WO2010057251A1 - Peptide conjugates for suppressing an immune response, methods of making and uses therefor - Google Patents
Peptide conjugates for suppressing an immune response, methods of making and uses therefor Download PDFInfo
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- WO2010057251A1 WO2010057251A1 PCT/AU2009/001501 AU2009001501W WO2010057251A1 WO 2010057251 A1 WO2010057251 A1 WO 2010057251A1 AU 2009001501 W AU2009001501 W AU 2009001501W WO 2010057251 A1 WO2010057251 A1 WO 2010057251A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6018—Lipids, e.g. in lipopeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates generally to compounds and methods for suppressing immune responses, including preventing, inhibiting, treating or decreasing unwanted or undesirable immune responses including autoimmune or allergic immune responses.
- the body may raise unwanted or undesirable immune responses in a number of situations, including autoimmune responses, transplant rejections and allergic immune responses.
- Lymphocyte development occurs during foetal development, and to a lesser extent throughout the remainder of life. Part of this development includes immunological tolerance; the process by which T and B lymphocytes (T and B cells) of the immune system, which recognise self antigens, are deleted before they develop into fully immunocompetent cells.
- T and B lymphocytes T and B cells
- autoimmune diseases occur when the body fails to recognise a self antigen, resulting in an immune response against the body's own cells and tissues. Any disease that results from such an immune response to a self antigen is termed an autoimmune disease.
- RA Rheumatoid arthritis
- MS multiple sclerosis
- Immunocompetent T cells will raise an immune response to foreign antigens including those within transplants containing nucleated cells.
- Matching the Major Histocompatibility Complex (MHC) type of the donor and recipient increases the success rate of grafts, but perfect matching is possible only when donor and recipient are related and even in these cases genetic differences at other loci still trigger rejection.
- MHC Major Histocompatibility Complex
- An allergic immune response occurs where the immune system reacts to a normally innocuous environmental antigen.
- the immune response results from interaction between the antigen and the antibody or T cells produced by earlier exposure to the same antigen.
- Allergic immune responses include type I (or immediate) hypersensitivity immune responses which are antibody (typically IgE) mediated, and type IV (delayed type) hypersensitivity immune responses which are cell mediated and antibody independent.
- IgE-mediated allergies include hayfever, skin inflammation (urticaria), food allergies, asthma and systemic anaphylaxis.
- Cell mediated allergic diseases include contact dermatitis, tuberculin reaction, and chronic transplant rejection.
- Antigens can be categorised as either class I or class II peptides depending on whether they attach to MHC class I molecules or MHC class II molecules.
- MHC class I molecules usually bind short peptides of 8-10 residues.
- Typical class I antigens are those derived from cytosolic pathogens. The length of peptides bound by MHC class II molecules is not constrained.
- Typical class II antigens are intravesicular pathogens, extracellular pathogens and extracellular toxins.
- MHC class I molecules are recognised by cytotoxic T cells which kill the cell bearing the antigen.
- MHC class II molecules are recognised by T helper cells which either activate effector molecules such as cytokines or activate B cells to secrete immunoglobulins. In this way, an immune response appropriate for the antigen is raised.
- Peptides which comprise sequences that prevent, inhibit, treat or decrease an unwanted or undesirable immune response are generally known. It is believed that many such peptides are based on known antigens but have modified sequences, normally at sites where T cell receptors or MHC anchor positions interact. These peptides can be classified as class I or class II peptides depending on which class of MHC molecule they interact with, although the mechanism of their interaction is not well understood yet. There is much interest in therapies based on these peptides as they will probably be disease- specific and therefore not associated with the side effects of some current immunosuppressive therapies.
- antigen presenting cells are typically pulsed or otherwise contacted with an immunosuppressive peptide. It is believed that the peptide attaches to appropriate MHC molecules on the surface of the cells and suppresses an unwanted or undesirable T helper cell-mediated immune response.
- CPPs Cell penetrating peptides
- BSA bovine serum albumin
- keyhole limpet hemocyanin have also been used to transport immunogenic peptides.
- Such proteins are not likely to be suitable carrier molecules for immunosuppressive peptides because, as for immunoglobulins, there is a risk that the patient will raise an immune reaction to the carrier protein, hi fact, commercially available BSA is often cationised so that it can provide an enhanced immunogenic response.
- WO 97/49425 describes a vaccine comprising an antigen and a carrier molecule that are linked through a labile bond.
- the application states that the site of attachment of the carrier molecule to the peptide affects the immunogenicity of the antigen.
- the inventors state that an S site of attachment yields a more immunogenic product than an N site of attachment. However, both were found to elicit class II immune responses, with the inventors noting that the N-attached carrier molecule induced a high antibody response.
- the present inventors surprisingly discovered that immunosuppressive peptides with lipid moieties attached at an S-site on the peptide are processed by the class II pathway for improved class II presentation, while immunosuppressive peptides with lipid moieties attached at an N-site on the peptide tend to form micelles and are processed through the class I pathway for improved class I presentation.
- the present inventors consider that better prophylactic and therapeutic immunosuppressive responses can be elicited through the class II pathway by attaching immunosuppressive peptides that are processed and presented through MHC class II molecules to a membrane permeating lipid moiety through a thioester linkage, and that better prophylactic and therapeutic immunosuppressive responses can be achieved through the class I pathway by attaching immunosuppressive peptides that are processed and presented through MHC class I molecules to a membrane permeating lipid molecule through an amide linkage.
- the present invention provides a peptide conjugate for suppressing or otherwise inhibiting an MHC class II response, the peptide conjugate comprising a peptide comprising an amino acid sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response and that is processed and presented by an MHC class II molecule, and a lipid moiety which is attached to the peptide through a thioester linkage, or a pharmaceutically acceptable salt thereof.
- the present invention provides a peptide conjugate for suppressing or otherwise inhibiting an MHC class I response, the peptide conjugate comprising a peptide comprising an amino acid sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response and that is processed and presented by an MHC class I molecule, and a lipid moiety which is attached to the peptide through an amide linkage, or a pharmaceutically acceptable salt thereof.
- the present invention provides a composition comprising the peptide conjugate of the present invention.
- the present invention provides a composition consisting essentially of the peptide conjugate of the present invention.
- the present invention provides a composition consisting of the peptide conjugate of the present invention.
- the present invention provides a composition comprising a peptide conjugate of the present invention but excluding a separate antigen that corresponds to the sequence of the peptide, which antigen elicits the unwanted or undesirable immune response.
- a method of making a peptide conjugate according to the first-mentioned aspect comprising linking a peptide comprising an amino acid sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response and that is processed and presented by an MHC class II molecule to a lipid moiety through a thioester linkage.
- a method of making a peptide conjugate according to the second-mentioned aspect comprising linking a peptide comprising an amino acid sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response and that is processed and presented by an MHC class I molecule to a lipid moiety through an amide linkage.
- the present invention provides a method for suppressing or otherwise inhibiting an unwanted or undesirable immune response in a subject, the method comprising administering to the subject the peptide conjugate or composition of the invention.
- the subject has or is afflicted with an autoimmune, an allergic immune or an allograft immune response.
- the method comprises identifying that the subject has an autoimmune, an allergic immune or an allograft immune response.
- Another aspect of the present invention relates to a method for suppressing or otherwise inhibiting an unwanted or undesirable immune response to a target antigen in a subject, the method comprising administering to the subject the peptide conjugate or composition of the invention, wherein the sequence of the peptide corresponds to the sequence of the target antigen.
- the subject has or is afflicted with an autoimmune, an allergic immune or an allograft immune response.
- the method comprises identifying that the subject has an autoimmune, an allergic immune or an allograft immune response.
- a further aspect of the present invention provides a method for preventing, inhibiting, treating or decreasing an autoimmune, an allergic immune or an allograft immune response in a subject, the method comprising administering to the subject the peptide conjugate or composition of the invention.
- the present invention provides a method for treating an autoimmune, an allergic immune or an allograft immune response in a subject, the method comprising administering to the subject the peptide conjugate or composition of the invention.
- the subject has or is afflicted with an autoimmune, an allergic immune or an allograft immune response.
- the method comprises identifying that the subject has an autoimmune, an allergic immune or an allograft immune response.
- Yet further aspect of the present invention provides a method for preventing, inhibiting, treating or decreasing an autoimmune, an allergic immune or an allograft immune response to a target antigen in a subject, the method comprising administering to the subject the peptide conjugate or composition of the invention, wherein the sequence of the peptide corresponds to the sequence of the target antigen.
- the present invention provides a method of treating an autoimmune, an allergic immune or an allograft immune response to a target antigen in a subject, the method comprising administering to the subject the peptide conjugate or composition of the invention, wherein the sequence of the peptide corresponds to the sequence of the target antigen.
- the subject has or is afflicted with an autoimmune, an allergic immune or an allograft immune response.
- the method comprises identifying that the subject has an autoimmune, an allergic immune or an allograft immune response.
- a peptide conjugate or a composition of the invention in the manufacture of a medicament for suppressing or otherwise inhibiting an unwanted or undesirable immune response in a subject, including an unwanted or undesirable immune response to a target antigen.
- a peptide conjugate or a composition of the invention in the manufacture of a medicament for preventing, inhibiting, treating or decreasing an autoimmune, an allergic immune or an allograft immune response in a subject, including an autoimmune, an allergic immune or an allograft immune response to a target antigen in a subject.
- the present invention provides a use of a peptide conjugate or a composition of the invention in the manufacture of a medicament for treating an autoimmune, an allergic immune or an allograft immune response in a subject, including an autoimmune, an allergic immune or an allograft immune response to a target antigen in a subject.
- the present invention provides a method for producing an immunosuppressive antigen presenting cell, the method comprising contacting an antigen presenting cell or antigen presenting cell precursor with the peptide conjugate or composition of the invention for a time and under conditions sufficient for the peptide or a processed form thereof to be presented by the antigen presenting cell or antigen presenting cell precursor.
- Figure 1 is a graphical representation of some of the results in
- Example 2 Mice were immunised with the peptide shown above each graph, and then lymph node cells extracted therefrom were tested for their proliferative responses to a nonacylated (circle), S -palm (square), or N-palm (triangle) forms of the same peptide. Each point on the graph represents the SI (mean ⁇ SD of three to five repetitions of each experiment) at a particular peptide concentration.
- Figure 2 shows some of the results in Example 3, where the uptake of different peptides by the macrophages was examined. Uptake of the peptides into the macrophages was monitored by flow cytometry (at 15 min) and by confocal microscopy after 1-, 5-, 15-, and 30-min incubation. The bar in this Figure represents 5 ⁇ m.
- the S- palmitoyalted and N-palmitoyalted peptides were taken up much more rapidly and to much higher concentrations into macrophages than were non-palmitoylated peptides, as indicated by the stronger staining of the palmitoylated peptides - the palmitoylated peptides could easily be visualised inside the cells after 1 min, whereas the non-palmitoylated peptides could barely be visualised, even after 30 min incubation. This is also seen in the flow cytometric plot at 15 min, where the fluorescence intensity of staining with the palmitoylated peptides is several logs higher than the non-palmitoylated peptide.
- FIG. 3 shows some results of Example 3, demonstrating that there were differences in the route of uptake of S-palm or N-palm peptides.
- S-palm peptide colocalised rapidly and strongly with endosomes.
- N-palm peptide did not colocalise strongly with endosomes in most cells.
- S-palm peptide colocalised strongly with lysosomes.
- N-palm peptide did not colocalise strongly with lysosomes in most cells, and there was no time-dependent increase of this localisation.
- the bars in the graphs in Figure 3 represent the percentage colocalisation (mean ⁇ SE) in at least 100 cells.
- nd means not done.
- N-palm peptide and S-palm peptide to endoplasmic reticulum (ER) were compared.
- the results show percentage colocalisation after incubation with biotinylated peptide for 30 or 60 minutes and staining to detect ER or peptide.
- Colocalisation of N-palm peptide and ER increased after 30 minutes of incubation, compared with colocalisation of S-palm peptide with ER.
- Figure 5 shows some results of Example 2 where S-palm peptide colocalised strongly with MHC class II, but only a small percentage colocalised with MHC class I (p ⁇ 0.0001). In contrast, N-palm peptide colocalised strongly with MHC class I and to a much lesser degree with MHC class II (p ⁇ 0.0001).
- raft refers to a graft containing cells, tissues, organisms etc that are of different genetic constitution to the recipient.
- the term "anergy” as used herein refers to a suppressed response, or a state of non-responsiveness, to a specified antigen or group of antigens by an immune system.
- T lymphocytes and B lymphocytes are anergic when they cannot respond to their specific antigen under optimal conditions of stimulation.
- antigen is meant all, or part of, a protein, peptide, or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal. Such antigens are also reactive with antibodies from animals immunised with that protein, peptide, or other molecule or macromolecule.
- the phrase “consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
- Consisting of is meant including, and limited to, whatever follows the phrase “consisting of.
- the phrase “consisting of” indicates that the listed elements are required or manadatory, and that no other elements may be present.
- peptide which encodes an amino acid sequence that displays substantial similarity to an amino acid sequence in a target antigen.
- the peptide will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to at least a portion of the target antigen.
- an effective amount in the context of preventing, inhibiting, treating or decreasing an immune response, is meant the administration of that amount of peptide compound to an individual in need thereof, either in a single dose or as part of a series, that is effective for achieving that prevention, inhibition, treatment or decrease.
- the effective amount will vary depending upon the health and physical condition of the individual, the taxonomic group of individual, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
- immunosuppressive peptide refers to a peptide that inhibits an immune response to an antigen or that causes the immune system to become unresponsive to an antigen. This includes peptides that cause insensitivity of T cells to T cell receptor-mediated stimulation. Representative “immunosuppressive peptides” may induce tolerance to an antigen, or stimulate suppressor cell (e.g., regulatory T cell) function, or induce ariergy in or clonal deletion of T cells in an antigen-specific manner.
- suppressor cell e.g., regulatory T cell
- isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
- membrane permeating lipid moiety refers to a moiety that is able to penetrate and pass through a membrane.
- the membrane permeating moiety is able to transport the peptide to which it is conjugated through a membrane.
- patient refers to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom prophylaxis or therapy is desired.
- Suitable vertebrate animals that fall within the scope of the present invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc), and fish.
- a preferred subject is a human suffering from an autoimmune disease, an allergic immune disease or who is an allo
- pharmaceutically acceptable excipient or diluent is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.
- pharmaceutically compatible salt refers to a salt which is toxicologically safe for human and animal administration.
- This salt may be selected from a group including hydrochlorides, hydrobromides, hydroiodides, sulfates, bisulfates, nitrates, citrates, tartrates, bitartrates, phosphates, malates, maleates, napsylates, fumarates, succinates, acetates, terephthalates, pamoates and pectinates.
- salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like.
- Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
- Peptide refers to a polymer of amino acid residues and to variants and synthetic analogues of the same.
- this term encompasses amino acid polymers in which all of the amino acid residues are naturally occurring and amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
- peptide conjugate refers to a compound that includes at least two moieties, a peptide moiety and a lipid moiety, that are joined or linked together by a covalent bond.
- prevention means to include prophylactic treatment, including but not limited to (1) preventing, inhibiting, or delaying the onset of, or the development of an autoimmune, an allergic immune or an allograft immune response, or (2) preventing, inhibiting, or delaying a symptom of the immune response.
- recombinant peptide is meant a peptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.
- treatment means to include therapeutic treatment, including but not limited to (1) relieving, altering, reversing, affecting, inhibiting the development of, inhibiting the progression of, ameliorating, or curing an autoimmune, an allergic immune or an allograft immune response, (2) relieving, altering, reversing, affecting, inhibiting the development of, inhibiting the progression of, ameliorating, or curing a symptom of the immune response, or (3) reducing the number of, or lengthening the time between relapses of the immune response, or reducing the symptoms of a relapse.
- the present invention contemplates peptide conjugates that comprise any peptide comprising an amino acid sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response. Many such peptides are known.
- altered peptide ligands are one class of such peptides. Interaction of T cell receptors with APLs has been shown to skew the cytokine profile of responding T cells, or to induce anergy. Therefore, it has been shown in experimental models that APLs can be used to stimulate or enhance a tolerogenic immune response mediated through T-cells and also have application in the prevention, inhibition, treatment or decrease of autoimmune, allergic immune or allograft immune responses.
- APLs are based on known antigens but have modified sequences, normally at sites where T cell receptors or MHC anchor positions interact with the antigen on which the APL is based.
- the sequence modification in the APL is usually determined by using a panel of peptides based on the known antigen, where at least one residue in each peptide is a conservative or non-conservative substitution of the residue in the known antigen. Each peptide is then tested for its ability to bind the MHC molecule of interest and to induce responses in T cells specific for the native antigen.
- APLs are based on antigens that are processed and presented through MHC class I molecules.
- APLs are based on antigens that are processed and presented through MHC class II molecules.
- APLs are derived from antigen sequences where the MHC anchoring amino acids are determined, for example by alanine scanning.
- the MHC anchoring amino acids may then be substituted with another amino acid or may be deleted from the sequence. If more than one amino acid is involved as an MHC anchor, more than one of these amino acids may be substituted or deleted.
- APLs are derived from CNS myelin proteins, such as proteolipid protein (PLP), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG).
- PPL proteolipid protein
- MBP myelin basic protein
- MOG myelin oligodendrocyte glycoprotein
- the peptide that comprises a sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response is a peptide, such as an APL, that comprises a sequence that includes at least one amino acid residue having a free thiol group that is capable of forming a thioester with the lipid moiety.
- the peptide contains at least one cysteine residue in its sequence.
- the peptide comprises one amino acid residue having a free thiol group in its sequence
- the free thiol group may be present in the side chain of an amino acid residue in the peptide.
- An amino acid residue having a free thiol group in its side chain may be a natural or common amino acid such as L-cysteine, or a non- natural or uncommon amino acid such as D-cysteine, L-homocysteine, D-homocysteine, L- penicillamine or D-penicillamine.
- the peptide that comprises a sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response is a peptide that does not normally include an amino acid residue having a free thiol group, but for the purposes of this invention has a further amino acid residue, in addition to the sequence, at the C-terminus or N-terminus of the peptide, having a free thiol group.
- the further amino acid residue is a cysteine, homocysteine or penicillamine residue, especially a cysteine residue.
- the peptide of the peptide conjugate that suppresses or otherwise inhibits an MHC class II response may also be capped with an N-terminal capping group and/or a C-terminal capping group. These groups may provide biostability to the peptide or may provide a free thiol group which may be acylated.
- the C-terminus may be capped with an amino group or substituted amino group to form an amidated C-terminus, including -CONH 2 , -CONH(alkyl), -CON(alkyl) 2 , -CONH-(CH 2 ) 1-3 SH wherein the -CO group is derived from the C-terminal carboxyl group and the term "alkyl" in each use independently refers to a saturated hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, 2-methylpropyl, tert-butyl, pentyl and hexyl.
- N-terminal capping groups include acyl groups including -C(O)(CH 2 ) 1-3 SH. If the N- or C-terminal capping group includes a thiol group, this group may be acylated in accordance with the invention. [0089] N-terminal and C-terminal capping groups may be introduced into the peptide by acylation or amidation as known in the art. Thiol containing N-terminal capping groups or C-terminal capping groups such as 3-mercaptopropionic acid or 2- mercaptoethylamine are commercially available.
- the peptide that comprises a sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response is a peptide, such as an APL, that comprises a sequence that includes at least one amino acid residue having a free amino group that is capable of forming an amide with the lipid moiety.
- the free amino group may be the amino group of the N-terminal amino acid or a free amino group on an amino acid side chain such as a lysine side chain.
- the peptide of the peptide conjugate that suppresses or otherwise inhibits an MHC class I response may also be capped with a C-terminal capping group and/or, when the N-terminal amino group is not part of the amide linkage with the lipid moiety of the conjugate, an N-terminal capping group. These groups may provide biostability to the peptide.
- the C-terminus may be capped with an amino group or substituted amino group to form an amidated C-terminus, including-CONH 2 , - CONH(alkyl), -CON(alkyl) 2 , where the -CO group is derived from the C-terminal carboxyl group and the term "alkyl" in each use independently refers to a saturated hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, 2- methylpropyl, tert-butyl, pentyl and hexyl.
- Suitable N-terminal capping groups include acyl groups including -C(O)(CH 2 ) 1-3 SH. If the N- or C-terminal capping group includes an amino group, this group may be acylated in accordance with the invention.
- N-terminal and C-terminal capping groups may be introduced into the peptide by acylation or amidation as known in the art.
- Thiol containing N-terminal capping groups or C-terminal capping groups such as 3-mercaptopropionic acid or 2- mercaptoethylamine are commercially available.
- the peptides of the peptide conjugate of this invention may be of any suitable size that can be utilised to suppress or inhibit unwanted or undesirable immune response. A number of factors can influence the choice of peptide size.
- the peptide has 6 to 60 amino acid residues, especially 10 to 50, 11 to 40, 12 to 30, 12 to 25 or 12 to 20 amino acid residues, more especially 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16 or 12 to 15 amino acid residues.
- the size of a peptide can be chosen such that it includes, or corresponds to the size of T cell epitopes and their processing requirements.
- class I-restricted T cell epitopes usually range between 8-10 amino acid residues in length, and that class II-restricted T cell epitopes usually range between 12 and 25 amino acid residues in length.
- the epitopes may or may not require natural flanking residues.
- Another important feature of class II-restricted epitopes is that they generally contain a 'core section' of 9-10 amino acid residues in the middle of the sequence which bind specifically to class II MHC molecules, and with flanking sequences either side of this 'core section' that stabilise binding by associating with conserved structures on either side of class II MHC molecules in a sequence independent manner.
- the functional region of class II-restricted epitopes is typically less than about 15 amino acid residues long.
- the size of the peptide is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 amino acid residues.
- the size of the peptide is no more than about 60, 50, 40, 30 amino acid residues.
- the size of the peptide is sufficient for presentation by an MHC class I molecule of an antigen presenting cell.
- the size of the peptide is sufficient for presentation by an MHC class II molecule of an antigen presenting cell.
- the present invention stems at least in part from the determination that the immunosuppressive effect of a peptide that comprises a sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response may be improved by attaching the peptide to a membrane permeating lipid moiety.
- the lipid moiety in the peptide conjugates of the present invention is therefore any suitable lipid moiety that allows the peptide to permeate or pass through membranes.
- the lipid moiety permeates membranes of antigen-presenting cells such as dendritic cells and macrophages.
- the lipid moiety comprises a saturated or monounsaturated fatty acid having 8 to 18 carbon atoms, especially 10 to 18 carbon atoms, 12 to 16 carbon atoms or 14 to 16 carbon atoms, more especially 14 to 16 carbon atoms, hi certain embodiments the fatty acid is a saturated fatty acid.
- Exemplary fatty acids include n- dodecanoic acid (lauric acid), n-tetradecanoic acid (myristic acid), n-hexadecanoic acid (palmitic acid), n-octadecanoic acid (stearic acid), hexadec-9-enoic acid (palmitoleic acid) and octadec-9-enoic acid (oleic acid).
- the fatty acid lipid moiety is selected from a myristoyl group or a palmitoyl group.
- the peptide conjugates comprise a peptide comprising a sequence that suppresses or otherwise inhibits an unwanted or undesirable immune response and a lipid moiety.
- the peptide and the lipid moiety may be linked by a covalent thioester linkage, or by an amide linkage.
- the thioester linkage is formed between a free thiol group on the peptide and a carboxylic acid on the lipid moiety.
- the amide linkage is formed between an amine group on the peptide and a carboxylic acid on the lipid moiety.
- the peptide conjugate comprises one lipid moiety. In other embodiments, the peptide conjugate comprises more than one lipid moiety linked to the peptide through thioester or amide linkages.
- the limit on the number and nature of lipid moieties attached to a peptide is the ability of the peptide conjugate to pass through a membrane without becoming anchored in the membrane.
- the peptide conjugate comprises one or two lipid moieties.
- the peptides of the peptide conjugates of the present invention may be prepared or obtained by methods known in the art including solution phase or solid phase synthesis (Jones, Amino Acid and Peptide Synthesis, Oxford Chemistry Primers, 1992), isolation from natural sources or preparation by recombinant methodology (Sambrook et al. Molecular Cloning: A laboratory manual, 2 nd Edition, Cold Spring Harbour Laboratory Press Plain view, New York, 1989).
- the peptides include at least one, especially one, thiol-containing residue, especially cysteine, in the peptide sequence.
- the at least one thiol-containing residue is incorporated in the sequence during solution phase, solid phase or recombinant synthesis or is present in the isolated naturally occurring peptide.
- the at least one thiol-containing residue is incorporated in the sequence at the N-terminus or C-terminus of the peptide during solution phase, solid phase or recombinant synthesis or after isolation of a naturally occurring peptide.
- the peptides include at least one residue having an amino-containing side chain, such as lysine.
- the at least one residue having an amino-containing side chain is incorporated in the sequence during solution phase, solid phase or recombinant synthesis or is present in the isolated naturally occurring peptide.
- the free amino group is the N-terminal amino group and the peptide is synthesised under normal solid phase, solution phase or recombinant synthesis or the naturally occurring peptide is isolated and if required, the N-terminal amino group deprotected of any protecting group that may have been introduced during synthesis or isolation.
- the peptides are synthesised using solid phase synthesis techniques using known strategies such as t-butoxycarbonyl (BOC) or 9- fluorenylmethoxycarbonyl (Fmoc), N-protection and deprotection strategies and carboxylic acid activation as known in the art.
- BOC t-butoxycarbonyl
- Fmoc 9- fluorenylmethoxycarbonyl
- Suitable side chain protection and deprotection strategies to use will depend on whether selective deprotection of side chain functional groups is required during or after synthesis.
- Suitable side chain protecting groups are known in the art, for example, in Greene & Wuts, Protective Groups in Organic Synthesis, 3 rd Edition, 1999, John Wiley & Sons.
- the peptides are synthesised using Fmoc solid phase synthesis and activation using coupling agents such as N-N'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), HBTU, benzotriazole-1-yl-oxy-tris- (dimethylamino)-phosphonium hexafluorophosphate (BOP), 3-(Diethoxy-phosphoryloxy)- 3H-benzo[d][l,2,3]-triazin-4-one (DEPBT), N,N'-diisopropylcarbodiimide (DIC), l-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl), 2-(lH-2-Azabenzotriazol-l- yl)-l,l,3,3-tetramethyluronium hexafluor
- coupling agents such as N
- the at least one thiol-containing residue is protected with a protecting group that may be selectively removed in the presence of other side chain or N-terminal protecting groups.
- Suitable thiol protecting groups are described in Greene & Wuts, ibid, and include, for example, methoxytrityl (Mmt) which is labile in 2% trifluoracetic acid (TFA) and t-butyl sulfenyl (StBu) which is labile in tributylphosphine.
- the peptide is synthesised using solid phase synthesis on a resin and the at least one thiol-containing residue thiol protecting group is selectively deprotected while the peptide is attached to the resin.
- the peptide conjugate is formed through thioacylation of the free thiol group with a carboxylic acid of the lipid moiety.
- Coupling of the peptide and the lipid moiety to form the peptide conjugate having a thioester linkage may be achieved by acylation or thioacylation methods known in the art.
- the free thiol group of the thio-containing amino acid residue may be reacted with an activated carboxylic acid, such as an acid chloride or anhydride, on the lipid moiety.
- the thiol group of the thiol-containing amino acid residue may be reacted with the carboxylic acid of the lipid moiety in the presence of a coupling agent such as those described above in relation to amide bond formation during peptide synthesis, especially BOP.
- the amino group may be protected with a protecting group that may be selectively removed in the presence of other side chain protecting groups.
- a protecting group that may be selectively removed in the presence of other side chain protecting groups.
- the free amino group may be protected during synthesis and selectively deprotected in the presence of other side chain or N-terminal protecting groups to allow selective formation of an amide bond with the lipid.
- the free amino group is the N-terminal amino group
- the N-terminal protecting group present during synthesis may be selectively deprotected in the presence of other protecting groups for selective formation of the amide bond with the lipid.
- Suitable amino protecting groups include Fmoc and BOC.
- the amino protecting group may need to be selected having consideration of other amino protecting groups used in the peptide.
- Coupling of the amine-containing peptide and the lipid moiety to form the peptide conjugate having an amide linkage may be achieved by N-acylation methods known in the art.
- the free amino group of the peptide may be reacted with an activated carboxylic acid of the lipid, such as an acid chloride or an anhydride.
- the free amino group of the peptide may be reacted with the carboxylic acid of the lipid moiety in the presence of a coupling agent such as those described above in relation to amide bond formation during peptide synthesis.
- the peptide and lipid may be reacted together to form the amide linkage while the peptide is still attached to the resin.
- Peptides may be cleaved from the resin used in the solid phase synthesis using standard techniques known in the art. For example, for cleavage of peptides prepared with Fmoc chemistry TFA or TFA compositions such as 95% TFA, 2.5% triisopropylsilane (TIS) AND 2.5% H 2 O may be used. This also results in concomitant deprotection of amino acid side chains. For peptides where BOC chemistry is used HF may be used for resin cleavage and removal of side chain protecting groups.
- the peptides or peptide conjugates may be isolated by precipitation and lyophylisation. Purification, if required, may be performed using reverse phase high performance liquid chromatography (RP-HPLC) and analysis may be performed using mass spectrometry, such as electrospray ionisation mass spectrometry.
- RP-HPLC reverse phase high performance liquid chromatography
- mass spectrometry such as electrospray ionisation mass spectrometry.
- compositions including immunosuppressive compositions such as vaccines, comprising the peptide conjugate of the present invention as the active ingredient in suppressing or otherwise inhibiting an unwanted or undesirable immune response.
- compositions including immunosuppressive compositions such as vaccines, consisting essentially of the peptide conjugate of the present invention as the active ingredient in suppressing or otherwise inhibiting an unwanted or undesirable immune response.
- compositions including immunosuppressive compositions such as vaccines, consisting of the peptide conjugate of the present invention as the active ingredient in suppressing or otherwise inhibiting an unwanted or undesirable immune response.
- immunosuppressive compositions such as vaccines, consisting of the peptide conjugate of the present invention as the active ingredient in suppressing or otherwise inhibiting an unwanted or undesirable immune response.
- these compositions may further comprise pharmaceutically acceptable excipients or diluents that are compatible with the active ingredient.
- Suitable excipients or diluents are those which are non-toxic to the individual receiving the composition, including, for example, water, isotonic saline with or without a physiologically compatible buffer like phosphate or HEPES, dextrose, glycerol, ethanol, or the like and combinations thereof.
- Carrying reagents, such as albumin and blood plasma fractions and nonactive thickening agents, may also be used.
- Non-active biological components to the extent that they are present in the composition, are especially derived from a syngeneic animal or human as that that will receive the composition, and are even more especially obtained previously from the subject to receive the composition.
- compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the immunosuppressive composition.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the immunosuppressive composition.
- adjuvants which may be effective include but are not limited to: surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl- N', N'bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; mineral gels such as aluminum phosphate, aluminum hydroxide or alum; peptides such as muramyl dipeptide and derivatives such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-
- the composition may comprise a peptide conjugate of the present invention in the form of a pharmaceutically acceptable salt, including acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like.
- Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
- compositions of the present invention uses routine methods known to persons skilled in the art. Techniques for formulation and administration of the compositions of the present invention may be found, for example, in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.
- compositions of the present invention are prepared as injectables, either as liquid solutions or suspensions, including vaccines. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
- Suitable administration routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, intradermal, transdermal, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
- devices or pharmaceutical compositions or compositions containing the peptide conjugate and suitable for sustained or intermittent release could be, in effect, implanted in the body or topically applied thereto for the relatively slow release of the peptide conjugate or composition of the invention into the body.
- the present invention also extends to methods for suppressing or otherwise inhibiting an unwanted or undesired immune response, including immune responses to a target antigen, in a subject by administering the peptide conjugates or compositions of the present invention, hi some embodiments, the immune response is a T- cell mediated response. In some other embodiments, the immune response is an antibody- mediated response.
- Also encapsulated by the present invention is a method for preventing, inhibiting, treating or decreasing an autoimmune, an allergic immune or an allograft immune response in a subject comprising administering an effective amount of a peptide conjugate or a composition of the present invention.
- the method may be performed before or once the subject displays symptoms of the immune response, including administering to the subject before or after the onset of symptoms.
- treatment commences 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 10 days, 1 month, 2 months, 6 months, 8 months, 12 months or 18 months after the onset of symptoms of the immune response.
- onset of symptoms includes the first time the subject has displayed any symptom of the immune response, or may be at a time when the subject displays symptoms of the immune response following a remission period where the subject did not display symptoms of the immune response following a period of displaying symptoms of the immune response.
- treatment commences when the subject is in remission, i.e. when the subject has previously displayed symptoms of the immune response but is not currently displaying symptoms of the immune response.
- Autoimmune responses that may be prevented, inhibited, treated or decreased by the present invention include tissue specific or systemic autoimmune diseases, including, but not limited to, Psoriasis, Acute disseminated encephalomyelitis (ADEM), Hashimoto's thyroiditis, Addison's disease, Idiopathic thrombocytopenic purpura, Ankylosing spondylitis, Lupus erythematosus, Antiphospholipid antibody syndrome (APS), Multiple sclerosis (MS), Autoimmune haemolytic anemia, Myasthenia gravis, Autoimmune hepatitis, Pemphigus, Bullous pemphigoid, Pernicious anaemia, Churg-Strauss Syndrome (or allergic granulomatosis), Polymyositis, Coeliac disease, Primary biliary cirrhosis, Dermatomyositis, Rheumatoid arthritis, Diabetes mellitus type 1 (IDDM), Sjo
- Allergic immune responses that may be prevented, inhibited, treated or decreased by the present invention include, but are not limited to both type I and type IV hypersensitivity immune responses such as hayfever, skin inflammation (urticaria), food allergies, asthma and systemic anaphylaxis, dermatitis, tuberculin reaction, and chronic transplant rejection.
- type I and type IV hypersensitivity immune responses such as hayfever, skin inflammation (urticaria), food allergies, asthma and systemic anaphylaxis, dermatitis, tuberculin reaction, and chronic transplant rejection.
- Allograft immune responses that may be prevented, inhibited, treated or decreased by the present invention include any unwanted or undesired immune response in a subject as the result of allograft transplantation to the subject.
- Suitable allograft transplants include organs and tissue including heart, lung, kidney, liver, pancreas, intestine, skin and stem cells, including hematopoietic stem cells and mesenchymal stem cells.
- the methods of the present invention may be achieved by in vivo administration of the peptide conjugates or compositions of the present invention as described above.
- the dosage of the peptide conjugate or composition of the present invention to be administered may depend on the subject to receive the peptide conjugate or composition inclusive of the age, sex, weight and general health condition thereof.
- the dosage will also take into consideration the pharmacokinetic properties of the peptide including the binding affinity of MHC class II molecules or MHC class I molecules to the peptide and the binding affinity of the T cell receptors to the peptide, the ability of the peptide conjugate to enter antigen presenting cells and pharmacokinetics compared to any antigen which is competing for the same MHC molecules and T cell receptor sites.
- precise amounts of the peptide conjugate for administration can also depend on the judgment of the practitioner.
- the physician or veterinarian may evaluate the subject's predisposition to the autoimmune, allergic immune or allograft immune response, or the progression of the immune response over time. In any event, those of skill in the art may readily determine suitable dosages of the agents of the invention without undue experimentation.
- the dosage of the peptide conjugate or composition administered to a patient should be sufficient to effect a beneficial response in the patient over time such as a reduction in the symptoms, reduced or no relapses in a patient, or the exhibition of symptoms or other parameters that indicate a beneficial response.
- a reduction in MRI lesions in a subject with multiple sclerosis is generally accepted in clinical trials as indicating a beneficial response to the treatment.
- Examples of usual patient dosages for systemic administration of the peptide conjugate stated in terms of patient body weight range from about 0.005-1.0 mg/kg, typically from about 0.01-0.5 mg/kg, more typically from about 0.05- 0.1.
- the dosages may be administered at suitable intervals such as to maintain an immunosuppressive effect, or boost the immunosuppressive response.
- intervals can be ascertained using routine procedures known to persons of skill in the art and can vary depending on the type of peptide conjugate employed and its formulation. For example, the interval may be daily, every other day, weekly, fortnightly, monthly, bimonthly, quarterly, half-yearly or yearly. 9. Ex vivo administration
- peptide conjugates or compositions of the present invention may be contacted ex vivo with antigen presenting cells or their precursors and the resulting mixture administered to the subject.
- the antigen presenting cells or precursors are derived or obtained from the subject receive the peptide conjugate or composition (i.e. an autologous antigen presenting cells).
- the antigen presenting cells or precursors are derived or obtained from a donor that is MHC matched or mismatched with the subject (i.e., an allogeneic antigen presenting cells).
- the donor is histocompatible with the subject.
- the antigen presenting cell is contacted with at least one peptide conjugate of the present invention which is suitably in soluble form in an amount and for a time sufficient for the peptide conjugate to be processed and the peptide (or processed form thereof) presented by the antigen presenting cells on their surface.
- the peptide conjugate is part of an immunomodulating composition including those described above.
- Antigen presenting cells or their precursors can be isolated by methods known to those of skill in the art. The source of such cells will differ depending upon the antigen presenting cell required for modulating a specified immune response, hi this context, the antigen presenting cell can be selected from dendritic cells, macrophages, monocytes and other cells of myeloid lineage.
- precursors of antigen presenting cells can be isolated from any tissue, but are most easily isolated from blood, cord blood or bone marrow (Sorg et al, 2001; Zheng et al, 2000). It is also possible to obtain suitable precursors from diseased tissues such as rheumatoid synovial tissue or fluid following biopsy or joint tap (Thomas et al, 1994a; Thomas et al, 1994b). Other examples include, but are not limited to liver, spleen, heart, kidney, gut and tonsil (Lu et al, 1994; Mcllroy et al, 2001; Vremec et al, 2000; Hart and Fabre, 1981; Hart and McKenzie, 1988; Pavli et al, 1990).
- Leukocytes isolated directly from tissue provide a major source of antigen presenting cell precursors. Typically, these precursors can only differentiate into antigen presenting cells by culturing in the presence or absence of various growth factors. According to the practice of the present invention, the antigen presenting cells may be so differentiated from crude mixtures or from partially or substantially purified preparations of precursors.
- Leukocytes peripheral blood mononuclear cells or PBMCs
- PBMCs peripheral blood mononuclear cells
- antigen presenting cells are present in peripheral blood as non-proliferating monocytes, which can be differentiated into specific antigen presenting cells, including macrophages and dendritic cells, by culturing in the presence of specific cytokines.
- Tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g., basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers.
- tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g., basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers.
- Langerhans cell precursors express CDl molecules as well as HLA-DR and can be purified on this basis.
- the antigen presenting cell precursor is a precursor of macrophages.
- these precursors can be obtained from monocytes of any source and can be differentiated into macrophages by prolonged incubation in the presence of medium and macrophage colony stimulating factor (M-CSF) (Erickson-Miller et al, 1990; Metcalf and Burgess, 1982).
- M-CSF medium and macrophage colony stimulating factor
- the antigen presenting cell precursor is a precursor of Langerhans cells.
- Langerhans cells can be generated from human monocytes or CD34 + bone marrow precursors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-4/TNF ⁇ and TGF ⁇ (Geissmann et al, 1998; Strobl et al, 1997a; Strobl et al, 1997b; Strobl et al, 1996).
- GM-CSF granulocyte/macrophage colony-stimulating factor
- IL-4/TNF ⁇ IL-4/TNF ⁇
- TGF ⁇ granulocyte/macrophage colony-stimulating factor
- the antigen presenting cell precursor is a precursor of dendritic cells.
- dendritic cell precursors can be obtained from peripheral blood, cord blood or bone marrow. These include monocytes, CD34 + stem cells, granulocytes, CD33 + CD1 Ic + DC precursors, and committed myeloid progenitors - described below.
- Monocytes can be purified by adherence to plastic for 1-2 hours in the presence of tissue culture medium ⁇ e.g., RPMI) and serum (e.g., human or foetal calf serum), or in serum-free medium (Anton et al, 1998; Araki et al, 2001; Mackensen et al, 2000; Nestle et al, 1998; Romani et al, 1996; Thurner et al, 1999). Monocytes can also be elutriated from peripheral blood (Garderet et al, 2001).
- tissue culture medium ⁇ e.g., RPMI
- serum e.g., human or foetal calf serum
- Monocytes can also be elutriated from peripheral blood (Garderet et al, 2001).
- Monocytes can also be purified by imrnunoaffinity techniques, including immunomagnetic selection, flow cytometric sorting or panning (Araki et al, 2001; Battye and Shortman, 1991), with anti-CD 14 antibodies to obtain CD14hi cells.
- the numbers (and therefore yield) of circulating monocytes can be enhanced by the in vivo use of various cytokines including GM-CSF (Groopman et al, 1987; Hill et al, 1995).
- Monocytes can be differentiated into dendritic cells by prolonged incubation in the presence of GM-CSF and IL-4 (Romani et al, 1994; Romani et al, 1996).
- a combination of GM-CSF and IL-4 at a concentration of each at between about 200 to about 2000 U/mL, more preferably between about 500 to about 1000 U/mL and even more preferably between about 800 U/mL (GM-CSF) and 1000 U/mL (IL-4) produces significant quantities of immature dendritic cells, i.e., antigen-capturing phagocytic dendritic cells.
- Other cytokines which promote differentiation of monocytes into antigen-capturing phagocytic dendritic cells include, for example, IL-13.
- Dendritic cells can also be generated from CD34 + bone marrow derived precursors in the presence of GM-CSF, TNF ⁇ ⁇ stem cell factor (SCF, c-kitL), or GM-CSF, IL-4 ⁇ flt3L (Bai et al, 2002; Chen et al, 2001; Loudovaris et al, 2001).
- CD34 + cells can be derived from a bone marrow aspirate or from blood and can be enriched as for monocytes using, for example, immunomagnetic selection or immunocolumns (Davis et al , 1994).
- CD34 + cells in blood can be enhanced by the in vivo use of various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001; Pulendran et al, 2000; Robinson et al, 2000).
- various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001; Pulendran et al, 2000; Robinson et al, 2000).
- DC can be generated from committed early myeloid progenitors in a similar fashion to CD34 + stem cells, in the presence of GM-CSF and IL-4/TNF.
- myeloid precursors infiltrate many tissues in inflammation, including rheumatoid arthritis synovial fluid (Santiago-Schwarz et al, 2001).
- Expansion of total body myeloid cells including circulating dendritic cell precursors and monocytes can be achieved with certain cytokines, including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro- GP) (Fleming et al, 2001 ; Pulendran et al, 2000; Robinson et al, 2000). Administration of such cytokines for several days to a human or other mammal would enable much larger numbers of precursors to be derived from peripheral blood or bone marrow for in vitro manipulation.
- cytokines including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro- GP)
- Dendritic cells can also be generated from peripheral blood neutrophil precursors in the presence of GM-CSF, IL-4 and TNF ⁇ (Kelly et al, 2001; Oehler et al, 1998). It should be noted that dendritic cells can also be generated, using similar methods, from acute myeloid leukaemia cells (Oehler et al, 2000).
- Transformed or immortalised dendritic cell lines may be produced using oncogenes such as v-myc or by myb.
- a second subset which lacks CD14, CD19, CD56 and CD3, known as plasmacytoid dendritic cell precursors, does not express CDl Ic, but does express CD123 (IL-3R chain) and HLA-DR (Farkas et al, 2001; Grouard et al, 1997; Rissoan et al, 1999). Most circulating CDl Ic + dendritic cell precursors are HLA-DR + , however some precursors may be HLA-DR-. The lack of MHC class II expression has been clearly demonstrated for peripheral blood dendritic cell precursors (del Hoyo et al , 2002).
- CD33 + CD1471o or CDl Ic + HLA-DR + , lineage marker- negative dendritic cell precursors described above can be differentiated into more mature antigen presenting cells by incubation for 18-36 h in culture medium or in monocyte conditioned medium (Thomas et al, 1993; Thomas and Lipsky, 1994; O'Doherty et al, 1993).
- peripheral blood dendritic cells are characterised by low density and so can be purified on density gradients, including metrizamide and Nycodenz (Freudenthal and Steinman, 1990; Vremec and Shortman, 1997), or by specific monoclonal antibodies, such as but not limited to the CMRF-44 mAb (Fearnley et al, 1999; Vuckovic et al, 1998).
- Plasmacytoid dendritic cells can be purified directly from peripheral blood on the basis of cell surface markers, and then incubated in the presence of IL-3 (Grouard et al, 1997; Rissoan et al, 1999).
- plasmacytoid DC can be derived from density gradients or CMRF- 44 selection of incubated peripheral blood cells as above.
- cytokines such as TNF- ⁇ , IL-6, IFN-cc, IL- l ⁇ , necrotic cells, re-adherence, whole bacteria, membrane components, RNA or polylC
- immature dendritic cells will become activated (Clark, 2002; Busher et al, 2002; Kaisho and Akira, 2002; Koski et al. , 2001).
- This process of dendritic cell activation is inhibited in the presence of NF- ⁇ B inhibitors (O'Sullivan and Thomas, 2002).
- the amount of peptide conjugate to be placed in contact with antigen presenting cells can be determined empirically by routine methods known to persons of skill in the art. Typically antigen presenting cells are incubated with the peptide conjugate of the present invention for about 1 to 6 hours at 37° C, although it is also possible to expose antigen presenting cells to peptide conjugate for the duration of incubation with growth factors. Generally, 0.001-1000 ⁇ g/mL of peptide conjugate is suitable for producing antigen presenting cells that are presenting the peptides on their surface through MHC I or MHC II molecules.
- peptide conjugate Usually 0.01-100 ⁇ g/mL of peptide conjugate is suitable for producing antigen presenting cells that are presenting the peptides on their surface through MHC I or MHC II molecules. Typically, 0.1-10 ⁇ g/mL of peptide conjugate is suitable for producing antigen presenting cells that are presenting the peptides on their surface through MHC I or MHC II molecules.
- the antigen presenting cells should be exposed to the peptide conjugate for a period of time sufficient for those cells to internalise the peptide.
- the time should be sufficient so that the antigen presenting cell also presents the peptide of the peptide conjugate or a processed form thereof on its surface.
- the time and dose of peptide conjugate necessary for the cells to internalise and present the processed peptide may be determined using pulse-chase protocols in which exposure to peptide conjugate is followed by a washout period and exposure to a read-out system e.g. , T cells reactive to the original antigen upon which the peptide of the peptide conjugate is based.
- a protocol may be used to prepare cells and peptide conjugate for suppressing or otherwise inhibiting an unwanted or undesired immune responses.
- Those of skill in the art will recognise in this regard that the length of time necessary for an antigen presenting cell to process and present the peptide will vary depending on the peptide or form of peptide in the peptide compound employed, its dose, and the antigen presenting cell employed, as well as the conditions under which peptide loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.
- the antigen presenting cells can be introduced into a patient by any means (e.g. , injection), which produces the desired immunosuppressive response to a peptide.
- the cells may be derived from the patient (i.e., autologous cells) or from an individual or individuals who are MHC-matched or -mismatched (i.e., allogeneic) with the patient.
- autologous cells are injected back into the patient from whom the source cells were obtained.
- the injection site may be subcutaneous, intraperitoneal, intramuscular, intradermal, or intravenous.
- the cells may be administered to a patient already suffering from the unwanted immune response or who is predisposed to the unwanted immune response in sufficient number to prevent or at least partially arrest the development, or to reduce or eliminate the onset of, that response.
- the number of cells injected into the patient in need of the treatment or prophylaxis may vary depending on inter alia, the peptide conjugate and size of the individual. This number may range for example between about 10 3 and 10 11 , and more preferably between about 10 5 and 10 7 cells (e.g., dendritic cells).
- Single or multiple administrations of the cells can be carried out with cell numbers and pattern being selected by the treating physician.
- the cells should be administered in a pharmaceutically acceptable excipient, which is non-toxic to the cells and the individual.
- excipient may be the growth medium in which the cells were grown, or any suitable buffering medium such as phosphate buffered saline.
- the cells may be administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment or prevention of unwanted immune responses for example but not limited to glucocorticoids, methotrexate, D- penicillamine, hydroxychloroquine, gold salts, sulfasalazine, TNF ⁇ or interleukin-1 inhibitors, and/or other forms of specific immunotherapy.
- Fmoc-1-amino acids were purchased from Nova biochem (Meudon,
- Palmitoyl chloride (Pam-Cl) and palmitic acid (Pam-OH) were purchased from Fluka (St-Quentic Fallavier, France). Dimethylformamide (DMF) was purchased from Merck (Briare Le Canal, France).
- the peptide (0.2 mmol) was synthesised manually on a Wang resin using the Fmoc/tBu strategy and BOP as coupling reagent. Typically, successive single couplings were performed with three equivalents of Fmoc amino acid and were monitored with the Kaiser colour test.
- Fmoc amino acids side-chain protecting groups were Lys(Dde), Asp(OtBu), His(Trt), Trp(Boc), Lys(Boc), Cys(Mmt). Boc-His(Boc)-OH was coupled as N- terminal amino acid and obtained by conversion of its DCHA salt.
- the peptide resin (300 mg) was treated with a solution of 2% TFA in dichloromethane (DCM) containing 5% TIS for 10 min in a glass reaction vessel equipped with a sintered glass filter using nitrogen for mixing. After filtration, the peptide-resin was washed with DCM. The deprotection and washing steps were repeated five times. The peptide-resin was finally washed with DCM.
- DCM dichloromethane
- Peptide resin 50 mg was introduced into a round bottom flask equipped with a condenser. A freshly prepared mixture of ⁇ -mercaptoethanol/DMF (1/1, v/v) (5 ml) was added. The mixture was heated overnight at 85 0 C. After filtration, the peptide resin was washed with DMF. [0163] Another example of carrying out deprotection of S-(tBu) cysteinyl residue is to use tributylphosphine.
- the peptide resin (50 mg) may be treated with a solution of tributylphosphine (100 equiv) and H2O (400 equiv) in DMF:DCM (1/1, 3ml) for 18 hours in a glass reaction vessel equipped with a sintered glass filter using nitrogen for mixing. After filtration, the peptide-resin may be washed with DCM and DMF.
- the biotinylated free thiol peptide resin (300 mg) was suspended in DMF (3 ml) containing palmitic acid (20 eq) and BOP (20 eq). Diisopropylethylamine (DIEA) (60 eq) was then added. After 1 hour of reaction at room temperature, the peptide resin was filtered off, washed with DMF, DCM, ether and then dried in a vacuum.
- DIEA Diisopropylethylamine
- N-terminal Fmoc protecting group Deprotection of the N-terminal Fmoc protecting group was achieved with 20% piperidine in DMF and treatment of the deprotected N-terminal amino group with palmitic acid in the presence of BOP. DIEA was then added. After 1 hour reaction at room temperature the peptide-resin was filtered off and washed with DMF, DCM and ether and dried under vacuum.
- Peptides were cleaved from the resin with the low odor mixture (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), 2.5% H2O). After evaporation of TFA, peptides were precipitated in ethyl ether and lyophilised after solubilisation in 10% acetic acid.
- TFA trifluoroacetic acid
- TMS triisopropylsilane
- H2O H2O
- MS ⁇ Multiple sclerosis
- MS is a chronic inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). It is a CD4 + mediated disease and has a strong association with MHC class II.
- CNS central nervous system
- EAE experimental autoimmune encephalomyelitis
- [0170J EAE can be induced in animal by injection of myelin proteins, such as
- EAE is also a useful model for aiding the development of new prophylaxis or treatment methods for MS.
- AU therapies approved for MS ameliorate EAE, and two approved medications: glatiramer acetate and Natalizumab, were developed directly from studies in EAE.
- acylation sites Cys 108, Cys 140, and Cys 183 are within the encephalitogenic PLP epitopes PLPl 04-117, PLP139-151, and PLP178-191 respectively (Greer et a 1996; Greer et ah, 2001). Reactivity to these epitopes has been found in some patients with MS.
- the Figure shows a zero mean score until day 10 (after PLPp immunisation) when the mean score demonstrates the start of signs the disease in the mice, peaking at approximately day 20 (after PLPp immunisation), following by a decrease in mean score until approximately day 38 and slight increase to what appears to be a constant mean score of 1 for the next 20 days.
- mice prior to onset of the disease, the mice are described as being in "pre-EAE onset stage" including at day 8 where PLPp induced IL-17-producing cells were detected in the drLN and in the spleen but not in the CNS.
- pre-EAE onset stage including at day 8 where PLPp induced IL-17-producing cells were detected in the drLN and in the spleen but not in the CNS.
- PLPp induced IL-17-producing cells were detected in the drLN and in the spleen but not in the CNS.
- PLPp induced IL-17-producing cells were detected in the drLN and in the spleen but not in the CNS.
- PLPp-specific IL-17 producing cells were detectable in the CNS 5 occurring there in higher frequencies than in the drLN and spleen.
- Npalm-PLP104-l 17 (palm)HN-K-T-T-I-C-G-K-G-L-S-A-T-V-T
- Npalm-PLP 139-151 (palm)HN-H-C-L-G-K- W-L-G-H-P-D-K-F
- mice were immunised with the above non-palmitoylated, S- palmitoylated or N-palmitoylated peptides in complete Freund's adjuvant to induce EAE.
- Lymph nodes from a different group of mice immunised in the same manner were removed 10 days after injection of the peptides and single cell suspensions of lymph node cells (LNC) were prepared.
- LNC LNC (3xlO 5 /well) were incubated at 37 0 C for 3 days in 96 well plates with 0-50 ⁇ g/ml antigen in a total volume of 200 ⁇ l (diluted in RPMI 1640 medium containing 10% FCS). Tritiated thymidine was added during the last 18 h of culture. Cells were harvested onto glass fibre filters, which were then put through a beta counter. Increased uptake of tritiated thymidine into cells indicated proliferation of the cells.
- mice immunised with S-palm-PLP 104-117 are significantly different from those for mice immunised with PLPl 04-117 (p ⁇ 0.001).
- the duration of the first episode of disease in mice immunised with ,S-patai-PLP139-151 is significantly different than that of mice immunised with PLP139-151 (p ⁇ 0.007).
- FIG. 1 is a graphical representation showing the proliferative responses of the LNC to the nonacylated and acylated PLP peptides.
- the peptide with which the mice had been immunised is shown above each graph.
- Each graph shows the proliferative responses of the LNC to the nonacylated (circle), S-palm (square), or N-palm (triangle) form of the same peptide.
- Each point on the graph represents the SI (mean ⁇ SD of three to five repetitions of each experiment) at a particular peptide concentration.
- mice immunised with the N- palmitoylated peptides induced a T cell response with a decreased CD4:CD8 ratio as shown in Table 4, and were not encephalitogenic.
- Table 4 CD4/CD8 ratios of activated LNC from mice (4 per group) immunised with nonacylated or palmitoylated peptides
- the cells were then incubated with murine antibodies against endosomes, lysosomes, endoplasmic reticulum, or MHC class I or II molecules for 1-3 hours at 37 0 C. After three washes in PBS, cells were incubated with streptavidin-Alexa 488 (1/400 dilution in PBS) or stre ⁇ tavidin-Cy3 (1/450 dilution in PBS) (both from Molecular Probes) and Texas Red-labeled rabbit anti-mouse IgG or IgM for 30 min in the dark at room temperature. After washing with PBS, coverslips were mounted in Aquapolymount medium.
- Immunofluorescence staining was monitored with a laser scanning microscope (LSM 510; Carl Zeiss Laboratories) equipped with a Plan-Apochromat 63 x oil DIC immersion lens (numerical aperture 1.4). Alexa 488 emission was excited using the 488-nm ray of the argon laser, whereas Texas Red was excited using the 543-nmline of the helium/neon laser. Emission signals of Alexa 488 and Texas Red were filtered with a LP 505-530 and a LP 560 filter, respectively. Quantification of colocalisation was performed on cells from 10 to 12 fields (at least 100 cells) with the colocalisation module of the Zeiss LSM Image Browser software.
- PC were washed and incubated with peptide for various times at 37 or 5°C. At the end of the incubation, they were fixed in freshly prepared 4% formaldehyde in PBS for 15 min at room temperature. After two washes with PBS, PC were permeabilised with 0.05% digitonin for 5 min at room temperature, washed twice more with PBS containing 1% FCS and 0.01% sodium azide, and double stained with PE-labeled F4/80 antibody to detect macrophages and FITC-streptavidin to detect the biotinylated peptide.
- the S- palmitoyalted and N-palmitoyalted peptides were taken up much more rapidly and to much higher concentrations into macrophages than were non-palmitoylated peptides, as indicated by the stronger staining of the palmitoylated peptides - the palmitoylated peptides could easily be visualised inside the cells after 1 min, whereas the non-pahnitoylated peptides could barely be visualised, even after 30 min incubation. This is also seen in the flow cytometric plot at 15 min, where the fluorescence intensity of staining with the palmitoylated peptides is several logs higher than the non-palmitoylated peptide.
- N-palm peptide but not S-palm peptide colocalised strongly with endoplasmic reticulum (ER) as shown in Figure 4.
- ER endoplasmic reticulum
- APL Q144 2HN-HCLGKQLGHPDKF-COOH lipoAPL S-palm Q144 2HN-HC(palm)LGKQLGHPDKF-COOH [0194]
- Example 1 The following peptides produced in Example 1 were used: native peptide F188 2HN-NTWTTCQSIAFPSK-COOH
- the APL Al 88 was protective when injected at a dose 5 times greater than the encephalitogenic peptide Fl 88.
- the S-palm APL Al 88 was protective when injected at a dose 5 times lower than the encephalitogenic peptide F 188.
- a 100 nM solution of Fl 88 peptide was emulsified in an equal volume of complete Freund's adjuvant containing an additional 4mg/ml Mycobacterium tuberculosis H37Ra. 200 ⁇ l of the emulsion (i.e. 50 nM F188) was then injected subcutaneously in a single site on the back of each SJL/J mouse. Each mouse also received 300 ng pertussis toxin intravenously on the same day as the peptide emulsion and then 3 days later.
- APL S-palm Al 88 2HN-NTWTTC(palm)QSIAAPSK-COOH [0213] SJL/J mice were injected subcutaneously with 1 OO ⁇ g S-palm-Fl 88 (S- palm-PLP178-191), i.e. 100 ⁇ l of a 1 mg/ml solution emulsified in an equal volume (i.e. 100 ⁇ l Complete Freund's Adjuvant containing an extra 4mg/mL Mycobacterium tuberculosis H37Ra. Each mouse also received 300 ng pertussis toxin intravenously on the same day as the peptide emulsion and then 3 days later.
- mice were immunised subcutaneously with 100 ⁇ l of a 1 mg/ml solution of either Al 88 or S-palm Al 88 emulsified in an equal volume of complete Freund's adjuvant. Lymph nodes were removed 10 days after injection of the peptides and single cell suspensions of lymph node cells (LNC) were prepared.
- LNC lymph node cells
- RNA analysis the LNC were stimulated for 4 hours in vitro in the presence of no antigen, or 20 ⁇ g/ml of Al 88 or the native peptide PLPl 78-191. After that time, cells were harvested and frozen for later RNA extraction.
- LNC were stimulated for 3 days in vitro in the presence of no antigen, or 20 ⁇ g/ml of Al 88 or the native peptide PLP178-191. They were then harvested and stained with antibodies for CD4 and CD25 prior to flow cytometric analysis.
- RNA extracted from LNC was then analysed by RT-PCR. Flow cytometric analysis of CD4+CD25+ T regulatory cells was also carried out on the LNC.
- mice significantly upregulated the transcription factor FoxP3, which is a marker of regulatory T cells, upon stimulation with either APL or native peptide.
- Example 9 describes a proposed experiment used to test the ability of an APL and a thiopalmitoylated form of the APL for preventing or treating type II collagen- induced arthritis using the mouse model described in Coutenay et al..
- DBA/1 mice will be immunised intraderrnally with 100 ⁇ g bovine type II collagen (CII) in Complete Freund's adjuvant. Each mouse will also receive a booster dose on day 21 by intraperitoneal injection of 100 ⁇ g of CII.
- CII bovine type II collagen
- mice will be treated with three inj ections each of 333 ⁇ g of APL intraperitoneally (total 1 mg) on days 24, 26, and 28 after the first immunisation with CII, using the same protocol as described in Wakamatsu et al .
- Wakamatsu et al the mice were shown to develop the disease 21-25 days (typically around day 24) after the initial immunisation of CII.
- Control mice will receive the injection vehicle according to the same schedule.
- S-palm APL will be tested in at the same concentration, and also at 100, 33 and 10 ⁇ g per injection, to determine whether lower doses of S-palm APL are more effective than the APL.
- the animals will be observed at daily intervals and evaluated for the severity or arthritis by scoring each paw.
- the score will range from 0 to 3 (0, no swelling or redness; 1, swelling or redness in one joint; 2, involvement of two or more joints; 3, severe arthritis of the entire paw and joints).
- the total score of each animal will be the sum score of all four paws.
- Example 10 describes a proposed experiment used to test the efficacy of an APL and a thiopalmitoylated form of the APL for preventing or treating type I diabetes in NOD mice.
- mice Female NOD mice will be used in these experiments. These mice provide a spontaneous model of autoimmunity, and typically develop frank diabetes between 15-20 weeks of age (Alleva et al, and Cameron et al).
- prediabetic female NOD mice i. e. , 4-week-old, insulitis-free
- mice will be treated with weekly subcutaneous injections of 400 ⁇ g APL for 12 weeks and then one injection every 2 weeks until mice reach 39 weeks of age.
- Control mice will receive the injection vehicle according to the same schedule.
- S-palm APL will be tested at the same concentration, and also at 100, 40 and 10 ⁇ g per injection, to determine whether lower doses of S-palm APL are more effective than the APL.
- Glucometer Bayer
- Mice with blood glucose levels equal to or greater than 200 mg/dl on two consecutive occasions will be considered diabetic.
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WO1997049425A1 (en) * | 1996-06-25 | 1997-12-31 | Stichting Instituut Voor Dierhouderij En Diergezondheid | Vaccine comprising antigens bound to carriers through labile bonds |
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Non-Patent Citations (1)
Title |
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GREER J. M. ET AL.: "Thiopalmitoylation of Myelin Proteolipid Protein Epitope.s,Enhances Immunogenicity and Encephalitogenicity", JOURNAL OF IMMUNOLOGY, vol. 166, no. 11, 2001, pages 6907 - 6913, XP008148532 * |
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