Patent Application
20100267651
The present invention relates to novel peptides designed to interfere with the function of the T-cell, such that the novel peptide can be used in the treatment of various inflammatory and autoimmune disease states. In particular, the peptide is useful in the treatment of disorders where T-cells are involved or recruited.
T-cells are a subgroup of cells which together with other immune cell types (polymorphonuclear, eosinophils, basophils, mast cells, B-, NK cells), constitute the cellular component of the immune system. Under physiological conditions T-cells function in immune surveillance and in the elimination of foreign antigen. However, under pathological conditions there is compelling evidence that T-cells play a major role in the causation and propagation of disease. In these disorders, breakdown of T-cell immunological tolerance, either central or peripheral is a fundamental process in the causation of autoimmune disease.
Central tolerance involves thymic deletion of self reactive cells (negative selection) and positive selection of T-cells with low affinity for self major histocompatibility complex antigens (MHC). In contrast, there are four, non-mutually exclusive hypotheses that have been proposed to explain peripheral T-cell tolerance which are involved in the prevention of tissue specific autoimmune disease. These include: anergy (loss of co-stimulatory signals, down regulation of receptors critical for T-cell activation), deletion of reactive T-cells, ignorance of the antigen by the immune system and suppression of autoreactive T-cells. Tolerance once induced does not necessarily persist indefinitely. A breakdown in any of these mechanisms may lead to auto-immune disease.
Autoimmune disease and other T-cell mediated disorders are characterised by the recruitment of T-cells to sites of inflammation. T-cells at these sites, coupled with their ability to produce and regulate cytokines and influence B-cell function, orchestrate the immune response and shape the final clinical outcome. An understanding of the process of T-cell antigen recognition and subsequent T-cell activation, leading to T-cell proliferation and differentiation, is therefore pivotal to both health and disease. Disturbance in this intricate structure-function relationship of the T-cell antigen receptor, harmonising antigen recognition with T-cell activation may provide the therapeutic means to deal with inflammation and T-cell mediated disorders.
The TCR is composed of at least seven transmembrane proteins'. The disulfide-linked (αβ-Ti) heterodimer is the clonotypic antigen recognition unit, while the invariant chains of CD3, consisting of ε, γ, δ, and ξ and η chains, are responsible for coupling the ligand binding to signalling pathways that result in T-cell activation and the elaboration of the cellular immune responses. Despite the gene diversity of the TCR chains, two structural features are common to all known subunits. Firstly, they are transmembrane proteins with a single transmembrane spanning domain—presumably alpha-helical. Secondly, all the TCR chains have the unusual feature of possessing a charged amino acid within the predicted transmembrane domain. The invariant chains have a single negative charge, conserved between the mouse and human, and the variant chains possess one (TCR-(β) or two (TCR-α) positive charges. Listed in Table 1 is the transmembrane sequence of TCR-α in a number of species showing that this region is highly conserved and that phylogenetically may subserve an important functional role. The octapeptide (bold) containing the hydrophilic amino acids arginine and lysine is identical between the species. The amino acid substitutions noted in the remaining portions of the transmembrane sequence are minor and conservative.
Studies on the assembly of the multicomponent TCR by Manolios et al2,3,4 showed that the stable interaction between TCR-α and CD3-δ and TCR-α and CD3-ε was localised to eight amino acids within the transmembrane domain of TCR-α and it was the charged amino acids arginine and lysine that were critical for this process. This finding exemplified the fact that amino acids within the transmembrane domain not only functioned to anchor proteins but were important in the assembly of subunit complexes and protein-protein interactions. For the first time it was found that the assembly of this complex receptor could hinge on only eight amino acids. The above system depended on the modification of complementary strand DNA (cDNA) to create a number of protein mutants. Chimeric cDNA molecules were transfected into COS cells to express the required protein. Coexpression of these chimeric proteins were used to evaluate the region of interaction. The technology involved cDNA manipulation, metabolic labelling, immunoprecipitation and gel electrophoresis. Transmembrane domains are small in size and proteins transversing this region are constrained to an alpha-helical configuration. These biophysical features coupled with the ability to engineer protein-protein interactions via transmembrane charge groups suggested a possible new approach to intervene and potentially disturb TCR function. The use of peptides as possible inhibitors of assembly, the recognition and application of this peptide sequence as a possible therapeutic agent to interfere with T-cell function was not a normal or obvious extension.
In co-pending International Patent Application No. PCT/AU96/00018 the present inventor developed peptides which disturb TCR function. The disclosure of this application is included herein by cross-references.
In the last decade a new age of therapeutics has developed with the so-called “Biologics”, that aim to target specific individual cells, and molecules within the cells, with the specific purpose of interrupting immunological networks and cascades thought to underlie the disease process. The disease model for rheumatoid arthritis has been exemplary in the design of biological agents and a number of different approaches have been devised and tested6. The model predicts that an initial arthritogenic peptide is presented to T-cells by an antigen presenting cell (APC) which causes activation of T cells and release of cytokines and proteases culminating in chronic inflammation and joint damage (
The trimolecular complex as the site for therapeutic intervention has been the subject of focus since the recent advances in the molecular characterisation of its constituents and has provided several approaches for immune intervention. The aim of therapy is to eliminate, prevent or downregulate the T-cell response by a variety of means (
(i) MAbs to T-cell antigens. The use of MAbs in the treatment of RA has been reviewed by a number of authors6,10,11. The MAbs tested were directed against a variety of antigens ranging from: (a) those present on all mature T-cells, and thought to be involved in the pathogenesis of RA (CD5, CDw52)12,13; (b) MAbs specific for T-cell subsets (CD4), which have the advantage of limited immunosuppressive effects14,15; and (c) to MAbs directed against T-cell activation antigens (IL-2 receptor) which may specifically suppress activated T-cells in response to antigen16,17. All the MAbs used are derived from rodents and only CAMPATH-1H has been “humanised” by recombinant cDNA techniques. Clinical studies indicate that these MAbs are well tolerated in patients and can induce a favourable clinical response. Side effects include an immune reaction to the rodent antibodies which may restrict recurrent use.
(ii) Anti-MHC therapy. Immunogenetic studies have demonstrated that the MHC molecules (DR1, DR4, Dw4 and DR4 Dw14) are important in RA susceptibility18. Since MHC molecules present antigenic peptides to T-cells they provide another target for immune intervention. The function of these molecules can be interfered with either by using MAbs (to the antigen binding sites)19 or high affinity binding of competitor peptides to the MHC groove (see below). MAbs directed against MHC molecules interfere with disease initiation in several animal models of autoimmunity20,21 and humans22.
(iii) Peptide competition. T-cell recognition of antigen can be disrupted by using high affinity MHC-binding peptides which block the antigen-binding site of MHC molecules and inhibit T-cell responses. By substitution of particular amino acid residues it is possible to generate “designer’ peptides, which have high affinity for MHC molecules but do not activate T-cells23. This therapy has the advantage of specificity without causing generalised immunosuppression.
(iv) T-cell vaccination. This form of therapy holds promise for those diseases which exhibit T-cell oligoclonality. The idea is to obtain pathogenic T-cell clones and vaccinate against these cells hoping to eliminate them from the available T-cell repertoire. Another more refined method of vaccination has been to synthesize peptides corresponding to the T-cell receptor sequences which are involved in antigen recognition. Autoimmune animal models vaccinated with such peptides support the view that it is possible to block functional T-cell clones by using synthetic peptides24,25. Whether these antiTCR strategies are applicable to rheumatoid disease depends on the oligoclonality of the autoreactive cells and their limited TCR usage. Although still controversial, evidence of a limited repertoire of TCR usage has been reported in RA26,27.
(v) Cytokine therapy. Synovial fluid analysis of patients with RA has shown the presence of a large number of cytokines including granulocyte-macrophage colony stimulating factor (GM-CSF), gamma-interferon (IFN-γ), interleukin-1 (IL-1) and tumour necrosis factor (TNF-α)28. Cytokines interact with cells to co-ordinate the immune and inflammatory response. They can be grouped as either pro-inflammatory or anti-inflammatory. IL-1 and TNF-α are in the former group and act synergistically. TNF-α is also one of the major cytokines regulating the expression of IL-128. Because of their central importance attempts to interfere with their regulation or production may have a positive effect on disease outcome29,30. Administration of IL-1 receptor antagonist to rats and mice with arthritis has reduced the severity of joint lesions and is in Phase II studies in human disease. Therapeutic use of MAbs to the IL-2 receptor has transient effects31. The receptors for a large group of cytokines have been cloned and sequenced (reviewed by Dower and Sims)32 and currently under clinical evaluation33. It may be that the soluble form of the cytokine receptors may be used to sequester the cytokines by a ligand type interaction and thereby reduce inflammation. Cyclosporin A modulates T-cell cytokine production and when given in several trials has given good clinical response. However the associated nephrotoxicity limits its use34.
(vi) The ability to disrupt cellular function by the use of peptides derived from protein sequences critical for receptor assembly, has only recently been published35 and is a new approach for the use of biologics, that could be included into the schema of biological mechanisms of action. That is, the disruption of cellular function by “disorganising” the assembly of receptors by use of peptides. By design, the peptide chosen corresponded to a common transmembrane sequence common to both CD4 and CD8 cells and currently other unique sites of TCR chain interaction are under investigation. In particular, interactions in the extra cellular domain between the antigen recognition chains, may prove useful in devising peptides for individual pathogenic T cell clones with specific Vα/Vβ usage.
The present inventor has now developed further novel peptides which disturb TCR function, presumably by interfering with assembly. These peptides are based on sequences from (i) the core peptide; (ii) peptides that correspond to alternative chain assembly regions; ie. CD3-δ, -ε, -γ chains; (iii) new sites of assembly, ie. interchain disulphide bond; and (iv) downstream sequences of core peptide. The present inventor has also found that these peptides have an effect on T-cell mediated inflammation. The efficacious clinical manifestations of the administered peptide would be a decrease in inflammation, e.g. as demonstrated by a decrease of arthritis in an adjuvant model of arthritis.
Accordingly, in a first aspect the present invention provides a peptide which inhibits TCR function, wherein the peptide is of the following formula:
R1-A-B-A-R2 in which
By “hydrophobic peptide sequence” we mean a sequence which includes at least 1 hydrophobic amino acid and which does not include a charged amino acid. Preferably, at least 50% of the amino acids make up the hydrophobic peptide sequence are hydrophobic amino acids. More preferably at least 80% of all amino acids which make up the hydrophobic peptide sequence are hydrophobic amino acids.
In a preferred embodiment of the present invention A is a peptide comprising from 2 to 6 amino acids.
In one preferred embodiment of the present invention the peptide sequence is derived from the TCR-a transmembrane chain. In one preferred aspect of this embodiment B is a positively charged amino acid. B is preferably lysine or arginine.
In yet a further preferred embodiment of the present invention the peptide comprises the sequence
In a further preferred embodiment the peptide sequence is derived from the TCR-α intracellular chain. In a preferred aspect of this embodiment the peptide comprises the sequence:
In a further preferred embodiment the peptide sequence is derived from the transmembrane CD3-δ, -ε, or -γ chain sequence. In this preferred embodiment B may be a negatively charged amino acid.
In yet a further embodiment, the peptide sequence is derived from the CD3-δ or -ε chain. In this preferred embodiment B may be aspartic acid. In a particularly preferred aspect of this embodiment the peptide comprises the following sequence:
In yet a further embodiment, the peptide sequence is derived from the CD3-γ chain. In this preferred embodiment B may be glutamic acid. In a particularly preferred aspect of this embodiment the peptide comprises the following sequence:
In a second aspect, the present invention provides a peptide which inhibits TCR function, wherein the peptide is derived from the TCR-α intracellular chain and comprises the formula:
It has also been found that the TCR-αβ interchain disulphide bond plays an important role in the T cell assembly and subsequent activation by antigenic peptide.
The present invention therefore also provides novel peptides which destabilise the interchain cysteine bond of the TCR-α and TCR-β chains and inhibit T-cell activation.
Accordingly, in a third aspect the present invention provides a peptide which inhibits TCR function, wherein the peptide is of the following formula:
R1-A-B-C—R2 in which
A is a peptide sequence of between 0 and 5 amino acids;
B is cysteine;
C is a peptide sequence of between 2 to 10 amino acids;
R1 is NH2; and
R2 is COOH.
In a preferred embodiment of the present invention A is a peptide sequence consisting of 5 amino acids.
In one embodiment the peptide is derived from the TCR-β chain. Preferably, C is a peptide consisting of 4 or 5 amino acids and includes at least one hydrophobic amino acid. In a preferred embodiment the peptide has the following sequence:
In another embodiment, the peptide is derived from the TCR-α chain. In this embodiment the peptide preferably has the following sequence:
A fourth aspect of the present invention provides a peptide that disturbs TCR function and that conforms to the following formula:
A-B-C-D-E in which:
In a preferred embodiment of the present invention, C is three or four hydrophobic amino acids. In a further preferred embodiment of the present invention, A is 2 hydrophobic amino acids and E is 1 to 3, and more preferably 1, hydrophobic amino acids. In yet a further preferred embodiment of the present invention, B is arginine and D is lysine or B is lysine and D is arginine. In yet a further preferred embodiment of the present invention, the peptide is: -Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 21), or Leu-Lys-Ile-Leu-Leu-Leu-Arg-Val (SEQ ID NO: 23), or Gly-Phe-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 27) or Phe-Lys-Ile-Leu-Leu-Leu-Arg-Val (SEQ ID NO: 28), or Leu-Arg-Leu-Leu-Leu-Lys-Val-OH (SEQ ID NO: 26).
It will be appreciated by those skilled in the art that a number of modifications may also be made to the peptides of the present invention without deleteriously affecting the biological activity of the peptide. This may be achieved by various changes, such as insertions and substitutions, either conservative or non-conservative in the peptide sequence where such changes do not substantially decrease the biological activity of the peptide.
Modifications of the peptides contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5′-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-bitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with dimethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid; 2-thienyl alanine and/or D-isomers of amino acids.
The peptides of the present invention may be synthesised using techniques well known to those skilled in this field. For example, the peptides may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Sheppard which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson and published by Blackwell Scientific Publications. Preferably a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross-linked with a small proportion of divinylbenzene (e.g. 1%) which is further swollen by lipophilic solvents such as dichloromethane or more polar solvents such as dimethylformamide (DMF). The polystyrene may be functionalised with chloromethyl or anionomethyl groups. Alternatively, cross-linked and functionalised polydimethyl-acrylamide gel is used which may be highly solvated and swollen by DMF and other dipolar aprolic solvents. Other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads. In a preferred for in, use may be made of commercial solid supports or resins which are selected from PAL-PEG, PAK-PEG, KA, KR or TGR.
In solid state synthesis, use is made of reversible blocking groups which have the dual function of masking unwanted reactivity in the α-amino, carboxy or side chain functional groups and of destroying the dipolar character of amino acids and peptides which render them inactive. Such functional groups can be selected from t-butyl esters of the structure RCO—OCMe3-CO—NHR which are known as t-butoxy carboxyl or ROC derivatives. Use may also be made of the corresponding benzyl esters having the structure RCO—OCH2—C6H5 and urethanes having the structure C6H5CH2O CO—NHR which are known as the benzyloxycarbonyl or Z-derivatives. Use may also be made of derivatives of fluorenyl methanol and especially the fluorenyl-methoxy carbonyl or Fmoc group. Each of these types of protecting group is capable of independent cleavage in the presence of one other so that frequent use is made, for example, of BOC-benzyl and Fmoc-tertiary butyl protection strategies.
Reference also should be made to a condensing agent to link the amino and carboxy groups of protected amino acids or peptides. This may be done by activating the carboxy group so that it reacts spontaneously with a free primary or secondary amine. Activated esters such as those derived from p-nitrophenol and pentafluorophenyl may be used for this purpose. Their reactivity may be increased by addition of catalysts such as i-hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page 215-216 of the abovementioned Nicholson reference) also may be used. Other acylating species are formed in situ by treatment of the carboxylic acid (i.e. the Na-protected amino acid or peptide) with a condensing reagent and are reacted immediately with the amino component (the carboxy or C-protected amino acid or peptide). Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 of the Nicholson reference), O'Benzotriazole-N,N,N′N′-tetra methyl-uronium hexafluorophosphate (HBTU) and its analogous tetrafluoroborate are frequently used condensing agents.
The attachment of the first amino acid to the solid phase support may be carried out using BOC-amino acids in any suitable manner. In one method BOC amino acids are attached to chloromethyl resin by warming the triethyl ammonium salts with the resin. Fmoc-amino acids may be coupled to the p-alkoxybenzyl alcohol resin in similar manner. Alternatively, use may be made of various linkage agents or “handles” to join the first amino acid to the resin. In this regard, p-hydroxymethyl phenylactic acid linked to aminomethyl polystyrene may be used for this purpose.
It may also be possible to add various groups to the peptide of the present invention to confer advantages such as increased potency or extended half life in vivo without substantially decreasing the biological activity of the peptide. It is intended that such modifications to the peptide of the present invention which do not result in a decrease in biological activity are within the scope of the present invention.
In a further aspect, the present invention provides a therapeutic composition that includes a peptide of the first, second, third, or fourth aspect of the present invention, plus a pharmaceutically acceptable carrier.
In a further aspect the present invention provides a method of treating a subject suffering from a disorder in which T-cells are involved or recruited, the method including administering to the subject a therapeutically effective amount of the peptide of the first, second, third or fourth aspect of the present invention.
The therapeutic composition may be administered by any appropriate route as will be recognised by those skilled in the art. Such routes include oral, transdermal, intranasal, parenteral, intraarticular and intraocular.
In yet a further aspect, the present invention consists in a method of delivering a chemical moiety to a cell including exposing the cell to the chemical moiety conjugated to a peptide of the first, second, third or fourth aspect of the invention.
In a preferred embodiment the chemical moiety is conjugated to the carboxy terminal of the peptide.
A non-exhaustive list of disorders in which T cells are involved/recruited include:
As used herein, the term “subject” is intended to cover both human and non-human animals.
The peptides of the present invention may be modified at the carboxy terminal without loss of activity. Accordingly, it is intended that the present invention includes within its scope peptides which include additional amino acids to the “core” sequence of the peptide of the present invention and which affect the T-cell antigen receptor.
It is envisaged that the peptides of the present invention are able to enter cells. Accordingly it is envisaged that, apart from its other uses, the peptide of the present invention could be used as a “carrier” to deliver other therapeutic agents to cells. This could be achieved, for example, by conjugating the therapeutic to be delivered into the cell to the peptide of the present invention.
As will be recognized from the above discussion, the peptide of the fourth aspect of the present invention is based on a portion of transmembrane domain of TCR-α. The complete murine sequence of this portion is NLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 29), whereas the corresponding human sequence is NLSVIGFRILLLKVAGFNLLMTL (SEQ ID NO: 30). There is complete sequence homology across a range of species in the last 15 amino acids of the TCR-alpha chain distal to the sequence upon the peptide of the present invention is based (shown in bold). Peptides including these additional 15 residues may have activity similar to the peptide of the present invention. The essential feature is that the peptide includes two positively charged amino acids separated by 3 to 5 hydrophobic amino acids.
As will be readily understood by those skilled in this field hydrophobic amino acids are Ala, Val, Leu, Ile, Pro, Phe, Tyr and Met; positively charged amino acids are Lys, Arg and His; and negatively charged amino acids are Asp and Glu.
In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples and figures in which:
a)—Schematic representation of antigen recognition by T-cells and subsequent downstream events. Possible sites of intervention include the trimolecular complex, T-cells, T-cell surface molecules, cytokines, recruitment of cells, and catalytic enzymes.
b)—Trimolecular complex with possible intervention sites.
FIG. 2—Effect of peptides on primed lymph node cells. Shown are means and standard errors (n=4). Peptide final concentrations were 100 p.g/ml and were delivered to the wells in 20 μl of 0.1% acetic acid.
FIG. 3—Effect of peptide/s on primed lymph node cells. Shown are the mean and standard error of four wells. Core peptide (CP) was either freshly dissolved (fresh) or in solution for at least three months at 4° C. (old).
FIG. 4—Effect of peptides on a rat T-cell line specific to MTB. Shown are mean and standard error of four wells. Peptides were 100 μM in the wells and stock solutions were 1 mM in 0.1% acetic acid.
FIG. 5—Effect of peptides on an MTB-specific T-cell line. Shown are means and standard errors (n=4). Peptide final concentrations were 100 μM except where stated. Core peptide (CP) at 0.1 mg/ml is 87 μM.
FIG. 6—Weight of treated and untreated rats. Shown are the means and standard errors of five rats in each group.
a)—Paw thickness in untreated rats. Each point represents the mean of both hind paws of each rat.
b)—Paw thickness in peptide-J treated rats. Each point represents the mean of both hind paws of each rat.
c)—Paw thickness in peptide-O treated rats. Each point represents the mean of both hind paws of each rat.
d)—Paw thickness in peptide-K treated rats. Each point represents the mean of both hind paws of each rat.
FIG. 8—Weight of untreated (MTB only) and treated (peptides N, M, P) rats. Shown are the means and standard errors of five rates in each group.
a)—Ankle thickness of untreated rats. Each point represents the thickness of individual ankle joints.
b)—Ankle thickness of peptide-M treated rats. Each point represents the thickness of individual ankle joints.
c)—Ankle thickness of peptide-N treated rats. Each point represents the thickness of individual ankle joints.
d)—Ankle thickness of peptide-P treated rats. Each point represents the thickness of individual ankle joints.
FIG. 10—Weight of peptide L-treated and untreated rats. Shown are the mean and standard errors of five rats in each group.
a)—Paw thickness in untreated rats. Each point represents the thickness of an individual hind paw.
b)—Paw thickness in peptide-L treated rats. Each point represents the thickness of individual hind paws.
c)—Ankle thickness of untreated rats. Each point represents the thickness of individual ankle joints.
d)—Ankle thickness of peptide-L treated rats. Each point represents the thickness of individual ankle joints.
a)—Shows the delayed induction and clinical severity of disease in animals treated or untreated with the core peptide of the fourth aspect of the invention.
b)—Shows the average weight of animals treated or untreated with the core peptide.
Peptide synthesis. Peptides were synthesised by solid phase synthesis using FMOC chemistry in the manual mode. Unprotected peptides were purchased from Auspep (Melbourne, Australia) with greater than 75% purity as assessed by HPLC. An example of an enclosed specification sheet is attached in the Appendix. The final concentration of peptide dissolved in 0.1% acetic acid used in cell culture ranged from 10 μM-200 μM. For in-vivo studies,
peptides were dissolved/suspended in squalane oil (2-, 6-, 10-, 15-, 19-, 23-hexamethyltetracosane).
Cells. The following cell lines were used: 2B4.11, a murine T-cell hybridoma that expresses a complete antigen receptor on the cell surface and produces IL-2 following antigen recognition (cytochrome-c); an interleukin-2 dependent T-cell line (CTLL) for conventional biological IL-2 assays; and the B-cell hybridoma cell line LK 35.2 (LK, I-Ek bearing) which acts as the antigen presenting cell. The hybridomas were grown in T-cell medium (RPMI-1640 media containing 10% foetal calf serum (FCS), gentamycin (80 μg/ml), glutamine (2 mM) and mercaptoethanol (0.002%)). The African green monkey kidney fibroblast cell line (COS) was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS.
Antigen presentation assay36. The mouse T-cell 2B4.11 hybridoma (2×104) was cultured in microtitre wells with LK35.2 antigen presenting B cells (2×104) and 50 μM pigeon cytochrome-c. After 16 hr 50 microlitres of assay supernatant was removed and assayed for the presence of IL-2. Serial twofold dilutions of the supernatant in media were cultured with the IL-2 dependent T-cell line CTLL. After 16 hr the CTLL cells were pulsed with 3H-thymidine for 4 hr and IL-2 measurements (IU/ml) determined. Peptides examined included: CP, A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, and P (Table 2). Peptide L was very insoluble and was not tested in vitro. The peptides were tested in the antigen presentation assay at final concentrations ranging from 10 μM to 200 μM.
Primed Lymph Node Cells (PLNC). Male Wistar rats were injected intradermally at the base of the tail with 1 mg of heat-killed Mycobacterium tuberculosis (MTB) suspended in 0.2 ml of squalane. When acute arthritis was well developed, after 10 to 16 days, rats were killed and the swollen popliteal lymph nodes were removed and a single cell suspension made by pressing the tissue through a fine sieve under aseptic conditions. Cells were washed in complete medium, resuspended and counted. Approximately 3.5×108 viable cells were obtained from two rats. The medium used was RPMI 1640 supplemented with 25 mM Hepes, penicillin (100 μg/ml), streptomycin (80 μg/ml), 2.5×10−5 M 2-mercaptoethanol and 2% pooled normal rat serum. The cells were pipetted into the wells of flat-bottom, 96 well microtitre plates at 2×105/well and a suspension of MTB was added to a final concentration of 100 μg/ml. Peptides were delivered to the wells in 20 μl volume giving final concentrations of 100 μg/ml peptides (or 100 μM) and 0.01% acetic acid, and a total of 200 μl per well. The plates were incubated at 37° C. in a humidified incubator at 5% CO2 for 3 days and then were pulsed with 1 μCi per well of 3H-thymidine in 25 ml of medium. After a further overnight incubation, the cultures were harvested using an automated cell harvester, and counted in a β-scintillation spectrometer.
T-Cell Lines. The method used was by Sedgwick et al (1989)37. PLNCs from MTB-immunised rats were cultured in 75 cm2 culture flasks at 5×106 per ml in a total of 50 ml containing 100 μg/ml MTB. After three days the cells were spun down and resuspended in 2 ml medium in a 15 ml centrifuge tube and were underlayered with 3 ml of Ficoll diatrizoate (9.9% Ficoll 400; 9.6% sodium diatrizoate), and centrifuged at 800 g for 20 minutes. The T-cell blasts were recovered from the interface, washed twice and resuspended at 2×105 per ml in medium supplemented with 10% FCS and 15% con A-stimulated spleen cell supernatant, as a source of IL-2. After four days culture in the rest phase, 2×105 T-cells per ml were restimulated with antigen and 107 syngeneic rat thymocytes per ml to act as antigen presenting cells. The latter had been inactivated by incubation with 25 μg/ml mitomycin C for 20 minutes at 37° C. and carefully washed three times. Cultures were in 75 cm2 flasks containing 50 ml and the antigen, MTB, was added at 100 μg/ml. Flasks were stood up vertically and cultured for 3 days. Again T-cell blasts were recovered by separation on Ficoll/diatrizoate, and the cycle was repeated. After 2-4 cycles, the cells were set in 96-well plates at 104 T-cells/well and 106 mitomycin-C-inactivated thymocytes, in 200 ml medium containing 100 μg/ml MTB and 2% rat serum. Additions of 20 μl were made to the wells containing peptides in 0.1% acetic acid. Cultures were incubated for three days, then 3H-thymidine (1 μCi in 25 ml medium) was added and the incubation continued overnight after which it was harvested and counted in the β-counter. Results are shown as count2 per minute (cpm) tritiated thymidine incorporation. The peptides tested in these assays for the ability to inhibit antigen-stimulated T-lymphocyte proliferation are shown in Table 2.
Adjuvant-induced arthritis in rats. Arthritis in rats was induced by a single intradermal injection of heat killed MTB in 200 μl squalane (adjuvant) at the base of the tail. Peptides (35 mg) were suspended in one millilitre squalane containing 5 mg of MTB. That is, there was 1 mg MTB and 7 mg peptide in 0.2 ml of squalane injected intradermally. At regular intervals for up to 28 days, animals were weighed and their arthritic condition assessed by measurement of ankle thickness and rear paw thickness (with a micrometer) and recording the number of arthritic joints involved. Rats were housed in standard cages after the initial tail injection and allowed access to unlimited water and pellet food. Rats generally developed arthritis 12-14 days after the injection. Consistent with previous reports, not all rats given MTB/squalane developed arthritis. In our case the success rate was more than 80% of MTB injected control rats developing arthritis. On day 29, the animals were sacrificed.
Initial experiments which attempted to demonstrate an effect of peptide on T-cell function in vitro used an antigen presenting assay. The mouse T-hybridoma 2B4, specific for the protein cytochrome c, was presented with antigen by the LK cell line, and the IL-2 content in the supernatant was bioassayed by measuring the proliferation of the IL-2 dependent line, CTLL5. As hybridomas can be phenotypically unstable, primary T-cells would be a better model and lymph node cells from rats immunised with heat killed MTB were used.
PLNC Experiment 1. The assay showed a strong inhibitory effect of core peptide on T-cell proliferation (
PLNC Experiment 2. In this experiment, the background counts in wells with no antigen were very high, above 10000 cpm (
T-Cell Line Experiment 1. The control, containing just the vehicle (20 μl 0.1% acetic acid) alone reduced the counts considerably compared with the untreated positive control, from over 50000 to approximately 30000 (
T-Cell Line Experiment 2. As in the previous experiment, T-cell line cells were adversely affected by the vehicle alone, with counts reflecting proliferation, reduced to about half of the positive control value (
Discussion. It has been shown in these experiments that T-cell receptor peptides can inhibit T-cell proliferation in response to challenge with the specific antigen to which the cells had been primed. This was shown both for primary lymph node cultures, and for T-cell lines established in culture. The most profound result was in the first experiment by CP which reduced proliferation by 90%. Peptides H also consistently reduced counts compared to the acetic acid vehicle control but not to the same extent. Peptide P was most inhibitory in
The solubility of the peptides were variable. At the concentration of the stock solutions, 1 mg/ml or 1 mM, most peptide solutions looked clear. Exceptions were peptides H, I, O P, which were turbid or had undissolved particles. Therefore, the true concentration of peptides in solution in the culture wells would be less than those nominated in the case of these partially soluble peptides. Core peptide could be dissolved at 2 mg/ml, but was not completely soluble at 5 mg/ml. When 20 μl of these stock solutions were added to the wells, 0.2 mg/ml CP was more inhibitory than 0.1 mg/ml, however, 0.5 mg/ml was less effective, as the peptide precipitated upon addition to the well. The vehicle for the peptides, except M and N, was 0.1% acetic acid which gave 0.01%, i.e., 1.75 mM in the wells. The HEPES-buffered medium effectively buffered this acidity, but in addition to the acetate concentration, the medium was effectively reduced in concentration to 90%. This did not adversely affect the antigen-stimulated proliferation of primary lymph node cell cultures (data not shown), but had a marked effect on cultures of T-cell lines, reducing tritiated thymidine incorporation by 50%. In these experiments, effects of peptides could still be determined by comparison with the vehicle control. The 0.05M sodium carbonate buffer, used to dissolve peptides M and N, was not as detrimental to line T-cells as acetic acid. Peptide L was not tested as it was extremely insoluble. Interestingly, the only peptide that reduced T-cell proliferation which was not a CP derivative was peptide P, and it also originated from the TCR alpha chain. Peptides K, M and N, from the beta, delta and gamma chains, were soluble in their respective buffers.
In summary the core peptide, representing the transmembrane domain of TCR alpha, and including the two charged amino acids, was effective at inhibiting antigen-stimulated T-cell proliferation of both PLNCs and line T-cells, in each experiment. The degree of inhibition varied between 50% and 90% in the different experiments. A peptide from the intracellular domain of the TCR-α chain, peptide P, also showed activity, but the peptides from the other TCR chains did not overtly inhibit proliferation of T-cells in these assays.
Peptides were examined in groups based on availability. As such the results are reported in four sections.
Methods. The first experiment consisted of 12 rats weighing approximately 190-210 grams that were purchased from the Perth Animal Resource Centre (ARC) and maintained in the Gore Hill Animal House facility. Used were core peptide (30 mg) suspended in adjuvant (0.6 ml squalane containing 7 mg MTB), core peptide Tris-monopalmitate (15 mg) suspended in 0.6 ml adjuvant, core peptide Tris-tripalmitate 20 mg/0.6 ml of adjuvant. PCT/AU96/000185 describes a method of lipid peptide conjugation.
Rats were divided into four groups, each group containing three rats. First group received adjuvant only (positive control), second group adjuvant with core peptide, third group core peptide.Tris.monopalmitate suspended in adjuvant, and last group core peptide.Tris.tripalmitate in adjuvant. Rats were injected with the above compounds in a 0.1 ml volume at the base of the tail. Baseline measurements of rat weight, paw width, and tail diameter were made on Day 0, and subsequently on day 4, 7, 9, 14, 16, 18, 21, 25 and 28. Arthritis was graded and animals sacrificed if there was marked swelling, redness and obvious discomfort. Not all rats given MTB developed arthritis. In general more than 80% of control rats developed arthritis.
Results. After 18 days all the control animals given adjuvant only had developed arthritis and had to be sacrificed. Two of the three core peptide treated animals (2/3) had no evidence of arthritis. Similarly, two of the three animals given core peptide.Tris.tripalmitate had no evidence of arthritis. Animals given core peptide.Tris.monopalmitate and adjuvant all developed arthritis. However, the onset and development of arthritis in this latter group was prolonged by 3-4 days and the clinical severity was much reduced (number of joints, paw swelling, loss of weight) compared to controls.
Experiments using adjuvant induced arthritis in rats showed that the peptide and its lipid conjugate had a protective effect on the induction of arthritis in this animal model. Results of repeat and subsequent experiments using a number of different peptides (7 mg/rat) and drugs are summarised in TABLE 3.
The results of the above experiments indicated that core peptide had an effect on inflammation both to delay its onset, decrease severity, and prevent onset of disease. These effects were similar to those obtained with the co-administration of cyclosporin and adjuvant. Cyclosporin is a well known and widely used immunosuppressive agent. There was no indiscriminate effect of peptide action. Best results were noted with core peptide and peptide B. In contrast there was no effect noted with peptide C or E having either no or negative charge group amino acids respectively. Extending the amino acids downstream towards the carboxy terminus had no negative effect. This observation confirms that carboxy modification can be performed without loss of biological activity. Therefore these peptides can be used as carrier peptides for the delivery of other chemical moieties.
(ii) Examination of peptide J, K, O.
Methods. The weight of the Wistar rats averaged 165 grams on day of injection (day 0). Each rat was injected intradermally, at the base of the tail, using a 21 gauge needle, with 1 mg MTB in 200 μl squalane, with or without 7 mg of one of the test peptides suspended in this volume. A glass syringe was used.
Results. Early symptoms were observed as soon as day 7 in two of the control (MTB only) rats, and these were killed because of severe arthritis on Day 11. Two more controls were killed on day 13, and the fifth, on day 17. All five of the untreated control rats developed acute arthritis.
(1) Weight.
(2) Paw thickness. Joint inflammation assessed as paw thickness in treated and untreated rats is shown in
Discussion. Peptide J and K sequences are derived from the extracellular domain of TCR-α and TCR-β chains, in the region of the disulphide bonds, respectively. They were comparable in efficacy and complement theoretical expectations that they should have similar effects (assuming similar levels of uptake by T-cells, etc.).
Peptide O was an extension of core peptide and included sequences from the carboxyl terminus of the TCR-α chain in the intracellular domain. Peptide O was most effective at ameliorating the development of MTB-induced arthritis and suggests that other downstream sequences from the core peptide may be important in influencing cell function. The core peptide is the smallest component of these sequences.
(iii) Examination of Peptides N, M and P
Methods. Rats received 1 mg of MTB in 0.2 ml squalane with or without peptides (7 mg). Single sites were used as noted above. Two of the control MTB rats developed arthritis early, two late, and one of the five remained well. This is consistent with the experimental model of 80% of MTB treated rats developing arthritis.
Results. (1) Weight.
(2) Ankle thickness.
(iv) Experiment with Peptide L
Methods. Same as above. Each rat was injected intradermally, at the base of the tail, using a 21 gauge needle, with 1 mg MTB in 200 μl squalane, with or without 7 mg of peptide L suspended in this volume. A glass syringe was used.
Results. (i) Weight. All of the control MTB group developed arthritis and had to be sacrificed by day 18 (
(ii) Joint involvement. Both paw and ankle thickness were significantly decreased compared to control
Primary T-cells from MTB-sensitised rats were used to test peptides. This was immediately successful and CP inhibited 3H-thymidine uptake. Results were consistent and repeatable, whether PLNC or in vitro propagated T-cell lines were used. CP was most effective followed by peptides P and H. It must be remembered that results in vitro are biased in favour of the more soluble peptides.
Adjuvant induced arthritis model. Table 4 summarises in vivo experiments which favours effectiveness of the peptides tested. Peptide J, O, N and L were very effective in the induction of disease. Similarly peptide K, M and P has a variable response in the delay of disease induction and severity.
Synthesis of Peptide. The first step to synthesize the peptide of the fourth aspect of the present invention was to synthesize a short hydrophobic peptide corresponding to the predetermined assembly sequence. The amino acid sequence of the competitive peptide is NH2-Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val-OH (SEQ ID NO: 21) hereafter referred to as “core peptide of the fourth aspect”. Subsequently a number of other peptides listed in Table 4 were synthesized (>95% purity, by Auspep Australia, Melbourne, Australia) and examined for their effect on T-cell function and inflammation.
Solubility. As will be recognized from the above discussion the peptide of the present invention is based on a portion of transmembrane domain of TCR-.alpha. The complete murine sequence of this portion is NLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 29), whereas the corresponding human sequence is NLSVIGFRILLLKVAGFNLLMTL (SEQ ID NO: 30). There is complete sequence homology across a range of species in the last 15 amino acids of the TCR-alpha chain distal to the sequence upon the peptide of the present invention is based (shown in bold). Peptides including these additional 15 residues may have activity similar to the peptide of the present invention. The essential feature is that the peptide includes two positively charged amino acids separated by 3 to 5 hydrophobic amino acids.
The core peptide for the fourth aspect and the other peptides listed above were noted to be hydrophobic and insoluble in aqueous solutions. A variety of solvents and carriers were tested. These included ethanol, dimethylsulphoxide (DMSO), dimethyl formamide (DMF), trifluoracetic acid (TFA), squalane oil (2,6,10,15,19,23-hexamethyltetracosane), and lipid conjugation by addition of palmitic acid to the core peptide via TRIS-conjugation (Whittaker R. G., Bender V. J. 1991) to increase solubility. The preferred solvent was DMSO and the final concentration used in cell cultures ranged from 0.1%-0.2%. Concentrations of DMSO greater than 1% was toxic to cells. Stock solutions of peptide and lipopeptide conjugates were dissolved in DMSO and used in a 1/1000 dilution.
The addition of peptide/lipopeptide in DMSO to aqueous solutions resulted in “fat” or “crystal” globules that settled to the bottom of the tissue culture flask and dissolved poorly. These globules could be seen by phase contrast microscopy, but were less obvious for lipid conjugates.
Core peptide containing C14-glycine (C14-peptide) was synthesized by Auspep Australia and used to study solubility. C14-peptide dissolved/suspended in DMSO was added to a final concentration of 100 μM to T-cell media (RPMI 1640 supplemented with 10% foetal calf serum and 0.3% mercaptoethanol: TCM) and shaken. The media was centrifuged and supernatant filtered through 0.2 μM filter or left unseparated. The total radioactivity in unseparated medium was 20.000 cpm, 1000 cpm after the medium was centrifuged and 500 cpm after the media was filtered. These experiments highlight the insoluble nature of the peptide of the fourth aspect in vitro and suggest that approximately 5% goes into solution.
Entry of Peptide into Cells. To examine if peptide enters cells, CH-peptide was added to a flask of 5×106 2B4.11 cells (T-cell hybridoma specific for cytochrome c) in a final concentration of 100 μM and 0.2% DMSO and incubated overnight. The adherent cells were washed four times with phosphate buffered saline (PBS) in the flask, solubilised with triton-containing buffer and radioactivity counted. The amount of radioactivity in the supernatant seas 70,000 cpm and 5000 cpm in the solubilised cells.
In a variation of the above experiment, 2B4.11 cells (7.5×104) were grown in Petri dishes containing 2 ml of TCM and a “Transwell” with 0.4 μM membrane was placed in the Petri dish. C14-peptide in a final concentration of 100 μM and 0.1% DMSO were added in the “Transwell” and after 24 hr and 48 hr incubation the counts determined on both sides of the filter and in the cells. Approximately 85% of radioactivity was retained in the “Transwell”, 8% in the Petri dish media and 7% within cells. The above experiments demonstrated that peptide was able to enter cells. Considering the low solubility of peptide (5%-10%) all of the available peptide in solution entered the cells (7%).
Intracellular Localization of Fluoresceinated Peptide in T-cells. Experiments suggested that the small percentage of peptide that goes into solution can enter/or be taken up by cells. To confirm this, core peptide covalently linked with fluorescein isothiocyanate (FITC) was added to T-cells and intracellular localization determined by visualization using confocal or conventional UV light microscopy.
Fluoresceinated labeled core peptide was prepared as follows: 10.25 mg of core peptide was dissolved in 0.5 ml dimethylformamide (DMF) and 2 μM of FITC in 0.5 ml of DMF was added drop wise with stirring, at room temperature. The pH was adjusted to 9 with N-methyl, N,N-diisopropylamine, and the reaction allowed to proceed for 1 hr. Semi-preparative HPLC was then used to separate FITC-peptide from free FITC, using a C-4 column (6 ml/min; buffer A, 0.1% TFA; buffer B, 80% acetonitrile, 20% water; 0.1% TFA; linear gradient of 40%-100% B). Fractions were monitored by analytical HPLC and the fractions containing pure fluoresceinated core peptide (FITC-peptide) pooled.
Two flasks of cultured 2B4.11 cells (5×106) were spun down and resuspended in PBS containing calcium and magnesium. To one flask, FITC dissolved in DMSO was added to a final concentration of 10 μM and to the other FITC-peptide 10 μM The final concentration of DMSO in both flasks was 0.1% previously shown to have no effects on T-cells. The cells were incubated at 37° C. for 30 min and then examined under the confocal microscope.
The observations can be summarized as follows: (i) FITC and FITC-peptide entered the cells; (ii) free HITC gave brighter fluorescence than FITC-peptide in the cells; (iii) the intracellular staining pattern was not different between the free FITC and FITC-peptide. Nuclear and especially bright nucleolar staining was observed; (iv) conjugation of peptide by FITC did not prevent entry of peptide into cells; (v) there was no “leaching” out of cells of FITC-peptide over a 5 hr period. These experiments demonstrate that FITC-peptide could be taken up by cells and localized intracellularly. In conjunction with experiments previously described showing intracellular uptake by C14-peptide it is evident that it is the inherent nature of the peptide sequence and not its conjugates (FITC, C14) that allows cellular entry.
Tris-fat Conjugation of Core Peptide Carboxyl Terminal. The effect of carboxyl conjugation of core peptide, as exemplified by lipid conjugation, on the ability of peptide to competitively inhibit the function of this crucial receptor was investigated. The efficacious clinical manifestations of the administered lipopeptide would be a decrease in inflammation e.g. as demonstrated by a decrease of arthritis in an adjuvant model of arthritis, as would be seen with peptide. In addition to the lipoconjugation of core peptide, a number of other lip peptides were synthesized and used as controls in subsequent experiments. The lipopeptides were synthesized according to the methods set out in Whittaker, R. G., Hayes, P. J., and Bender, V. J. (1993) Peptide Research 6, 125 and Australian Patent No. 649242. The disclosure of these references is incorporated by herein by cross reference
Preparation of Fluorescein Labeled (SEQ ID NO. 32) Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val-Gly-Tris-mono- and tri-palmitates. To a solution of each of the deprotected lipopeptides (15 and 6 mg) in DCM (1 ml) a solution of FITC (4 mg 10 μmole) in DMF (500 μl) was added with stirring. The apparent pH of the reaction was maintained at 9.0 by the addition of triethyl amine (TEA). The fluorescein-labeled mono, and tri-palmitoyl derivative of the peptide were purified by semi-preparative HPLC (C4 column, System B). The purified compounds were evaporated to dryness and lyophilised from tert. butyalcohol to give the fluorescein labeled peptide monopalmitate (RtB, 7.83) and tripalmitate (RtB, 9.85) which were tested as described below.
TLC of the fluorescein-labeled lipopeptides (DCM: MeOH, 95:5) showed the absence of free FITC and free Gly-Tris-monopalmitate and Gly-Tris-tripalmitate (used in lipopeptide synthesis) (by ninhydrin staining).
Solid Phase Peptide Synthesis. Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ ID NO: 21) (core peptide) and its fully protected form, Boc-Gly-Leu-Arg(PMC)-Ile-Leu-Leu-Leu-Lys(Boc)-Val-OH (SEQ ID NO: 21) (and the C14-labeled peptide) were supplied by Auspep Pty Ltd. Both were synthesized by the FMOC-chemistry in the manual mode.
It also will be understood readily by those skilled in the art that there are a number of well known linkers that can be used to join compounds (such as peptides) with a carboxyl group to an amino group. These include:
a) a linker with an amino group to the compound and a carboxyl group to the Tris (or amino acid if present) such as an amino acid or antibiotic.
b) a linker with an amino group to the compound and a sulphonic acid group to the Tris (or amino acid if present) such as 2-aminoethanesulphonic acid (taurine).
c) a linker with a hydroxyl group to the compound and a carboxyl group to the Tris (or amino acid if present) such as glycolic acid, lactic acid etc.
d) a linker with a hydroxyl group to the compound and a sulphonic acid group to the Tris (or amino acid if present) such as 2-hydroxyethanesulphonic acid (isethonic acid).
e) a linker with a hydroxyl group to the compound and a reactive halide group to the Tris (or amino acid if present) such as 2-chloroethanol.
f) other examples of potentially suitable linkers between a compound with a reactive carboxyl and the amino group of Tris (or amino acid if present) include the compound families exemplified by p-hydroxybenzaldehyde, 2-chloroacetic acid, 1,2-dibromoethane and ethyleneoxide.
Linkers could also contain disulphide groups that would reduce to liberate modified peptide intracellularly.
Localization of FITC Conjugated Lipopeptides in COS Cells. Using con-focal microscopy, the ability of FITC-conjugated lipopeptides to enter non-T cells (COS cells-fibroblasts) was examined.
Materials: Stock concentration in DMSO-Core peptide.Tris.monopalmitate. FITC (MW 1862) 10 mM; core peptide.Tris.dipalmitate.FITC (MW 2334) 10 mM; core peptide.Tris.tripalmitate.FITC (MW 2806) 10 mM; glycine.Tris.monopalmitate.FITC (MW 805) 10 mM; glycine.Tris.tripalmitate.FITC (MW 1286) 10 mM; FITC, (MW 390) 6.4 mM.
Method: COS cells were grown on cover slips until 80% confluent, washed twice with PBS and incubated with FITC conjugated lipopeptides for 15 min or 2 hr. The final concentration of lipopeptides was 10 μM and 6.4 μM for FITC, for each time point respectively. Cells were washed twice with PBS, mounted with PBS/glycerol and examined with confocal microscopy.
Results: Experiments indicated that fluorescein-conjugated lipopeptides can transmigrate across cell membranes and localize to within the cellular cytoplasm, reaching as far as the endoplasmic reticulum (ER), where protein synthesis takes place. The extent of cellular penetration was influenced by the lipid moiety attached to the peptide. Of the lipopeptides the monopaletate had the greater ability to infiltrate within the fibroblasts and T-cells so far examined (see below). The ER is the best site to try and effect assembly. Once all the chains have assembled and transported to the cell surface it may be much harder to disrupt the receptor at the cell surface membrane. Targeting peptides to the ER is an ideal site to disrupt the TCR complex.
Localization of FITC Conjugated Lipopeptides in T-cells. Using con-focal microscopy, the ability of FITC conjugated lipopeptides to enter T-cells was examined.
Materials: Lipopeptides as above. 2B4.11 T-cell hybridoma cell line.
Method: 2B4.11 T-cells were grown in TCM and resuspended in a concentration of 8×105 cells/ml. Viability >95% using trypan blue. One ml of cells was added to polypropylene tubes and washed twice with PBS. Cells were resuspended in PBS and one microliter of stock FITC-conjugated lipopeptides added for 30 min. Cells were washed with PBS, mounted with PBS/glycerol, and viewed using confocal microscopy.
Results: Similar to that of COS cells (see above). Results showed that lipopeptides were able to enter T-cells. The lipoconjugation of peptide does not prevent entry of peptides into cells and has the potential use of being used as a carrier vehicle to increase solubility.
Effect of Peptides and Lipopeptides on TCR Assembly and Cell Surface Expression on T-Cells Using Flow Cytometry Analysis.
Materials: The T-cell hybridoma 2B4.11 which expresses a complete TCR on the cell surface was used as a positive control to assess the effects of peptides on TCR expression. The cells were grown in TCM. The n-deficient variant 21.2.2 and the β- and ξ-deficient cell line 3.12.29, derived by repetitive subcloning of 2B4.11 cells (Sussman et al., 1988) and lacking TCR expression were used as negative controls.
Peptides tested included core peptide, lipopeptides and a peptide from tumor necrosis factor receptor termed 558 (used as a negative control). The final concentration of each substance used in incubation was 10 μM.
Antibodies: The following antibodies were used for immuno-precipitation and flow cytometry analysis: Mouse IgG2a monoclonal antibody (MAb) against TCR-α chain of the T-cell hybridoma 2B4 (A2B4-2, Samelson et al., 1983), MAb against 2B4.11 TCR-β chain (K)25), hamster IgG anti-CD3-ε MAb (145-2C11 [2C11], Leo et al., 1987), rabbit anti-CD3-ε polyclonal antiserum raised against purified mouse CD3-ε (127, Minami et al., 1987), anti-CD3-δ polyclonal antibody (R9) raised in goat immunized with a COOH-terminal peptide of the mouse CD3-δ chain (Samelson et al., 1986).
Method: FACS analysis: 1×106 (2B4.11, 21.2.2) cells were incubated with a number of separate peptides and lipopeptides in a final concentration of 10 μM overnight. The cells were then washed with PBS and incubated with 50 μl primary antibody (A2B4 or 2C11) for 30 mins at 4° C. Cells were washed twice in PBS and 0.1% BSA and incubated for an additional 30 min at 4° C. with FITC-labeled second antibody. Cells were washed two additional times with PBS and 0.1% BSA prior to analysis on a Becton-Dickson FACS Analyser or Becton-Dickson FACS Scan.
Results: The expression of TCR on 2B4.11 cells treated with core peptide control peptide, or lipopeptides did not alter the cell surface expression of the receptor. These experiments have been repeated with higher concentration of core peptide (100 μM) and longer incubation times ranging from 1-10 days and the results have been the same showing no change in T-cell surface antigen receptor expression.
The following experiments were performed to assess the in vitro effects of peptides/lipopeptides on T-cell function.
Antigen Presentation Assay I: An antigen presentation assay (described below) examined the ability of a number of peptides to inhibit T-cell activation following antigen recognition, by measuring the product of T-cell activation, Interleukin-2 (IL-2).
Material: The following cell lines were used: 2B4.11, a T-cell hybridoma that expresses a complete antigen receptor on the cell surface (Samelson et al., 1983) and produces IL-2 following antigen recognition (cytochrome c). Interleukin-2 dependent T-cell line (CTLL) for conventional biological IL-2 assays; and the B-cell hybridoma cell line LK 35.2 (LK, I-Ek bearing; Kappler et al., 1982) which acts as the antigen presenting cell. The hybridomas were grown in TCM. Cytochrome c (Sigma, USA) was added in the media to give a final concentration of 50 μM in the antigen presenting assay.
Peptides examined included: core peptide, seven other control peptides from a variety of sources having an equivalent length to core peptide and peptides A, B, C, D, E and F. The final concentration of the peptides in the antigen presentation assay was examined at several levels ranging from 10 μM to 100 μM.
Method: For T-cell antigen stimulation 2×104 LK35.2 cells were co-cultured with 50 μM pigeon cytochrome c dissolved in PBS and 2×104 2B4.11. T-cells for 16 hr. The assay was done in triplicate. Supernatants were recovered and IL-2 content determined by CTLL proliferation. The incorporation of 3H-thymidine is directly proportional to the amount of IL-2 present in the supernatant. The ability of different peptides to inhibit IL-2 production was examined. In addition to measuring 3H-thymidine incorporation, IL-2 measurements (IU/ml) were also determined.
Results. In assays where either cytochrome c (antigen) or LK cells (antigen presenting cells) were omitted there was no IL-2 production. The lack of T-cell activation under such conditions indicated that there was no lipopolysaccharide (LPS) or endotoxin in the solutions which may have non-specifically stimulated the T-cells. The combination of all three constituents of the assay at the concentrations shown above resulted in the production of IL-2 as measured by high 3H-thymidine incorporation by CTLL cells (22,000 cpm). When core peptide or other analogues were added to the assay system the amount of IL-2 produced varied respectively. All peptides tested at 10 μM had no effect on IL-2 production. The best effect was noted with core peptide at 100 μM and peptide C (100 μM) leading to a 15%-30% reduction in IL-2 production compared to control. This was reproducible on at least three separate occasions. Peptides A, B, D, E and F had a variable and minor effect on T-cell activation. The seven control peptides with equivalent length but no sequence homology to the peptide had no effect on IL-2 production.
Preincubation of core peptide and other peptides at 37° C. in TCM prior to addition in the antigen presenting assay, improved solubility and activity and was reflected as an additional incremental increase above baseline activity noted with freshly prepared peptide.
Antigen Presentation Assay II. The following experiments were performed to assess the in vitro effects of lipopeptides on T-cell function.
Material: Lipopeptides examined included: core peptide Tris-monopalmitate (100 μM and 0.1% DMSO) and core peptide Tris-tripalmitate (100 μM and 0.2% DMSO). The final concentration of the two lipopeptides in the antigen stimulation assay is shown in brackets respectively.
Method. As described in the Effect of Peptides and Lipopeptides on TCR Assembly and Cell Surface Expression on T-Cells using Flow Cytometry Analysis.
Results. Initially, when core peptide Tris-tripalmitate (100 μM and 0.2% DMSO) was added, there was a reduction in T-cell activation by 75% (highest count 5.190 cpm cf 22,000 cpm for control). The addition of core peptide Tris-monopalmitate (100 μM and 0.1% DMSO) had a profound effect on IL-2 production, with only 137 cpm recorded (similar to background). The concentration of 0.1% DMSO and 0.2% DMSO used in the test system was examined and not found to influence IL-2 production. Subsequent experiments have confirmed these findings and show an IL-2 reduction of 86%-92% compared to control. Palmitic acid alone (100 μM) used to conjugate the peptide, added to the antigen presenting assay system did not affect IL-2 production.
The following experiments examined the ability of core peptide to circulate within the animal following administration, and the effects on experimentally induced inflammation.
Distribution of C14-core Peptide. To examine the distribution of subcutaneously injected peptide in mice, C14-core peptide (5 mg/mouse) was dissolved in 150 microliters of squalane oil and injected at the base of the tail of Balbic mice. After 24 hr, counts were measured from pulped organs. Distribution of the peptide was noted in thymus (5%), spleen (7%), blood (3%) and a large proportion in lymph nodes (28%), kidney (30%) and liver (28%).
Experiments were extended to examine the ability of core peptide to prevent disease in animal models of inflammation. Three in vivo experimental models including adjuvant induced arthritis in rats, cyclophosphamide induced diabetes in NOD mice, and experimental allergic neuritis in rats were used. In these models, encompassing two species, core peptide was able to influence the degree of inflammation.
Adjuvant Induced Arthritis in Rats. The rat adjuvant arthritis model is a classic model of inflammation which has been used extensively by a number of laboratories to study disease progression and effects of potential new anti-inflammatory drugs thereon over the last 30 years (Pearson et al., 1961; Cremer et al., 1990; Holmdahl and Kvick., 1992; Cannon et al., 1993). This model has also been widely used by researchers at the Royal North Shore Hospital over the last 10 years and procedures have been established for the study of this model of inflammation. All procedures on the animals were carried out under halothane/oxygen/nitrous oxide anaesthesia (2% v/v halothane in 1 liter/min O2 and 2 liters/minN2O). Rats were injected intradermally at the base of the tail with a minimal adjuvant dose (1 mg heat killed Mycobacterium tuberculosis [MTB9 in 100 μl squalane) once and only once. The method for co-administrating test samples with MTB was first described by Whitehouse et al., 1990. At regular intervals between days 0-28, animals were weighed and their arthritic condition assessed by measurement of maximum tail thickness and rear paw thickness (with a micrometer screw gauge). Rats were housed in holding bins after the initial tail injection and allowed access to unlimited water and pellet food. On day 29 the animals were sacrificed.
Materials. The first experiment consisted of 12 rats weighing approximately 190-210 grams that were purchased from the Perth Animal Resource Centre (ARC) and maintained in the Gore Hill Animal House facility. Used were core peptide (30 mg) suspended in adjuvant (0.6 ml squalane containing 7 mg MTB), core peptide Tris-monopalmitate (15 mg) suspended in 0.6 ml adjuvant, core peptide Tris-tripalmitate 20 mg/0.6 ml of adjuvant.
Rats were divided into four groups, each group containing three rats. First group received adjuvant only (positive control), second group adjuvant with core peptide, third group core peptide.Tris.monopalmitate suspended in adjuvant, and last group core peptide.Tris.tripalmitate in adjuvant. Rats were injected with the above compounds in a 0.1 ml volume at the base of the tail. Baseline measurements of rat weight, paw width, and tail diameter were made on Day 0, and subsequently on day 4, 7, 9, 14, 16, 18, 21, 25 and 28. Arthritis was graded and animals sacrificed if there was marked swelling, redness and obvious discomfort. Not all rats given MTB developed arthritis. In general more than 80% of control rats developed arthritis.
Results. After 18 days all the control animals given adjuvant only had developed arthritis and had to be sacrificed. Two of the three core peptide treated animals (2/3) had no evidence of arthritis. Similarly, two of the three animals given core peptide.Tris.tripalmnitate had no evidence of arthritis. Animals given core peptide.Tris.monopalmitate and adjuvant all developed arthritis. However, the onset and development of arthritis in this latter group was prolonged by 3-4 days and the clinical severity was much reduced (number of joints, paw swelling, loss of weight) compared to controls.
Experiments using adjuvant induced arthritis in rats showed that the peptide and its lipid conjugate had a protective effect on the induction of arthritis in this animal model. Results of repeat and subsequent experiments using a number of different peptides (7 mg/rat) and drugs are summarized in TABLE 5.
The results of the above experiments indicated that core peptide had an effect on inflammation both to delay its onset, decrease severity, and prevent onset of disease. These effects were similar to those obtained with the co-administration of cyclosporin and adjuvant. Cyclosporin is a well known and widely used immunosuppressive agent. There was no indiscriminate effect of peptide action. Best results were noted with core peptide, peptide C and F. Core peptide and peptide C each have charged amino acid groups at the same site but the amino acids reversed. This indicated that it was the charge group rather than the particular amino acid that was important. In contrast there was no effect noted with peptide B or D having either no or negative charge group amino acids respectively. Extending the amino acids downstream towards the carboxy terminus had no negative effect. This observation confirms that carboxy modification can be performed without loss of biological activity. Therefore these peptides can be used as carrier peptides for the delivery of other chemical moieties. Increasing the amino acid number between the two polar charge groups (peptide E) resulted in lower efficacy. Decreasing the number of amino acids between the charged groups (peptide F) had no negative effect.
Dosage Effect of Core Peptide. Based on previous experiments by Whitehouse et al (personal communication) an initial dose of 5-7 mg/rat was given. To evaluate a lower limit, a number of different core peptide concentrations were examined. The results (TABLE 6) indicated that in addition to a specific action of core peptide it was limited by its effects by dosage.
Tail Diameter Measurements. A feature of adjuvant induced arthritis is the development of inflammation in the tail. Tail measurements (mm) between saline injected rats and core peptide treated rats were not statistically significant. Tail diameters from MTB treated rats however were significantly increased (p<0.001) compared to saline and core peptide treated rats (p<0.001), TABLE 7.
In addition to the oedema noted in the tails, MTB alone given to rats caused ulceration and inflammation at the site of injection that was not present with rats given core peptide or saline.
Experimental Allergic Neuritis (EAN). To further confirm the ability of core peptide to delay and diminish the severity of disease induced by T-cells a different model (experimental allergic neuritis) was tested “blind” by independent experimenters (Associate Professor Pollard and Mr J Taylor) at a different institution (University of Sydney).
Materials and Methods: Animals—Male Lewis rats weighing between 239-451 grams were obtained from the Bosch Animal House, University of Sydney colony or from ARC, Perth. All experiments were conducted in accordance with experimental guidelines approved by the Animal Care and Ethics Committee of the University of Sydney.
Induction of EAN. Lewis rats were immunized in each hind footpad with 50-75 μl of bovine peripheral nerve myelin (PNM) emulsified in complete Freunds adjuvant. The immunization emulsion consisted of equal volumes of saline and incomplete Freunds adjuvant (Sigma, USA) mixed with bovine PNM and MTB (strain H37RA, DIFCO) added at 15 mg/ml and 5 mg/ml respectively. Where animals received ovalbumin/peptide the peptide was added to the immunization emulsion at 70 mg/ml. These experiments were performed with the experimenters having no knowledge of what peptide was in the immunisation emulsion.
Animals were observed at least every second day post immunisation for clinical signs and were scored using the following scale: 0, normal; 0.5, weak tail; 1, flaccid tail; 1.5, limp tail and ataxia in hind legs; 2, paraparesis; 2.5, limp tail and severe paraparesis; 3, paraplegia; 3.5, limp tail and paraplegia and forelimb paresis; 4, quadraparesis; 4.5 limp tail and quadraplegia; 5, dead.
Peripheral Nerve Myelin Isolation. Bovine PNM was prepared essentially as described by Norton and Podulso (1973).
Results. A representative example is shown in
Diabetes in NOD/Lt (F) Mice. In yet another model of T-cell mediated disease the effects of subcutaneously injected core peptide on the induction of diabetes in NOD/Lt (F) mice was tested “blind” by an independent experimenter (Prof L Harrison, WEHI, Melbourne).
A cellular autoimmune process that selectively destroys the pancreatic islet beta cells is thought to be responsible for the development of insulin-dependent diabetes mellitus (IDDM) in humans and in the spontaneous animal models including the NOD mouse (Leiter et al., 1987). A common histopathological feature associated with the development of IDDM is insulitis, the presence within and around the islets of mononuclear cells consisting predominantly of T lymphocytes and to a lesser extent macrophages (Foulis et al., 1986). Experimental strategies aimed at suppressing cellular autoimmunity such as neonatal thymectomy, administration of cyclosporin A or administration of anti-T lymphocyte antibodies prevent the development of diabetes (Campbell et al., 1991).
Animals —NOD/Lt(F) mice 10 weeks old at experimental Day 0. This is a high-incidence strain commonly used as an animal model for diabetes.
Materials: Core peptide dissolved in squalane at a stock concentration of 3.33 mg/ml. Ovalbumin used as a control was suspended in squalane at a stock concentration of 3.33 mg/ml. Peptide and ovalbumin were “solubilised” just prior to injection, with vortexing. A total of 250 μg (75 μl) was injected subcutaneously on the right flank on Day −1, 0 and 1. Mice were given intraperitoneal cyclophosphamide in water at 300 mg/kg on Day 0. Blood glucose measurements were taken on Day 0, 10, 14 and 21. There were 16 mice in the treatment group and 16 in the ovalbumin control group.
Results. Experiments demonstrate a protective effect of core peptide on the induction of autoimmune beta cell destruction which manifests as diabetes (TABLE 8). This finding again confirms the general immunosuppressive ability of core peptide in a different T-cell mediated disease model.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
With regard to the fourth aspect of this invention, the present inventor has shown that the peptides of the present invention are able to inhibit T-cell mediated immune responses in at least three different models by a mechanism previously unreported.
Additionally, the experiments described for the fourth aspect herein were conducted primarily with the core peptide, for the fourth aspect, that was deliberately chosen to be homologous with the known sequence of the mouse and rat TCR alpha chain. However, for clinical treatment of humans a peptide containing a phenylalanine residue instead of a leucine towards the amino terminus of the core peptide may be preferable to maximize homology with the known human sequence.
Dis; 49: 452-459, (1990)
| Number | Date | Country | Kind |
|---|---|---|---|
| PO 0389/96 | Jun 1996 | AU | national |
| PO 0390/96 | Jun 1996 | AU | national |
| PO 0391/96 | Jun 1996 | AU | national |
| PO 0392/96 | Jun 1996 | AU | national |
| PO 0393/96 | Jun 1996 | AU | national |
| PO 0394/96 | Jun 1996 | AU | national |
This application is a continuation of U.S. patent application Ser. No. 10/912,551, filed Aug. 6, 2004, which is a continuation of U.S. patent application Ser. No. 09/202,305, filed Mar. 22, 1999, which is now issued as U.S. Pat. No. 7,192,928, issued Mar. 20, 2007, which is the National Stage of International Application No. PCT/AU97/00367, filed Jun. 11, 1997, which claims the benefit of Australian applications: PO 0390/96 filed Jun. 11, 2006; PO 0392/96 filed Jun. 11, 2006; PO 0393/96 filed Jun. 11, 2006; PO 0391/96 filed Jun. 11, 2006; PO 0389/96 filed Jun. 11, 2006; and PO 0394/96 filed Jun. 11, 2006, each of which U.S., international and Australian applications is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10912551 | Aug 2004 | US |
| Child | 12621793 | US | |
| Parent | 09202305 | Mar 1999 | US |
| Child | 10912551 | US |
