), or MBP72-85 (30 μg) plus the cyclic analogue c-Ala81MBP72-85 (500 μg) or [Arg91,Ala96]MBP87-99 (n=5) in 200 μl of an emulsion containing equal volumes of peptide diluted in sterile saline and Freund's complete adjuvant (Difco, USA) containing 4 mg/ml heat-killed M. tuberculosis (H37Ra) (Difco). Immunization was performed subcutaneously in the two hind foot pads and repeated 7 days later with the same dosage. Rats were examined daily for clinical signs of EAE and scored as following: 0, no clinical disease; 0.5, weight loss; 1, tail weakness; 2, paraparesis of hindlimbs; 3, paraplegia of hindlimbs; 4, paraplegia with forelimb weakness, moribund; 5, death. PBS/CFA-injected animals served as negative controls. For histological analyses, mice were anaesthetized with ether, bled and perfused with PBS (pH 7.4) (PBS) followed by 4% paraformaldehyde in PBS. Spinal cord was dissected out and fixed overnight in 4% paraformaldehyde in PBS at 4° C. before been embedded in paraffin. Paraffin sections were stained with Luxol fast blue and Nuclear Fast Red for visualization of demyelination and inflammation respectively.
a. Animal Handling
Female C57BL/6 pathogen-free mice were purchased from the Hellenic Pasteur Institute, Athens, and housed in the animal facility of the B′ Neurological Department of AHEPA University Hospital, Aristotle University Medical School, Thessaloniki, Greece. All experimental procedures were in accordance with the National Institute of Health guidelines. Mice were fed a regular diet and given water without antibiotics.
b. EAE Induction and Evaluation of Clinical Disease.
Female C57BL/6 female mice, 10 weeks-old, were divided into 2 different groups depending on the peptides used to prepare the emulsion of injection: group A (or control group, n=9) was immunized using the linear myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55) (Sigma) and group B (n=8) was immunized using a 1:1 mixture of the linear MOG35-55 and the cyclo(83-99)MBP83-99 peptide analog. Both groups were immunized with subcutaneous injections at the left para-lumbar region of emulsions composed of either 300 μg of linear MOG35-55 (group A) or 300 μg of linear MOG35-55 plus 300 μg of cyclo(83-99)MBP83-99 (group B) emulsified in 200 μl of a solution containing 100 μl complete Freund's adjuvant (CFA) containing 4 mg/ml Mycobacterium tuberculosis H37RA and 100 μl filtered phosphate buffered saline (PBS) (day 0). Additionally, on day 0, both mice groups were also given i.p. 400 ng of Pertussis toxin (Sigma) diluted in 0.5 ml of filtered PBS and 48 hours later, on day 2, another 200 ng of Pertussis toxin diluted again in 0.5 ml of PBS. An additional injection of the emulsified peptides, as described previously, was also delivered 7 days later into the right para-lumbar region.
All animals were examined daily and evaluated for clinical signs of disease. The first clinical signs appear on day 12-16 post-immunization, depending on the development and severity of EAE disease. The clinical status of the mice was graded as follows: 0: without clinical disease; 1: flail tail; 2: tail paralysis; 3: hind limb weakness sufficient to impair righting; 4: paraplegia; 5: paraplegia with forelimb paresis or plegia; 6: death from EAE.
Two experimental sets were conducted using exactly the same experimental procedures: the first experiment evaluated the acute phase of the disease and animals were sacrificed at day 17 post disease induction and the second experiment evaluated the chronic clinical course of the disease and thus animals were sacrificed at day 46 post disease induction.
On day 17 (acute phase of EAE) and day 46 post-EAE-induction (chronic phase of EAE) animals were subjected to transcardial perfusion with 4% paraformaldehyde in PBS. Brains and spinal cords were removed, post-fixed in the same fixative for approximately 20 hours, routinely processed for paraffin embedding and sectioned at 6 pin. Sections from animals of acute and chronic phases of the disease, were then stained using the following methods: a) a modified Bielschowsky silver impregnation staining method combined with haematoxylin, for the simultaneous evaluation of axonal injury, axonal loss and inflammatory processes in EAE as previously described in detail [Lourbopoulos et, al., 2007]; b) Luxol fast blue staining counterstained with Nuclear fast Red for the detection of demyelinating areas within the CNS of animals, using routine histopathological protocols.
Pathological evaluation was performed under a light microscope (Olympus Axioplan-2) by two blinded investigators and photos were taken using a CCD camera (Nikon). Five randomly selected longitudinal sections per tissue (brains and spinal cords) were evaluated as follows: for each animal, each section was evaluated under 20× or 40× optical fields (depending on the object of study) so as to cover the entire area of the section. Initial study of pathology revealed that spinal cords had the majority of lesions (compared to brains) and thus further detailed study was focused on the spinal cord sections of the animals.
Depending on the object of interest the following scales were used to perform the evaluation: (a) For axonal injury (AI), the following scale was used: 0=no AI, 1+=scattered dystrophic injured axons without any spheroid or ovoid, 2+=mild AI with the presence of at least one spheroid or ovoid, 3+=moderate AI, 4+=severe AI; generally injured axons were defined and identified as spheroids and ovoids (which represent axonotmesis), and dilated (dystrophic) axons which represent injured axons not yet being cut. (b) For axonal loss (AL): 0=normal axonal density, 1+=<25% AL, 2+=mild AL (26-50%), 3+=moderate to severe (51-75%) AL, 4+=severe AL (>75%); various degrees of decreased density of axons was attributed to axonal loss and evaluated accordingly. (c) For infiltrations: number of infiltrating cells per optical field and number of infiltrating cells per infiltration. (d) Demyelination was evaluated under 40× optical fields; using a prefrontal grid, we measured the area of demyelination and the total area of white matter in each optical field and then subtracted the % of demyelination present in each optical field.
Statistical analysis of the data was performed using the SPSS 11.5 software. For scale clinical, histopathological and in vitro proliferation data we initially tested their normality using Shapiro-Wilk and Kolmogorov-Smirnov tests in order to assess their validity for student's t-test application. Wherever data were found to violate the normality assumption and a logarithmic transformation could not be applied, we used the non-parametric equivalent Mann-Whitney U test for comparison of the two groups (treated and control in each EAE). Values within the text are expressed as mean±SD. For nominal or ordinal data a Pearson chi-square test or Fisher's exact test was used for comparison of the two groups, depending on the tables' properties. Kaplan-Meier survival analysis was performed in the clinical course of EAE with “disease onset” as the end-point of the analysis; disease onset was defined as the first day with clinical score of “1”. Mean Maximal Score (MMS) was calculated for each group using the maximal scores from each animal of each group. Area Under the Curve (AUC) was calculated for each animal using the formula:
The ability of linear MBP83-99, [A91]MBP83-99, [E91]MBP83-99, [F91]MBP83-99, [Y91]MBP83-99, [R91,A96]MBP83-99, cyclo(83-99)MBP83-99 and cyclo(83-99)[A91]MBP83-99 peptides conjugated to reduced mannan, to induce T cell responses after 2 injections was measured using IFN-γ and IL-4 cytokine secretion by ELISpot analysis. Mice immunized with linear and cyclo(83-99)MBP83-99 and linear [F91]MBP83-99 generated weak IFN-γ secreting T cells; all other peptides were negative (
The production of total IgG antibody responses in mice immunized with linear MBP83-99, [A91]MBP83-99, [E91]MBP83-99, [F91]MBP83-99, [Y91]MBP83-99, [R91,A96]MBP83-99 and cyclo(83-99)MBP83-99-[A91]MBP83-99 peptides conjugated to reduced mannan, were measured using ELISA. High IgG antibody levels were generated in mice immunized with both linear and cyclo(83-99)MBP83-99 peptides (titer >1/25,600) (
Since high levels of antibodies were generated to MBP83-99 peptides and its mutant analogues, we determined whether sera from these mice cross reacted with the native MBP protein. Mice immunized with linear or cyclo(83-99)MBP83-99 and [A91, R96]MBP83-99, peptides highly cross reacted with native MBP protein, mice immunized with linear or cyclo(83-99)[A91]MBP83-99 and [E91]MBP83-99 reacted moderately, whereas mice immunized with [F91]MBP83-99 or [Y91]MBP83-99 did not react with native MBP protein at 1/200 and 1/1,600 sera dilution (
ELISpot assays are an ex vivo 18 h assay that measures IFN-γ secretion by T cells. It does not require expansion of cell cultures, as it detects specifically activated effector cells (both CD4 and CD8 cytokine producing terminal effectors). The sensitivity of the assay is higher than limiting dilution analysis, FACscan analysis, or ELISA methods and can reliably detect precursor frequencies of antigen specific effectors of 1 in every 0.5 million cells. It is therefore an appropriate method to detect antigen specific cells. Here, we measured IFN-γ production generated by the native MBP83-99 peptide and assessed the % inhibition (antagonize) of IFN-γ produced in the presence of mutant peptide analogues.
Cyclic head-to-tail MBP83-99 single and double mutant analogues were tested for their ability to inhibit IFN-γ responses induced by the native agonist MBP83-99 peptide. Mice were immunized with the native agonist MBP83-99 peptide emulsified in complete Freund's adjuvant (CFA). Spleen cells were isolated 25 days later, and the ability of cyclic peptides to inhibit (antagonize) IFN-γ production was assessed in in vitro antagonism ELISpot assays (
Cyclo(83-99)MBP83-99 and Cyclo(83-99)[A91]MBP83-99 Compared to their Linear Counterparts Enhance IL-4 Production when Emulsified in CFA
The ability of the antagonist cyclo(83-99)[A91]MBP83-99 and the native agonist cyclo(83-99)MBP83-99 analogues to induce T cell responses after one injection, in comparison to linear [A91]MBP83-99 and linear MBP83-99, was determined in SJL/J mice. Spleen cells were isolated 28 days after injection, and ELISpot assay was performed to measure IFN-γ and IL-4 secretion (
Cyclo(83-99)MBP83-99 and Cyclo(83-99)[A91]MBP83-99 Compared to their Linear Counterparts Enhance Antibody Production when Emulsified in CFA
The production of specific IgG antibody responses in mice immunized with linear MBP83-99, linear [A91]MBP83-99, cyclo(83-99)MBP83-99, and cyclo(83-99)[A91]MBP83-99 peptides emulsified in CFA was measured using ELISA. Linear MBP83-99 peptide did not generate IgG antibodies in SJL/J mice; however, antibodies were generated to cyclo(83-99)MBP83-99 (
Cyclization does not Affect T Cell Proliferation to Native MBP83-99 Peptide
Proliferation assays were used to detect the level of antigen specific T cells by measuring the [3H]thymidine uptake of T cells proliferating in the presence of peptides on days 1-6. Spleen cells from mice immunized with linear agonist MBP83-99 peptide were isolated 28 days after immunization and assessed by T cell proliferation assay. T cells proliferated to MBP83-99 peptide, reaching a peak by day 5 of up to 25 000 cpm (
The ability of linear and cyclic agonist MBP83-99, and linear and cyclic antagonist [A91]MBP83-99 peptides conjugated to reduced mannan, to induce T cell responses after two injections was measured using IFN-γ and IL-4 cytokine secretion by ELISpot analysis. Mice immunized with linear and cyclic agonist MBP83-99 peptides generated IFN-γ secreting T cells; however, both linear and cyclic antagonist [A91]MBP83-99 peptides were negative (
Cyclization does not Affect Antibody Responses when Cyclic MBP83-99 Peptide Analogues are Conjugated to Reduced Mannan
The production of total IgG antibody responses in mice immunized with linear agonist MBP83-99, linear antagonist [A91]MBP83-99, cyclo(83-99)MBP83-99, and cyclo(83-99)[A91]MBP83-99 peptide analogues, conjugated to reduced mannan, were measured using ELISA. High IgG antibody levels were generated in mice immunized with linear MBP83-99 and cyclo(83-99)MBP83-99 peptide analogues (titer up to at least 1/25600) (
To test the effect of a preventive administration of cyclo(83-99)MBP83-99 at EAE induction 2 experimental sets were designed, each one with 2 experimental groups as previously described at materials and methods: the first experiment was designed to study the effect of cyclo(83-99)MBP83-99 at the pathological procedures at acute phase of EAE and the second one to study the effect of the peptide up to the chronic phase of EAE.
For the study the acute phase, EAE was induced as described previously. The animals developed a severe and aggressive disease with and with fast and steep disease onset. The developed EAE disease was a highly homogenous one since variability in clinical scores was very low. All animals were sacrificed at day 17, i.e. 5 days post their first clinical sign of EAE (acute phase). The mean clinical score was statistical significant different at days 15, 16 and 17 (Student's t-test, p<0.05). No animals died from EAE, in either group, until the day of sacrifice. Mean Maximal Clinical Scores (MMS) were not calculated for this experiment since animals were not allowed to live long enough to reach their maximal scores. On the other hand, mean Areas Under the Curve (mAUC) for each group have been calculated and are significantly different between the 2 groups: group B appeared to have a lower mAUC (group B mAUC=13.32±2.55 compared to control group mAUC=18.042±3.34; Mann-Whitney U test p=0.014).
Mean day of disease onset (first definite clinical sign or score “1”) was not different between the 2 groups (mean day of disease onset for control=11.67±1.21 and Group B=12.00±0.81; Kaplan-Meier survival analysis, log rank p=0.7309). Thus, clinical data from acute phase indicate that MBP83-99 preventive co-administration conferred a mild clinical benefit in a severe and aggressive form of MOG-EAE.
For the study the chronic phase of EAE, animals were allowed to survive until day 46 post EAE induction (chronic phase). This second EAE experiment was slightly different from the first one since the developed EAE was a moderate one, with less steep slope of onset. Moreover, this EAE had a higher variation concerning the clinical scores of each animal in either group. Animals that received the cyclo(83-99)MBP83-99 peptide had a lower mean clinical score throughout the entire observation period but this failed to reach statistical significance (Student's t-test, p>0.05). No animals died from EAE in either group. Mean Maximal Clinical Scores (MMS) were not statistically different between the 2 groups despite a lower MMS of group B (control group MMS=3.125±1.45 and group B MMS=2.375±1.45; Student's t-test p=0.32); mean Areas Under the Curve (mAUC) for each group were also not significantly different although group B had a lower AUC (group B mAUC=51.08±36.58 compared to control group mAUC=66.54±39.38; Student's t-test p=0.429). Mean day of disease onset (first definite clinical sign or score “1”) was not significant different between the 2 groups (mean day of disease onset for control=18.5±11.23 and Group B=17.00±11.74; Kaplan-Meier survival analysis, log rank p=0.1925).
Study of inflammatory processes at acute phase of EAE showed no quantitative differences of inflammatory processes the between the control group and group B when the following parameters were evaluated: the number of infiltrating cells per optical field (control group: 309.4±219.9, group B: 290.8±277.7. Student's t-test, p=0.593) and inflammatory cells per infiltration (control group: 55.75±37.4, group B: 53.72±62.0. Student's t-test, p=0.780).
However, in the experiment of chronic phase, the inflammatory process in the spinal cords of each group were different: both groups reduced there inflammatory burden within their tissues but the residual inflammation was significantly more in the control group as this was depicted by the infiltrating cells per optical field (control group: 33.18±67.03, group B: 10.71±39.0. Student's t-test, p=0.001) and the numbers of inflammatory cells per infiltration (control group: 15.76±28.5, group B: 6.0±15.8. Student's t-test, p=0.000).
These data indicate that the cyclo(83-99)MBP83-99 co-administration at disease onset produces a quantitatively similar inflammatory load within the spinal cords of the animals, which does not remain during the chronic phase since we observed significantly less inflammatory cells within the spinal cord parenchyma of the cyclo(83-99)MBP83-99 treated animals.
Study of axonal pathology revealed a mild but statistical significant beneficial effect of cyclo(83-99)MBP83-99 treatment on axonal injury and a mild effect on axonal loss at the acute phase of disease (Pearson's chi-square tests for axonal injury and axonal loss respectively: p=0.006 and p=0.000, refer to tables below).
However, during chronic phase axonal injury was not different between the 2 groups (Fisher's exact test, p=0.104), but axonal loss was found to be less in group B compared to control ((Fisher's exact test, p=0.000).
0%
These data indicate that cyclo(83-99)MBP83-99 co-administration at disease onset slightly deviates the axonopathic process within the spinal cords of the animals, resulting in a mild, but statistical significant, reduction of axonal injury during acute phase. This reduced axonal injury is followed by less axonal loss within the white bundles of the spinal cord during the acute phase of EAE and is additionally contributing to less axonal loss during the chronic phase of the disease.
Study of demyelinating process revealed a significant protective effect of the cyclo(83-99)MBP83-99 treatment on myelin integrity. Demyelination was minimal in group B during acute and chronic phase of EAE compared to control group which exhibited a high percentage of demyelination of the white matter of spinal cords (Acute phase: control group=20.73±16,53 and group B=9.58±10.5 Student's t-test, p=0.000; Chronic phase: control group=14.92±15.31 and group B=7.65±11.4 Student's t-test, p=0.000).
Multiple Sclerosis (MS) is a demyelinating and inflammatory disease of the central nervous system with autoimmune pathology. There is increasing evidence that T cell responses to candidate myelin antigens, such as myelin basic protein (MBP), may play an important role in the pathogenesis of the disease. It has been demonstrated that T cell responses to MBP are associated with preferential recognition of the 83-99 immunodominant region of MBP83-99 (ENPVVHFFKNIVTPRTP; SEQ ID NO: 2).
The peptide analogues of the invention are based on the encephalitogenic peptide MBP83-99, (ENPVVHFFK91NIVTP96RTP; SEQ ID NO: 2) with the principal TCR contact residues at positions 91 and/or 96 replaced to give [A91]MBP83-99, [E91]MBP83-99, [F91]MBP83-99, [Y91]MBP83-99, [R91,A96]MBP83-99. Cyclo(83-99)83-99 and cyclo[A91]MBP83-99 derivatives were also synthesized. Peptides were conjugated to reduced mannan via a KLH linker and injected into mice. Reduced mannan was used as a carrier to divert immune responses to Th2. The use of reduced mannan to divert the immune responses towards Th2 to MBP peptides constitutes a novel strategy for the immunotherapy of MS. The immune responses induced in mice are summarized in Table 2.
The agonist peptide MBP83-99 conjugated to reduced mannan induced high levels of IFN-γ, low levels of IL-4 and generated antibodies which were cross reactive with the native MBP protein. Substitution of K91 to A91, E91 or Y91 diverted IFNγ responses to high IL-4 responses in immunized mice. In addition, immunization with reduced mannan-[Y91]MBP83-99 analogue induced moderate levels of IL-10 cytokine production and very weak T cell proliferative responses. Immunization with [A91]MBP83-99, [E91]MBP83-99 or [Y91]MBP83-99 analogues generated IgG1 antibodies, and antibodies generated to [A91]MBP83-99 and [E91]MBP83-99 cross reacted weakly with native MBP protein. However, antibodies produced to [Y91]MBP83-99 peptide did not cross react with native MBP protein. [F91]MBP83-99 peptide conjugated to reduced mannan induced IFN-γ, IL-4 and IL-10 responses, strong T cell proliferative responses and weak antibody responses. Double mutations in the peptide analogue [R91, A96]MBP83-99 when conjugated to reduced mannan was able to generate high levels of IL-4 and no IFN-γ, however the IgG1 antibodies generated were cross reactive with the native protein. Cyclo(83-99)MBP83-99 and cyclo(83-99)[A91]MBP83-99 did not induce desired responses. It is clear that [A91]MBP83-99, [E91]MBP83-99 and [Y91]MBP83-99 peptides gave the best cytokine profile with [Y91]MBP83-99 being the most promising, (since, in addition to cytokine, had no reactivity to native MBP protein) as a therapeutic peptide against MS.
It is of interest that when all peptide analogues were conjugated to oxidized mannan, no IFN-γ or IL-4 responses were generated. However, moderate antibody levels were induced (not shown). Oxidized mannan has been shown to preferentially generate CD8 responses, and thus, was unable to induce responses to the CD4 epitope of MBP83-99 and its analogues. In addition, immunization of SJL/J mice with linear MBP83-99, [A91]MBP83-99, [E91]MBP83-99, [F91]MBP83-99, [Y91]MBP83-99, [R91,A96]MBP83-99, cyclo(83-99)MBP83-99 and cyclo(83-99)[A91]MBP83-99 induced either IFN-γ and IL-4 secretion or antibody production. In addition, T cells against the agonist peptide only weakly proliferated in the presence of [Y91]MBP83-99 or [R91, A96]MBP83-99 analogues and all peptide analogues were able to inhibit (antagonize) IFN-γ production in vitro by T cells against the agonist MBP83-99 peptide. Together, the cytokine profile, antibody data, proliferation and antagonism experiments indicate that the [R91, A96]MBP83-99 peptide analogue shows promise for further therapeutic studies when emulsified in complete Freund's adjuvant (Table 3). As can be seen in the summary table (Table 3), emulsifying all the peptide analogues in complete Freund's adjuvant was able to downregulate IFN-γ secretion but was unable to induce IL-4 cytokine (except [R91, A96]MBP83-99), However, when the peptide analogues were conjugated to reduced mannan-KLH very strong IL-4 cytokine and little to no IFN-γ was generated, in addition to IL-10 (Table 2). Thus, conjugating MBP83-99 peptide analogues to reduced mannan was able to divert the immune response from Th1 to Th2 and thus constitutes a novel approach to the immunotherapy of MS.
Clinical data suggest that the preventive co-administration of the cyclo(83-99)MBP83-99 peptide in 1:1 ratio has a mild beneficial effect, which is not as strong as the one observed in analogous experiments in Lewis rats' MBP-EAE [Tselios T et al., 2002, Tselios, et al., 2000]. Results suggest that the observed benefit in C57BL/6 mice' MOG-EAE is not always statistically significant and is dependent on the clinical characteristics of the produced EAE (inter-group variability, disease severity and aggressiveness).
Despite the fact that the cyclo(83-99)MBP83-99 peptide seems to lower the disease severity and clinical score, this effect does not always reach statistical significance. Careful study of both experimental sets (acute and chronic) lead to the conclusion that cyclo(83-99)MBP83-99 co-administration with the linear MOG35-55 peptide has a mild beneficial effect which is strongly affected by the group size and variance of the clinical scores of each EAE experiment [Fleming, et. al., 2005]. Thus, when the induced EAE is highly homogenous and has a sufficient sample size then the mild benefit from the cyclo(83-99)MBP83-99 peptide administration is statistically significant; on the other hand, when the induced EAE is not very homogenous and has high variability concerning the clinical scores (as this was the case with the chronic experimental set), then the mild benefit from the cyclo(83-99)MBP83-99 peptide administration does reach statistical significance.
As far as histopathology is concerned, the results indicate that there is a quantitative similar inflammatory burden within the spinal cords of the 2 groups (control and cyclo(83-99)MBP83-99 or group B) during the acute phase; this inflammatory load is cleared in both groups, as they progress to chronic phase, but the clearance seems to be more efficiently in group B and leads to reduced inflammatory cells within their spinal cords. When demyelination was studied, the animals in group B were found to have significant less demyelinated axons compared to controls, both in acute and chronic phases of the disease. When axonopathic processes were studied, axonal injury and axonal loss were found to be significantly milder in group B during acute phase, a fact that seems to lead to an increased preservation of axons in group B, through progression of EAE to chronic phase, as this is indicated by the reduced axonal loss in group B at chronic phase. Thus, a relatively preserved axonal density was observed in group B during chronic phase which seems to conform to the mild clinical benefit of cyclo(83-99)MBP83-99 co-administration during chronic phase.
The aforesaid results cannot be explained simply through the inflammatory load of the spinal cords of the animals since there is no difference between the absolute numbers of infiltrating cells within the CNS parenchyma during acute phase of EAE. However, it is probable that the equal numbers of infiltrating cells during acute phase of the disease do not necessary correspond to equal numbers of “reactive” MOG-specific T-lymphocytes within the parenchyma. If this is to be true and there are less MOG-reactive T-cells and more cyclo(83-99)MBP83-99 specific but not reactive T-cells, then it could probably result in reduced demyelination, reduced axonopathic processes and increased clearance of the inflammatory cells as the disease progressed to chronicity [Lassmann et. al., 2007, Abromson-Leeman, et al., 2005, Bauer J et al., 1998, Berger T et al., 1997, Suvannavejh G C et al., 2000, Pender M P and Rist M J 2001].
Thus, as a conclusion, the cyclo(83-99)MBP83-99 peptide does indeed produce some beneficial effect in the C57BL/6 MOG-EAE. However, this effect is not as strong as in MBP-EAE in Lewis rats.
In the modeled H2 I-As complex of the MBP peptide, the residues V87, F90, N92, and T95 are anchored in MHC pockets P1, P4, P6 and P9, respectively11, 12. The residue K91 is pointing up from the MHC groove. The models indicate that mutations of the K91 residue do not cause major disruptions to the structures and to the intermolecular interactions between the peptide and the MHC cleft (
With respect to hydrogen bonding interactions, the most pronounced effect includes the loss of contact made by the nitrogen atoms of K91 and F90 (MBP). Also, [A91, A96]MBP87-96 does not display the contacts to the MHC residues N82(B), Y67(B), and Y68(A), observed in the complex with the wild type peptide. However, compensatory interactions are observed to the MHC residues T77(B) and D57(B). It must be noted that [A91, A96]MBP87-96 displays the greatest deviation from the intermolecular hydrogen bond interactions of the wild type complex, compared to all other mutants we have studied previously11, 12. Interestingly, the differences in the van der Waals (vdW) interactions made by the native peptide vs. [A91, A96]MBP87-96 were less pronounced than was observed with other analogs11, 12. In this case, the loss of contact made by K91 is once more observed. To balance, the analog exhibits the A91 (MBP) interactions with F11(B) (MHC). Other losses of contacts by [A91, A96]MBP87-96 include V87 to Y9(A), H81(B) and N82(B), F89 to Y9(A), and V94 to T65(A). All these are compensated by the interactions, observed in the mutant complex, but not exhibited by the native MBP peptide itself: V87 to F54(A), F89 to V78(B), and V94 to Y61(B), respectively. Based on the novel interactions noted by [A91, A96]MBP87-96 and [R91, A96]MBP87-96 peptide with H2 I-As, we assessed their immunological profile in SJL/J (H2s) mice compared to the native peptide.
The ability of the native MBP87-99 peptide and the double mutant analog [R91, A96]MBP87-99 to induce immune responses in SJL/J mice was assessed by IFN-γ and IL-4 ELISpot assay. High levels of IFN-γ were generated to MBP87-99 peptide which were decreased by 30% (p<0.01) when mice were immunized with [R91, A96]MBP87-99 peptide emulsified in CFA (
[A91, A96]MBP87-99 Peptide Conjugated to Reduced Mannan Diverts Immune Responses from Th1 (IFN-γ) to Th2 (IL-4)
The ability of the linear MBP87-99 or [A91, A96]MBP87-99 peptides either emulsified in CFA or conjugated to reduced mannan, to induce T cell responses after 1 or 2 injections respectively was measured using IFN-γ and IL-4 cytokine secretion by ELISpot analysis. Mice immunized with MBP87-99 peptide induced high levels of IFN-γ secreting T cells when immunized with either CFA or reduced mannan (p<0.01) (
T Cells from SJL/J Mice Immunized with [A91, A96]MBP87-99 Emulsified in CFA or Conjugated to Reduced Mannan do not Cross-React with the Native MBP87-99 Peptide
T cells after immunization with the linear mutant [A91, A96]MBP87-99 peptide emulsified in CFA or conjugated to reduced mannan were examined if they cross-reacted with the native MBP87-99 peptide using ELISpot assay. T cells from mice immunized with linear [A91, A96]MBP87-99 peptide emulsified in CFA (
The production of total IgG antibody responses in mice immunized with MBP87-99 or [A91, A96]MBP87-99 peptide analogs emulsified in CFA (
Since higher levels of antibodies were generated to linear [A91, A96]MBP87-99 peptide when conjugated to reduced mannan, it was determined whether sera from these mice cross-reacted with the native MBP87-99 peptide. Sera from mice immunized with MBP87-99 reacted with MBP87-99 peptide (positive control), while antibodies from mice immunized with linear [A91, A96]MBP87-99 conjugated to reduced mannan did not cross-react with the native MBP87-99 peptide at 1/500 and 1/1,000 sera dilutions (
Activation of CD4+ T cells is initiated by the interaction between the TCR and a peptide-antigen that is presented by MHC class II molecules, and, the engagement of co-stimulatory molecules of antigen presenting cells33. This process is followed by T cell proliferation, stimulation of reactive T cells specific to the antigen and secretion of relevant cytokines. Many studies have shown that peptides with mutations at critical TCR contact residues, results in altered T cell function34-36. In particular, altered peptide ligands (or mutant peptides) have been found to shift the balance of immune responses from Th1 to Th234, 37, 38. Th1 responses (IFN-γ) involve proinflammatory cytokines that mediate autoimmune diseases, and, Th2 responses (IL-4, IL-10) reduce IFN-γ secretion and other inflammatory cytokines, preventing autoimmunity15, 39, 40.
The development of safe and effective vaccines and immunotherapeutic approaches against MS is actively being studied. To date, a number of clinical trials have been undertaken in MS patients with altered peptide ligands41, 42. Even though the peptides were demonstrated to induce appropriate responses in pre-clinical studies, these clinical trials were discontinued due to adverse reactions41, 42. In some patients, unanticipated cross-reactions were stimulated by the peptide analogs against the native peptide/protein41, 42. Thus, further pre-clinical testing is required and new modified peptides need to be designed in order to develop an effective vaccine for MS. In the cancer setting, it was demonstrated that the mutation of the HLA-A2 derived peptide (from carcinoembryonic antigen) at position 6 generated a superagonist. It induced CD8+ T cells that cross reacted with high concentrations of the native peptide, however, did not recognize carcinoembryonic antigen expressing cancer cells43. Thus, there is a need for extensive analysis of tumor cross recognition prior to any clinical usage of APL as vaccines. Therefore, we designed and synthesized two linear mutant peptides, based on the short immunodominant epitope MBP87-99, with mutations at the positions K91 and P96 (K91 was modified to R91 or A91 and P96 to A96). The mutant analogs were injected in SJL/J mice in order to examine their ability to shift immune responses from Th1 to Th2, and whether T cells and antibodies cross reacted with the native peptide. An adjuvant (CFA) or a suitable carrier (reduced mannan) were used.
Immunization of SJL/J mice with native MBP87-99 peptide emulsified in CFA generated high levels of IFN-γ, whilst the double mutant analog [R91, A96]MBP87-99 decreased IFN-γ secretion by 30%. These findings are similar to those previously published with the longer double mutant peptide [R91, A96]MBP83-9911. It is of interest that the levels of IFN-γ were further decreased (70%) when [R91, A96]MBP87-99 peptide was conjugated to the carrier reduced mannan. Furthermore, immunization of mice with the native MBP87-99 peptide either emulsified in CFA or conjugated to reduced mannan did not induce IL-4 cytokine secreting T cells. Thus, substitution of K91 and P96 with R91 and A96, respectively, could decrease IFN-γ levels, generate high levels of IL-4 when [R91, A96]MBP87-99 peptide was conjugated to reduced mannan, but there was still secretion of IFN-γ. By contrast, immunization with the double mutant analog [A91, A96]MBP87-99 conjugated to reduced mannan induced high levels of IL-4 and no IFN-γ was detected. Substitution of residues 91 and 96 to Ala ([A91, A96]MBP87-99) has the ability to generate IL-4 cytokine, however, with the appropriate carrier, it is able to divert immune responses from IFN-γ to IL-4. Overall, the use of adjuvant (CFA) generates both Th1 and Th2 responses and does not seem to be beneficial, however, the use of an appropriate carrier such as reduced mannan conjugated to [A91, A96]MBP87-99 was able to divert immune responses from Th1 to Th2. Most importantly, T cells secreting IFN-γ and IgG antibodies generated to the double mutant [A91, A96]MBP87-99 peptide did not cross-react with the native MBP87-99 peptide. It is clear that the double mutant [A91, A96]MBP87-99 peptide analog is a promising candidate for further studies for the immunotherapy of MS.
Likewise, we recently demonstrated using single amino acid mutations of the longer peptide MBP83-99, that a single amino acid change of K91 to Y91 ([Y91]MBP83-99) when conjugated to reduced mannan also diverted immune responses from Th1 to Th212, similar to [A91, A96]MBP87-99 peptide conjugated to reduced mannan. It is clear that the peptide sequence and length and the type of adjuvant/carrier used are important for eliciting the desired immune response to a peptide-based vaccine.
Modeling of the ([A91, A96]MBP87-96) and ([R91, A96]MBP87-96) analogs in complex with H2 I-As revealed that the side-chains of the mutated residues are exposed to make contacts with TCR and the mutations do not cause major disruptions to the complex structures and to the intermolecular interactions between the peptides and the MHC cleft. It was noted that the backbone of [R91, A96]MBP87-96, [A91, A96]MBP87-96 and native MBP87-96 peptides overlapped very closely and only minor conformational changes were observed in the amino acids at positions 91 and 96 (
Peptides causing antagonism have been shown to have fewer hydrogen bond contacts between the peptide side chains and the CDR3 loops of the TCR44. Loss of hydrogen bond contact can cause agonist or super-agonist (hyper-stimulatory APL) peptides to become antagonists. For example, a single amino acid mutation of vesicular stomatitis virus peptide VSV8 (RGYVYQGL (SEQ ID NO: 29) to RGYVYEGL (SEQ ID NO: 30) leads to antagonism of T cell hybridomas specific to native VSV8. The crystal structure of this APL with H-2Kb demonstrated that a minor peptide modification induced profound biological effect45. The TCR, which recognizes VSV8 (RGYVYQGL; SEQ ID NO: 29) peptide and its APL (RGYVYEGL; SEQ ID NO: 30), was mutated by a single amino acid at the CDR3β loop, and this was able to modulate the TCR-antagonistic properties of an APL46. These examples validate the results of our modeling studies with respect to MBP mutant peptides. Namely, that even subtle conformational changes are sufficient to have profound biological effects.
Molecular modeling was carried out using HyperChem modeling package (HyperCube Inc., version 7.52) as previously described11. OPLS force field was used for molecular mechanics geometry optimization. The MHC-peptide complexes were generated based on the crystal structure of I-Au complex with MBP1-11 peptide (PDB code 1K2D). This template was chosen upon the consideration of nine relevant crystal structures, based on the combination of sequence identity to the target, crystal structure, resolution, and the degree of disruption to the peptide interaction residues, upon mutation12. Alignment of the MBP peptide within the MHC cleft was carried out based on the analysis of all possible MBP positions and the following preferred binding register was deduced12
To produce mutated MBP peptides, the lysine residue at position 91 and the proline residue at position 96 were mutated to A91 and A96 ([A91, A96]MBP87-96) and R91 and A96 ([R91, A96]MBP87-96), respectively. The complexes were then optimized and the intermolecular interactions in the complexes were studied using the program LigPlot47.
Peptides MBP87-99 (VHFFKNIVTPRTP; SEQ ID NO: 33), [R91, A96]MBP87-99 (VHFFRNIVTARTP; SEQ ID NO: 34) and [A91, A96]MBP87-99 (VHFFANIVTARTP; SEQ ID NO: 7) were prepared on 2-chlorotrityl chloride resin (CLTR-C1) using Fmoc/tBu methodology22, 48-51. Preparative HPLC for MBP87-99, [R91, A96]MBP87-99 and [A91, A96]MBP87-99 peptide analogs were performed using a Lichrosorb RP-18 reversed phase semipreparative column with 7 μm packing material. The peptides were >95% pure as analyzed by analytical RP-HPLC and ESI-MS (
MBP87-99, [R91, A96]MBP87-99 or [A91, A96]MBP87-99 peptides were conjugated to keyhole limpet hemocyanin (KLH) via glutaraldehyde which acts as a linker between mannan and peptide.27 14 mg mannan (from Saccharomyces cerevisiae, SIGMA, St Louis, USA) was dissolved in 1 ml sodium phosphate buffer (pH 6.0), followed by the addition of 100 μl of 0.1 M sodium periodate (dissolved in pH 6.0 phosphate buffer) and incubated at 4° C. for 1 hour (h) in the dark. Ethanediol (10 μl) was added to the mixture and incubated for 30 minutes (min) at 4° C. The resultant mixture (oxidized mannan) was passed through a PD-10 column (Sephadex G-25 M column, Amersham Biosciences, Sweden) pre-equilibrated in phosphate buffer (pH 9.0) and 2 ml solution comprising oxidized mannan collected. One mg each of MBP87-99-KLH, [A91, A96]MBP87-99-KLH or [R91, A96]MBP87-99-KLH peptides were added to 2 ml of an oxidized mannan solution and incubated overnight (O/N) at room temperature (RT) in the dark. Conjugation occurs via Schiff base formation between free amino groups of KLH and oxidized mannan. Reduced mannan-KLH-MBP87-99, reduced mannan-KLH-[R91, A96]MBP87-99 or reduced mannan-KLH-[A91, A96]MBP87-99 complexes were prepared by adding 1.0 mg sodium borohydride for 6-8 h at RT in the dark and were used without further purification as previously described12, 23, 30, 31. MBP peptide analogs were previously characterized by capillary electropheresis for conjugation to mannan52 and by SDS PAGE gel, staining with Coomassie, Silver or Schiff's reagent (data not shown). Peptides were 100% conjugated to reduced mannan.
Female 6-8 week old SJL/J mice used in this study, were purchased from Walter and Eliza Hall Institute (Victoria, Australia) and housed at the Biological Research Laboratory at Burnet Institute, Austin campus, Heidelberg, Australia.
MBP87-99, [R91, A96]MBP87-99 or [A91, A96]MBP87-99 peptides were dissolved in PBS and emulsified in an equal volume of complete Freund's adjuvant (CFA) which contained 1.0 mg/ml of heat killed Mycobacterium tuberculosis H37RA (Sigma, Victoria, Australia). SJL/J mice were given one subcutaneous injection containing 50 μg peptide into the base of tail.
SJL/J mice were immunized twice on days 0 and 14 intradermally (base of tail) with 50 μg MBP87-99-KLH-reduced mannan, [R91, A96]MBP87-99-KLH-reduced mannan or [A91, A96]MBP87-99-KLH-reduced mannan conjugates.
ELISpot detects specific T cell responses to antigens by measuring the secretion of specific cytokines from individual cells. Spleen cells from immunized SJL/J mice were isolated 28 days after CFA immunization or 14 days after the last immunization with reduced mannan-peptides and assessed by ELISpot for IFN-γ and IL-4 secretion by T cells. IFN-γ ELISpot assay was performed on MultiScreen-IP Filter Plate (MAIP S4510) with hydrophobic PVDF filters (Millipore, UK), while IL-4 ELISpot assays were performed on MultiScreen-HA Filter Plate (MAHA S4510) with mixed cellulose esters filters (Millipore, UK). MAIP S4510 plates were pre-weted with 50 μl of 70% ethanol, washed 5 times with 200 μl of sterile phosphate-buffered saline (PBS) and coated with 70 μl of 5 μg/ml anti-IFN-γ capture antibody, AN18 (Mabtech, Australia) in PBS and incubated O/N at 4° C. 70 μl of 5 μg/ml anti-IL-4 capture antibody (Mabtech, Australia) was added directly to MAHA S4510 plates and incubated O/N at 4° C. without 70% ethanol treatment. Following 5 washes with PBS, plates were blocked by adding 200 μl of culture media (supplemented with 2.5% fetal calf serum) and incubated for 2 h at 37° C. The blocking media was discarded and 10 μg/ml MBP87-99, [R91, A96]MBP87-99 or [A91, A96]MBP87-99 recall peptides were added into each defined well. ConA (1.0 μg/ml) was used as an internal positive control and no peptide (cells alone) as a negative control. Triplicate wells were set up for each condition. Spleen cells (0.5 million cells) in 100 μl culture media were seeded into each well and incubated at 37° C. for 18 h for IFN-γ or 24 for IL-4, respectively. Plates were washed 5 times with PBS/0.05% Tween 20 followed by 5 times with PBS and incubated for 2 h at RT with anti-mouse IFN-γ or IL-4 monoclonal antibody-biotin. Plates were washed and streptavidin-ALP was added at 1.0 μg/ml and incubated for a further 2 h at RT. Spots of activity were detected using a colorimetric AP kit (Biorad, Hercules, Calif. USA) and counted using an AID ELISpot plate reader (Autoimmun Diagnostika GmbH, Germany). Data are presented as mean spot forming units (SFU) per 0.5 million cells +/−standard error of the mean (SEM).
Blood was collected and sera isolated from mice prior to and 4 weeks after immunization with CFA or 14 days after the last immunization with reduced mannan conjugates. MBP87-99 or [A91, A96]MBP87-99 peptides conjugated to BSA or KLH were coated onto polyvinyl chloride (PVC) microtiter plates at 10 μg/ml in 0.2 M NaHCO3 buffer, pH 9.6, 0/N at 4° C. Non-specific binding was blocked with 2% BSA for 1.0 h at RT. After washing (0.05 Tween 20/PBS), serial dilutions of sera were added and incubated for a further 2 h at RT. The plates were washed and bound antibody was detected using HRP-conjugated sheep anti-mouse antibody (1/1000 dilution in PBS) (Amersham, UK) and developed using 2,2′-azino-di(3-ethylbenzthiazoline)6-sulfonic acid (ABTS) (Sigma, UK). Absorbance at 405 nm was recorded using a Fluostar Optima microplate reader (BMG labtech, Offenburg, Germany).
Mean values were compared using the Student's two-tailed t-test. P value threshold of p<0.01 indicates a statistically significant difference.
The ability of the linear MBP87-99 or cyclo(87-99)[A91, A96]MBP87-99 peptides either emulsified in complete Freund's adjuvant (CFA) or conjugated to reduced mannan, to induce T cell responses after 1 or 2 injections respectively was measured using IFN-γ and IL-4 cytokine secretion by ELISpot analysis. In general, mice immunized with MBP87-99 peptide induced high levels of IFN-γ secreting T cells when immunized with either CFA or reduced mannan (
T Cells and Antibodies from SJL/J Mice Immunized with Cyclo(87-99)[A91 A96]MBP87-99 Emulsified in CFA or Conjugated to Reduced Mannan do not Cross-React with the Native MBP87-99 Peptide
While cyclo(87-99)[A91, A96]MBP87-99 peptide emulsified in CFA or conjugated to reduced mannan generated IFN-γ responses, we examined whether the IFN-γ producing T cells cross-reacted with the native MBP87-99 peptide using ELISpot assay. SJL/J mice were immunized with cyclo(87-99)[A91, A96]MBP87-99 peptide and assessed IFN-γ production against MBP87-99 peptide. T cells from mice immunized with cyclo(87-99)[A91, A96]MBP87-99 emulsified CFA (
The production of total IgG antibody responses in mice immunized with MBP87-99 or cyclo(87-99)[A91, A96]MBP87-99 peptide analogs emulsified in CFA (
These findings are of importance in the clinical setting, as cross reactive T cells and antibodies to the native peptide were induced in clinical trials with altered peptide ligands of MBP peptide which were the reason for premature termination of the trials11, 12. It is clear that cyclization alters the conformation of the peptide, thus, being presented in an altered form by the MHC, and hence, the T cells that are induced recognize a different conformation compared to the native form. We had previously demonstrated that cyclic peptides bind to human MHC class II, HLA-DR413, and, a cartoon model of how cyclic peptides may be presented by murine MHC was shown3.
Peptides MBP87-99 (VHFFKNIVTPRTP; SEQ ID NO: 33) and cyclic double mutant peptide with Ala mutations at positions 91 and 96, cyclo(87-99)[A91, A96]MBP87-99 (cyclo, head-to-tail, VHFFANIVTARTP; SEQ ID NO: 8) were prepared on 2-chlorotrityl chloride resin (CLTR-C1) using Fmoc/tBu methodology. Head-to-tail cyclization of MBP87-99[A91, A96] peptide was achieved using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxy-7-azabenzotriazole, 2,4,6 collidine in DMF solution, allowing fast reactions and high yield cyclization products. Results indicate that HOAt, a 4-nitrogen containing variant, is a very effective coupling additive, more efficient than HOBt for solution or solid phase synthesis14-17. Preparative HPLC for peptide analogs were performed using a Lichrosorb RP-18 reversed phase semi-preparative column with 7 μm packing material. The peptides were >95% pure as analyzed by mass spectrometry (ESI-MS).
MBP87-99 or cyclo(83-99)[A91,A96]MBP87-99 peptides were conjugated to keyhole limpet hemocyanin (KLH) via glutaraldehyde; KLH acts as a linker between mannan and peptide18. Conjugation procedures were previously published3-9,18. Briefly, mannan (from Saccharomyces cerevisiae, SIGMA, St Louis, USA) was dissolved pH 6.0 sodium phosphate buffer, followed by the addition of sodium periodate and incubated for 1 hour at 4° C. Ethanediol was added to the mixture and incubated for 30 mins at 4° C. The mixture (oxidized mannan) was passed through a PD-10 column (Sephadex G-25 M column, Amersham Biosciences, Sweden) pre-equilibrated in pH 9.0 phosphate buffer. Oxidized mannan was eluted, to which peptides were added and allowed to react overnight at room temperature in the dark. Conjugation occurs via Schiff base formation between free amino groups of KLH and oxidized mannan. Reduced mannan-KLH-MBP87-99 or reduced mannan-KLH-cyclo(83-99)[A91, A96]MBP87-99 complexes were prepared by adding sodium borohydride. MBP peptide analogs were previously characterized by capillary electrophoresis for conjugation to mannan19 and by SDS PAGE gel, staining with Coomassie, Silver or Schiff's reagent (data not shown). Female 6-8 week old SJL/J mice were immunized twice with 50 μg MBP87-99 or cyclo(83-99)[A91, A96]MBP87-99-KLH-reduced mannan conjugates on days 0, 14.
MBP87-99 or cyclo(87-99)[A91, A96]MBP87-99 peptides were dissolved in PBS and emulsified in an equal volume of CFA (Sigma, VIC Australia). SJL/J mice were given one subcutaneous injection containing 50 μg of MBP87-99 or cyclo(87-99)[A91, A96]MBP87-99 peptide.
ELISpot assays for IFN-γ and IL-4 secretion by T cells and ELISA assays for binding of sera to native MBP protein or peptide were carried out as previously described3, 4, 20, 21. Mean values were compared using the Student's two-tailed t-test. P value threshold of p<0.01 indicates a statistically significant difference.
Various modifications and variations of the described aspects 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 of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20070100697 | Nov 2007 | GR | national |
| 0724878.4 | Dec 2007 | GB | national |
| 0810553.8 | Jun 2008 | GB | national |
| 2008904757 | Sep 2008 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/003493 | 11/20/2008 | WO | 00 | 3/1/2011 |
