WO2006109285A1

WO2006109285A1 - Methods and compounds for the treatment of autoimmune diseases and chronic inflammatory conditions

Info

Publication number: WO2006109285A1
Application number: PCT/IE2006/000032
Authority: WO
Inventors: Kingston Mills; Brian Keogh; Peter Mcguirk
Original assignee: The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin
Priority date: 2005-04-14
Filing date: 2006-04-18
Publication date: 2006-10-19
Also published as: GB0507557D0

Abstract

Described is a method for modulating an immune response in order to treat conditions such as autoimmune disease. Specifically it has been shown that the adoptive transfer of dendritic cells primed with filamentous haemagglutinin can serve to prevent the onset and development of EAE, a murine model for multiple sclerosis. Such a finding can be used to provide a therapy which can be used to treat autoimmune diseases and immune-mediated disorders .

Description

"Methods and compounds for the treatment of autoimmune diseases and chronic inflammatory conditions"

Field of the Invention

The present invention relates to a novel method of treatment for autoimmune and chronic inflammatory conditions. More specifically there is provided a method of treating autoimmune and chronic inflammatory conditions involving the adoptive transfer of dendritic cells which have been modulated with filamentous haemagglutinin (FHA) . The invention further extends to the use of such modulated dendritic cells.

Background to the Invention Dendritic Cells

Dendritic cells (DCs) are very effective antigen- presenting cells (APCs) with the unique ability to prime naive T lymphocytes to novel antigens. Dendritic cells are produced in the bone marrow and migrate via the blood stream into virtually all tissues in the body. These cells are usually found in the structural compartment of such lymphoid organs as the thymus, lymph nodes, and spleen.

However, they are also found in the bloodstream and other tissues of the body. Dendritic cells capture antigen when appropriate and migrate to draining lymph nodes where an immune response is initiated.

Dendritic cells process and present the antigen via major histocompatibility complex (MHC) -peptide complexes to antigen-specific naive T lymphocytes. Both myeloid and lymphoid dendritic cell populations have utility in this invention.

Dendritic cells in the peripheral blood are identified within the HLA-DR⁺, lineage (CD3, CDl4, CDl9, CD56) negative (Lin^~) blood mononuclear cell population. The precursors for the peripheral epithelial (CDla^hi) and dermal (CDla¹⁰) dendritic cells are identified within myeloid blood CDlIc⁺ Dendritic cells .

Immune-mediated Diseases

Inflammatory Bowel Disease (IBD) includes two major pathologies; ulcerative colitis and Crohn's disease. Both are characterised by an exaggerated response of the mucosal immune system to stimuli originated from the intestinal flora. In both ulcerative colitis and Crohn's disease, tumour necrosis factor alpha (TNF-alpha) has been invoked as one of the major factors in the chronicity of the inflammation.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases in humans . These autoimmune diseases are inflammatory conditions of the intestine mediated by CD4⁺ T cells. Regulatory T cells (Tr cells) prevent the development of autoimmune diseases in normal individuals. Injection of CD45RB^high (naxve) T cells can induce colitis in severe combined immunodeficient (SCID) mice, which can be prevented by co-transfer of CD45RB^low or CD4⁺ CD25⁺ regulatory T cells (7) . Furthermore elimination of CD45RB^low or CD4⁺ CD25⁺ regulatory T cells leads to spontaneous development of various autoimmune diseases in otherwise normal mice or rats .

Autoimmune diseases Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system. Individuals with this disease have autoreactive T cells (T cells that recognize self antigens) , which together with interleukin (IL) -lβ and tumour necrosis factor (TNF) oc, participate in the formation of inflammatory lesions along the myelin sheath of nerve fibres . The cerebrospinal fluid (CSF) of patients with MS contains activated T cells, which infiltrate the brain tissue and cause the characteristic inflammatory lesions, destroying the myelin.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for MS. It is induced in mice or rats by injection of mylein basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG) or peptides thereof with complete Freund's adjuvant. The disease can also be induced by transfer of MBP or MOG-specific T cells that secrete IFN-γ (called ThI cells) . The animals develop cellular infiltration of the myelin sheaths of the central nervous system, resulting in demyelination and eventually paralysis. The clinical signs and pathological changes resemble MS.

A method of preventing the development of multiple sclerosis in a prophylactic sense would be highly advantageous in order to prevent development and progression of the condition. Such a method of treatment would not only improve significantly the quality of life of an individual with the condition, but would also confer significant costs savings in relation to the cost of medical care and medicaments which would be required if normal progression of the disease was to occur.

Summary of the Invention

The inventors of the present invention have made the surprising discovery that the adoptive transfer of dendritic cells primed with filamentous haemagglutinin can serve to prevent the onset and development of multiple sclerosis . Such a finding can be used to provide a therapy which can be used to treat autoimmune diseases and immune-mediated disorders . According to a first aspect of the present invention there is provided a method for eliciting an immune response in a subject suitable, the response being characterised in that it provides treatment of an immune-mediated disease, the method comprising the steps of:

- exposing isolated dendritic cells to an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof ex-v±vo for a sufficient time and under condition suitable to modulate the function of the dendritic cells,

- administering the dendritic cells to the subject whereby the immune response generated in the subject is sufficient to prevent the onset or progression of the immune-mediated disease disease.

In one embodiment of this aspect of the invention the dendritic cells are autologous to the subject.

In one embodiment the dendritic cells are immature dendritic cells. Alternatively the dendritic cells are mature dendritic cells.

In a further embodiment the dendritic cells may be administered along with a Toll-Like Receptor (TLR) agonist. Any suitable TLR agonist may be administered. The TLR agonist may be specific to any defined human Toll like receptor. In specific embodiments, the TLR agonist has^" specϊficity for^""" TLR2 , TLR4 or TLR9. In further embodiments the TLR agonist may be selected from any one or more of LPS, CpG motifs, dsRNA, PoIy(IrC), lipoteichoic acid, heat shock proteins, lipid A, flagellin and Pam- 3Cys .

In a yet further embodiment, a self-antigen may be further optionally co-administered to the individual along with the treated dendritic cells. An example of a self antigen is MOG or myelin basic protein.

According to a second aspect of the present invention there is provided a composition for modulating an immune response in a individual suffering from an immune mediated condition, the composition comprising dendritic cells which have been activated by being pulsed with FHA for use in the modulation of an immune response characteristic or causative of an autoimmune disease or immune mediated condition.

In one embodiment the composition further comprises a self antigen. In a further embodiment, the composition comprises a Toll-like receptor ligand.

In a further aspect of the present invention, the immune mediate condition is an autoimmune disease such as multiple sclerosis, rheumatoid arthritis, Crohn's disease or psoriasis.

According to a third aspect of the present invention there is provided a pharmaceutical compos^~iti^~bn for the treatment of an autoimmune disease or an immune mediated condition, said composition comprising dendritic cells activated in the presence of FHA along with a pharmaceutically acceptable carrier or diluent.

According to a fifth aspect of the present invention there is provided the use of dendritic cells which have been pulsed with FHA in the preparation of a medicament for modulating the immune response in an individual who suffers from an autoimmune disease or immune mediated condition.

In one embodiment the dendritic cells are derived from naϊve dendritic cells which have been pulsed with FHA.

In a further embodiment the medicament further comprises a Toll-like receptor agonist. In a still further embodiment the medicament comprises a self antigen. According to a sixth aspect of the present invention there is provided the use of dendritic cells which have been pulsed with FHA for use in medicine. According to a seventh aspect of the present invention, there is provided dendritic cells which have been exposed ex vivo to FHA for use in medicine.

In one embodiment the dendritic cells which are exposed to the FHA are naϊve dendritic cells.

A yet further aspect of the present invention provides dendritic cells which have been exposed ex vivo to FHA for use in the treatment of an immune- mediated disorder.

In one embodiment the dendritic cells which are exposed to the FHA are immature dendritic cells.

In another embodiment the dendritic cells which are exposed to the FHA are mature dendritic cells.

Preferably the immune-mediated disorder is an autoimmune disease, most preferably multiple sclerosis, rheumatoid arthritis, psoriasis or Crohn's disease.

A yet further aspect relates to the use of dendritic cells which have been exposed ex vivo to FHA in the preparation of a medicament for the treatment of an autoimmune disease. Preferably the immune-mediated disorder is an autoimmune disease, most preferably multiple sclerosis . In further embodiment the autoimmune disease is rheumatoid arthritis, psoriasis or Crohn's disease.

In one embodiment the subject is a mammal. In a preferred embodiment, the subject is a human.

In one embodiment, the immune-mediated disease is an autoimmune disease. In a particular embodiment, the autoimmune disease is multiple sclerosis.

As defined herein the term "immune-mediated disease" is taken to include; multiple sclerosis, Crohn's disease, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis and psoriasis.

Also within the definition of "immune-mediated disease" are other immune-mediated disorders such as one or more of diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, - cutaneous^" lupus erythematosus^",^""'" scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, Alzheimer's disease and coeliac disease, colitis, asthma or atopic disease.

The present invention further provides for the use of dendritic cells which have been modulated following exposure to FHA.

Accordingly, a yet further aspect of the present invention provides the use of a dendritic cell which has been primed with an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof in the treatment of an autoimmune disease.

A further aspect of the invention provides the use of a dendritic cell which has been primed with an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof in the preparation of a medicament for the treatment of an autoimmune disease or chronic inflammatory condition.

Typically the dendritic cell is immature, though alternatively it may be a mature dendritic cell.

In addition to priming a dendritic cell with FHA alone, a self antigen, or other antigen associated with an autoimmune disease may be used, in combination, to prime the dendritic cell.

In an alternative embodiment of this aspect of the invention the dendritic cells may be replaced by any other suitable antigen presenting cell. Such suitable antigen presenting cells are macrophages, monocytes and B-cells .

Accordingly, in a still further aspect of the present invention there is provided a method for eliciting an immune response in a subject suitable for the treatment of an autoimmune disease, the method comprising the steps of:

- providing dendritic cells,

- modulating the function of the dendritic cells by exposing the dendritic cells to an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof along with an antigen associated with the autoimmune disease to be treated under conditions suitable to modulate the function of the dendritic cells, - administering the dendritic cells to the subject whereby the immune response generated suppresses the onset and/or progression of an autoimmune disease.

Alternatively, the dendritic cells may be derived from an individual who is MHC (major histocompatability complex) matched in order to avoid rejection of the administered dendritic cells.

Further, a dendritic type cell line (which is also

MHC matched) could be used.

In one embodiment, the dendritic cells would be pulsed with the self antigen before, during or after

FHA stimulation.

In a further embodiment, the modulation of the dendritic cells occurs ex-vivo.

Typically the dendritic cells are immature, though alternatively they may be mature dendritic cells.

Typically the subject is a human.

In one embodiment, the antigen associated with the autoimmune disease may be a self antigen. In one embodiment the antigen is MOG (myelin oligodendrocyte glycoprotein) . Alternatively the antigen may ^"be selec£eS^"from^~~aήy one ^"or ^"more ^"of W

13

glutamic acid decarboxylase 65 (GAD 65) , native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, thyroid stimulating hormone (TSH) receptor, Japanese 5 cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, and dust mite antigens and feline antigens for animal, histocompatibility antigens, antigens involved in graft rejection and an altered peptide ligand. The antigens involved in 10 graft rejection comprise antigenic components of the graft to be transplanted into, for example, the heart, lung, liver, pancreas, kidney of graft recipient and neural graft components .

15 The self antigen may also be selected from any one or more of a myelin protein, beta amyloid protein, amyloid precursor protein and collagen and peptide thereof .

20 Preferably the myelin protein is myelin basic protein or peptide thereof. The myelin basic protein is myelin oligodendrocyte glycoprotein synthetic peptide, most preferably a MOG peptide (35-55) .

25

In a further embodiment, a Toll-like receptor agonist (TLR) is co-administered along with the modulated dendritic cells. The TLR agonist may be specific to any defined human Toll like receptor

30 which has specificity for any known Toll-like receptor, for examples any one of TLRl through to TLR-I-3-. -In specific embodiments^", the ^"TLR^""agό^~nTst^~h^"as^~ W

14

specificity for TLR2 , TLR4 or TLR9. In further embodiments the TLR agonist may be selected from any one or more of LPS, CpG motifs, dsRNA, PoIy(IiC), lipoteichoic acid, heat shock proteins, lipid A, 5 flagellin and Pam-3Cys.

In one embodiment the autoimmune disease is multiple sclerosis. In further embodiments, the condition is rheumatoid arthritis, Crohn's disease or psoriasis. 10 In a yet further embodiment, the autoimmune disease is an immune-mediated condition such as any of those listed hereinbefore.

A still further aspect of the invention relates to 15 the use of dendritic cells which have been primed with FHA along with a self-antigen relating to a specific autoimmune disease, in the preparation of a medicament for the treatment of an autoimmune disease. 20

In one embodiment the medicament further comprises a

Toll-like receptor agonist.

Where a self antigen is provided in the medicament, 25 it is typically preferred that the autoimmune disease which will be treated will be the same as the autoimmune disease from which the antigen is derived from.

30 A yet further aspect of the present invention provides a method of preventing the onset and/or progression of an immune-mediated disease, the method comprising the steps of:

- isolating immature antigen presenting cells, preferably dendritic cells from a subject, - exposing the isolated antigen presenting cells, preferably dendritic cells to FHA,

- culturing the dendritic cells under conditions such that they undergo maturation, and

- re-administering the FHA stimulated mature antigen presenting cells, preferably dendritic cells to a subject.

In a preferred example the antigen presenting cells, preferably dendritic cells which are re-administered are autologous to the subject.

In a further embodiment, the immature dendritic antigen presenting cells, preferably dendritic cells are exposed to FHA along with a self antigen or antigen which is associated with the immune-mediated disease to be treated.

The antigen presenting cells are preferably dendritic cells, however may further be monocytes, macrophages or B-cells.

In a preferred embodiment the maturation of immature dendritic cells is performed in the presence of cytokines such as TNF-alpha, IL-6, IL-I.

A yet further aspect of the present invention provides for a -composition for^{^}the^~t^"reatme^"ήt of ^' multiple sclerosis, the composition comprising a dendritic cell which has been primed with FHA.

In a further embodiment of this aspect of the 5 invention, the dendritic cell has been primed in the presence of a self-antigen along with FHA.

In a further aspect of the present invention there is provided a method of treating multiple sclerosis 0 comprising the steps of:

- modulating the activity of dendritic cells through the exposure of said dendritic cells to FHA,

- administering the modulated dendritic cells 15 to a subject in need of treatment.

Use of dendritic cells which have been exposed ex vivo to FHA in the preparation of a medicament for the treatment or prevention of autoimmune disease or 0 chronic inflammatory, or other immune-mediated condition-

Typically, the administration of the modified dendritic cells results in the down regulation of 25 IL-17 production. Further, the administration of the modified dendritic cells results in the down regulation of interferon gamma.

A further still aspect of the present invention

30 provides a method of preventing the generation of neutralising antibodies against a biologic used in - - the-r-apy-,- -the -method -comprising the^' steps ^"of : - isolating immature dendritic cells from a subject,

- exposing the isolated dendritic cells to FHA along with the biologic, - culturing the dendritic cells under conditions such that they undergo maturation, and

- re-administering the FHA/biologic stimulated mature dendritic cells to a subject.

In one embodiment the biologic is an antibody, such as a monoclonal antibody, for example a humanised antibody or a chimeric antibody. In a further embodiment the biologic is a type I interferon, or Factor VIII or anti-TNF-α.

A further still aspect of the present invention provides for the use of dendritic cells which have been exposed ex vivo to FHA and a biologic in the preparation of a medicament for the treatment or prevention of neutralising antibody formation against said biologic.

In one embodiment the dendritic cells may be exposed to two or more biologies .

In a preferred example the dendritic cells which are re-administered are autologous to the subject.

Alternatively the dendritic cells are from a donor and are MHC matched to the subject to whom they are re-administered. In a further embodiment, the immature dendritic cells are exposed to FHA along with a self antigen or antigen which is associated with the immune- mediated disease to be treated as well as at least one biologic.

As herein defined a 'biologic' is a drug prepared from animal tissue or some other living source, this may include, but is not limited to a virus, serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or other similar product used to prevent, treat or cure disease or injury. The biologic may be any biologic against which neutralising antibodies may be raised or naturally produced biologically active agent etc. Further the biologic can be an antibody, in particular a monoclonal antibody, a binding member with specificity for a target ligand, a protein or a fragment thereof .

Examples of such biologies include monoclonal antibodies such as Infliximab, recombinant interferon products, and in particular type I interferon products such as interferon alpha-2a, recombinant interferon alpha-2b, and interferon beta, or the like. Although set-forth with regard to the use of dendritic cells, the present invention can use any- suitable antigen presenting cell in order to mediate the effects defined herein. Accordingly in a further aspect of the present invention, there is provided the use of any antigen presenting cell in place of the dendritic cell defined in any of the foregoing aspects of the present invention.

The term "antigen presenting cell" which may be abbreviated as APC refers to a cell that processes antigens for presentation to T lymphocytes. Antigen-presenting cells, including but not limited to macrophages, monocytes, dendritic cells (for example, cutaneous epidermal Langerhans cells, dermal dendritic cells, and dendritic cells resident in lymph nodes and spleen) , and B cells, can be obtained by production in vitro from stem cells and from progenitor cells found in human peripheral blood and bone marrow.

In one embodiment, the APCs are isolated from a subject that is also the intended recipient of the APCs (autologous embodiment) .

For use in the methods of the present invention, antigen presenting cells comprise in one embodiment dendritic cells, including, but not limited to Langerhans cells, follicular DCs, and lymphoid DCs. The methods of the present invention can also be used to enhance the maturation of other antigen presenting cells, including, but not limited to macrophages , monocytes , and B cells .

In all aspects of this invention, the filamentous haemagglutinin (FHA) is the wild-type FHA molecule, for example as derived from Bordetella pertussis or Bordetella bronchisepetica or Bordetella parapertussis . However the term FHA also encompasses related molecules from other bacteria. Related molecules may include proteins from other bacteria with sequences homologous to those in FHA. "FHA" also encompasses, fragments , analogues and derivatives of FHA, including synthetic (e.g., recombinant) forms of FHA. Such synthetic molecules may be altered, removed or purified from its naturally occurring state through human intervention .

Where the form of the FHA molecule provided is not the naturally occurring, full length form, then preferably the amino acid sequence has at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, or 90%, 95%, 98% or 99% homology to amino acid sequence of the full length naturally occurring 'wild type' form.

A derivative of FHA as disclosed herein may be, in certain embodiments, the same length or shorter than the wild type FHA peptide. In other embodiments, the peptide sequence or a variant thereof may comprise a larger peptide. As is well understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for 'conservative variation', such as substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as lysine or glutamic acid for aspartic acid, or glutamine for asparagine .

Analogues of, and for use in, the invention as defined herein means a peptide modified by varying the amino acid sequence e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.

Such analogues of the amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids, while providing a peptide capable of inducing an alteration in the cytokine profile of a neural cell, and in particular a cell of the hippocampus.

Analogues also include derivatives of FHA, including FHA being linked to a coupling partner, e.g. a label, a drug, a toxin and/or a carrier or transport molecule.

In another embodiment, the term FHA also encompasses the use of non-peptide mimetics of FHA, which may be used in the invention. Such mimetics of FHA may be prepared,- either- wholly or ^"partly,^~" by chemical synthesis. Generation of the peptides in this way- can be performed by methods which are well known to the person skilled in the art.

Treatment / Therapy

The term 'treatment' is used herein to refer to any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment) . Treatment may include curative, alleviation or prophylactic effects. The term "therapy" is used interchangeably with "treatment".

Administration The modified matured dendritic cells may be administered alone but can alternatively be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.

The dendritic cells of the invention may be administered to a patient in need of treatment via any suitable route. The precise dose will depend upon a number of factors, including the precise nature of the form of FHA to be administered. Preferably the route of administration will be the same as that of the biologic.

A preferred route of administration is parenterally

(including subcutaneous, intramuscular, intravenous-) -.—Further^'"suitable routes^" of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual) , vaginal, intradermal, intrathecal and epidural) administration or administration via oral or nasal inhalation, by means of, for example a nebuliser or inhaler.

For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, Lactated Ringer's injection.

Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R. , Lippincott Williams & Wilkins; 20th edition (December 15, 2000) ISBN 0- 912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H. C. et al . 7^th Edition ISBN 0-683305-72-7 the entire disclosures of which are herein incorporated by reference.

Dose

The composition is preferably administered to an -individual in—a -"therapeutically effective amount", this being sufficient to show benefit to the individual .

Definitions Unless otherwise defined, all technical and scientific terms used herein ^"have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers .

The term "dendritic cell" refers to an antigen presenting cell that can elicit a T cell or B cell response. Thus, the term "dendritic cell" refers to a cell expressing major histocompatability complex (MHC) molecules to which antigens are bound. Intracellular antigens are processed in the cytosol and presented on MHC class I molecules, which then activate cytotoxic T lymphocytes (CTLs).

Extracellular antigens that have been endocytosed are typically presented on MHC class II molecules, which then stimulate T helper cells.

The terms "immature dendritic cells" and "immature

DCs" are used interchangeably to refer to DCs that have not matured to a state wherein they are capable of eliciting a T cell or B cell response.

The terms "mature dendritic cells" and "mature DCs" are used interchangeably to refer to immature DCs that have undergone maturation.

Immature and mature DCs can be identified using any one of several methods, including: (a) immunophenotypic analysis; (b) ability to migrate to a lymph node; (c) antigen capture capability; and (d) ability to present antigen and activate T cells, as described further herein below.

The term autoimmune disease refers to one of a number of unrelated disorders caused by inflammation and destruction of tissues by the body's own immune system and involves the generation of cellular or humoral immune responses against components or products of its own tissue, treating them as foreign.

Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.

The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention, and further, with reference to the figures .

Brief description of the drawings

Figure 1 shows two graphs, A and B, illustrating that immunotherapy with dendritic cells from mice injected with FHA (filamentous haemagglutinin) or with MOG (myelin oligodendrocyte glycoprotein) and FHA confers protection against EAE (experimental autoimmune encephalomyelitis) ,

Figure 2 shows two bar charts, A and B, showing that the results of the transfer of DCs from mice injected with FHA or FHA and MOG suppresses MOG-specific proliferation by spleen and lymph node cells in mice induced to develop EAE,

Figure 3, graphs A and B show transfer of dendritic cells from mice injected with FHA or with a combination of FHA and MOG suppresses MOG-specific IFN-γ production by spleen (graph A) and lymph node (graph B) cells in mice induced to develop EAE, and

Figure 4 shows that transfer of DCs from mice injected with FHA and MOG to mice induced to develop EAE results in lower MOG-specific IL-17 production when compared with DC from mice injected with MOG^" alone. Filamentous haemagglutinin (FHA)

Bordetella pertussis causes a protracted and severe disease, which is often complicated by secondary infection and pneumonia, and can have a lethal outcome in young children. Recovery from infection is associated with the development of B. pertussis- specific ThI cells and these cells play a critical role in clearance of the bacteria from the respiratory tract. However, antigen-specific ThI responses in the lung and local lymph nodes, are severely suppressed during the acute phase of infection. B. pertussis has evolved a number of strategies to circumvent protective immune responses.

The virulence factor, filamentous haemagglutinin (FHA) from B. pertussis, is capable of inhibiting LPS-driven IL-12 production by macrophages, IL-12 and IFN-γ production in a murine model of septic shock and ThI responses to an unrelated pathogen, influenza virus, when administered simultaneously to the respiratory tract. FHA is considered to function primarily as an adhesin, mediating binding of B. pertussis to the β2-integrins (CR3,

CDllb/CDl8, αMβ2) via binding to leukocyte response integrin (αvβ3, CD61) and the integrin-associated protein (CD47) complex (5) . FHA may also contribute to suppressed ThI responses during acute infection with B. pertussis by the induction of T cells with regulatory activity, as a result of its interaction - ^" with cells of the innate immune system. EXAMPLES

Example 1 - Immunotherapy with dendritic cells provided by adoptive transfer Materials and Methods :

Groups of female C57BL/6 mice were immunised subcutaneously with PBS, MOG₃₅-₅₅ (50μg) , FHA (5μg) or MOG₃₅-₅₅ (50μg) and FHA (5μg) . Five hours later, mice were sacrificed and inguinal lymph nodes were harvested.

CDlIc⁺ dendritic cells were purified by magnetic cell sorting (MACS) and transferred into female C57BL/6 mice that had EAE (experimental autoimmune encephalomyelitis) , a mouse model of multiple sclerosis, induced seven days previous.

EAE was induced as follows. Mice received 150μg of MOG₃₅_₅₅ emulsified in CFA supplemented with 5 mg/ml of Mycobacteria tuberculosis H37RA subcutaneously in the flank. At the same time, mice were treated with 500ng of PT intraperitoneally in PBS. Mice also received 500ng PT in PBS at 2 days.

Mice were monitored daily for signs of EAE, which usually appeared at days 9-13. Mice were scored as follows, 0 = no clinical score, 1 = loss of tail tone, 2 = wobbly gait, 3 = hind limb weakness, 4 = hind limb paralysis, 5 = tetraparalysis/death. For animals with no clinical signs, onset was arbitrarily calculated as one day after the experiment was -terminatedr -Thre^~~dis^~ease^{" "}Index ^"was^" calculated by dividing the sum of the daily disease scores by the average day of disease onset and multiplying by 100.

Results:

The results as shown in graphs A and B of Figure 1 show that dendritic cells from mice injected with FHA or with a combination of FHA and MOG confer protection against EAE when transferred to mice at the onset of clinical symptoms of EAE.

Dendritic cells from mice injected with MOG alone had a small protective effect which was most pronounced later in disease progression. In contrast dendritic cells from mice injected with FHA or with a combination of FHA and MOG showed a delay in the onset of symptoms, which were eventually least severe in mice given dendritic cells from FHA- treated mice.

Example 2 - Transfer of DC from mice injected with FHA or FHA and MOG Materials and methods :

Groups of female C57BL/6 mice were immunised subcutaneousIy with PBS, MOG₃₅-₅₅ (50μg) , FHA (5μg) or MOG₃₅-₅₅ (50μg) and FHA (5μg) . Five hours later, mice were sacrificed and inguinal lymph nodes were harvested. CDlIc⁺ dendritic cells were purified on MACS columns and transferred into female C57BL/6 mice that had EAE induced seven days earlier. EAE was induced as follows. Mice received 150μg MOG₃₅-₅₅ emulsified in CFA supplemented with 5 mg/ml of Mycobacteria tuberculosis H37RA subcutaneously in the flank. At the same time, mice were treated with 500ng of PT intraperitoneally in PBS. Mice also received 500ng PT in PBS at 2 days.

Mice were sacrificed 27 days after induction of EAE and spleen and lymph node were stimulated with medium only, MOG (25 or 100 μg/ml) and MOG-specific proliferation was determined by ³H-thymidine incorporation .

Results : The results as shown in graphs A and B of Figure 2 show that treatment with dendritic cells from mice injected with FHA only or with a combination of MOG and FHA suppresses MOG-specific proliferation by spleen (graph A) and lymph node (graph B) cells in mice immunised with MOG and CFA plus PT to induce

EAE.

Further, it can be seen from graph B that in the lymph node, injection with FHA alone provided a higher level of inhibition of proliferation than MOG and FHA.

Example 3 - Effect of transfer of dendritic cells from mice injected with FHA or FHA and MOG on MOG- specific IFN-γ production by spleen and lymph node cells in mice induced to develop EAE

Material and methods : Groups of female C57BL/6 mice were immunised subcutaneousIy with PBS, MOG₃₅_₅₅ (50μg) , FHA (5μg) or MOG₃₅-₅₅ (50μg) and FHA (5μg) . Five hours later, mice were sacrificed and inguinal lymph nodes were harvested. CDlIc⁺ dendritic cells were purified on MACS columns and transferred into female C57BL/6 mice that had EAE induced seven days earlier.

EAE was induced as follows. Mice received 150μg MOG35-55 emulsified in CFA supplemented with 5 mg/ml of Mycobacteria tuberculosis H37RA subcutaneously in the flank. At the same time, mice were treated with 500ng of PT intraperitoneally in PBS. Mice also received 500ng PT in PBS at 2 days.

Mice were sacrificed 27 days after induction of EAE and spleen and lymph node were stimulated with medium only, MOG (25 or 100 μg/ml) and IFN-γ concentrations in MOG-stimulated spleen and lymph node supernatants were determined by ELISA using antibodies from BD Pharmingen.

Results :

The results, as shown in graph A and B of Figure 3, show that treatment with dendritic cells from mice injected with FHA only or with a combination of MOG and FHA suppresses MOG-specific IFN-γ production by spleen cells from mice immunised with MOG and CFA plus PT to induce EAE.

Furthermore, when compared with dendritic cells from mice injected with MOG only, transfer of dendritic cells from mice injected with a combination of FHA and MOG or with FHA only suppresses MOG-specific IFN-γ production by lymph node cells from mice immunised with MOG and CFA plus PT to induce EAE.

Example 4 - Effect of transfer of DCs from mice injected with FHA and MOG to mice induced to develop EAE on MOG-specific IL-17 production (when compared with DC from mice injected with MOG alone) Materials and methods :

Groups of female C57BL/6 mice were immunised subcutaneously with PBS, MOG₃₅-₅₅ (50μg) , FHA (5μg) or MOG₃₅-₅₅ (50μg) and FHA (5μg) . Five hours later, mice were sacrificed and inguinal lymph nodes were harvested. CDlIc⁺ dendritic cells were purified on MACS columns and transferred into female C57BL/6 mice that had EAE induced seven days earlier.

EAE was induced as follows . Mice received 150μg MOG₃₅-₅₅ emulsified in CFA supplemented with 5 mg/ml of Mycobacteria tuberculosis H37RA subcutaneously in the flank. At the same time, mice were treated with 500ng of PT intraperitoneally in PBS. Mice also received 500ng PT in PBS at 2 days.

Mice were sacrificed 27 days after induction of EAE and spleen and lymph node were stimulated with medium only, MOG (25 or 100 μg/ml) and IL-17 concentrations in MOG-stimulated spleen and lymph node supernatants were determined by ELISA using a Duoset from R&D Systems. W

33

Results :

The results, as shown in Figure 4, show that when compared with dendritic cells from mice injected with MOG only, transfer of dendritic cells from mice 5 injected with MOG in the presence of FHA suppresses MOG-specific IL-I7 production by lymph node cells from mice immunised with MOG and CFA plus PT to induce EAE.

10 IL-17 production is induced in T-cells by IL-23. These results shown above could be important in supporting the role of IL-17 and IL-23 as being mediators of the onset and development of multiple sclerosis and other autoimmune disease and chronic

15 inflammatory conditions.

The importance of IL-17 and IL-23 in the onset and the development of these conditions suggests that the adoptive transfer of dendritic cells primed with

20 either FHA alone or with a combination of FHA and

MOG can be shown to reduce IL-17 expression. This finding provides a further route to therapy in relation to the treatment or prevention of multiple sclerosis through the down regulation of the 25 expression of IL-17 or through blocking or inhibiting the activity of IL-17 through a blocking molecule such as an antibody or the like.

All documents referred to in this specification are

30 herein incorporated by reference. Various modifications and variations to the described - - - embodiments - of the- inventions ^"will^""Be^"" apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments . Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

Claims :

1. A method for eliciting an immune response in a subject suitable for the treatment of an immune- mediated disease, the method comprising the steps of:

- exposing isolated dendritic cells to an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof ex-vivo for a sufficient time and under condition suitable in order to modulate the function of the dendritic cells,

- administering the dendritic cells to the subject whereby the immune response generated in the subject is sufficient to prevent the onset or progression of the immune-mediated disease.

2. The method of claim 1 wherein the dendritic cells are autologous to the subject.

3. The method of claim 1 or claim 2 wherein the dendritic cells are immature dendritic cells .

4. The method of claim 1 or claim 2 wherein the dendritic cells are mature dendritic cells.

5. The method of any one of claims 1 to 4 wherein the dendritic cells are co-administered along with a

Toll-Like Receptor (TLR) agonist which is specific to^" any^" Toll-like receptor.

6. The method of any one of claims 1 to 5 wherein the dendritic cells are co-administered along with a self antigen.

7. The method of any one of claims 1 to 5 wherein the subject is a human.

8. The method according to any preceding claim wherein the immune-mediated disease is an autoimmune disease .

9. The method according to claim 8 wherein the autoimmune disease is multiple sclerosis .

10. The method according to claim 8 wherein the autoimmune disease is selected from the group consisting of; Crohn's disease, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis and psoriasis.

11. A composition for modulating an immune response in a individual suffering from an immune mediated condition, the composition comprising dendritic cells which have been activated by being pulsed with FHA for use in the modulation of an immune response characteristic or causative of an autoimmune disease or immune-mediated condition.

12. A composition as claimed in claim 12 which further comprises a self antigen.

13. A composition as claimed in claim 11 or 12 which further comprises a Toll-like receptor agonist .

14. A composition as claimed in claim 11 wherein the autoimmune disease such as multiple sclerosis, rheumatoid arthritis, Crohn's disease or psoriasis.

15. A pharmaceutical composition for the treatment of an autoimmune disease or an immune mediated condition, said composition comprising dendritic cells activated in the presence of FHA along with a pharmaceutically acceptable carrier or diluent.

16. A pharmaceutical composition as claimed in claim 15 which further comprises a self antigen.

17. A pharmaceutical composition as claimed in claim 15 or claim 16 which further comprises a ToIl- like receptor ligand.

18. Use of dendritic cells which have been pulsed with an agent comprising filamentous haemagglutinin

(FHA) or a derivative or mutant or fragment or variant or peptide thereof in the preparation of a medicament for modulating the immune response in an individual who suffers from an autoimmune disease or immune mediated condition.

19. Use as claimed in claim 18 wherein the dendritic cells are derived from naϊve dendritic cells which-have been-"pul-S^"e^"d^"wrth^' FHA^".^{' "}

20. Use as claimed in claim 18 wherein the medicament further comprises a Toll-like receptor agonist.

5

21. Use as claimed in claim 18 or claim 19 wherein the medicament further comprises a self antigen.

22. Dendritic cells which have been pulsed with FHA 0 for use in medicine.

23. The use of an antigen presenting cell which has been primed with an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or 5 fragment or variant or peptide thereof in the treatment of an autoimmune disease.

24. Use of an antigen presenting cell according to claim 23 where in the antigen presenting cell is a 0 macrophage, a monocyte or a B-cell.

25. A method for eliciting an immune response in a subject suitable for the treatment of an autoimmune disease, the method comprising the steps of: 5 - providing dendritic cells,

- modulating the function of the dendritic cells by exposing the dendritic cells to an agent comprising filamentous haem.agglutinin (FHA) or a derivative or mutant or fragment 0 or variant or peptide thereof along with an antigen associated with the autoimmune

- --- - -- disease -to- be treated^~τrnder^{" "}conditions^"" suitable to modulate the function of the dendritic cells, - administering the dendritic cells to the subject whereby the immune response generated in the subject is sufficient to suppress the onset and/or progression of an autoimmune disease.

26. The method of claim 25 wherein the dendritic cells are autologous to the subject.

27. The method of claim 25 wherein the modulation of the dendritic cells occurs ex-vivo.

28. The method of claim 25 wherein the dendritic cells are immature.

29. The method of claim 25 wherein the dendritic cells are mature.

30. The method according to any one of claims 25 to 29 wherein the subject is a human.

31. The method according to any one of claims 25 to 30 wherein the self antigen is selected from the group comprising MOG (myelin oligodendrocyte glycoprotein) , glutamic acid decarboxylase 65 (GAD 65) , native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, thyroid stimulating hormone (TSH) receptor, Japanese cedar pollen - antigens-, -ragweed-poll^~en^~antigens^', ^' rye^{"" "}grass pollen antigens, and dust mite antigens and feline antigens for animal, histocompatibility antigens, antigens involved in graft rejection and an altered peptide ligand.

32. Use of dendritic cells which have been primed an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof along with a self-antigen relating to a specific autoimmune disease, for the treatment of an autoimmune disease.

33. The use of dendritic cells according to claim 32 wherein the autoimmune disease which is treated is the same as the autoimmune disease from which the self antigen is derived from.

34. A method of preventing the onset and/or progression of an immune-mediated disease, the method comprising the steps of:

- isolating immature dendritic cells from a subject,

- exposing the isolated dendritic cells to FHA,

- re-administering the FHA stimulated mature dendritic cells to a subject.

35. The method of claim 34 wherein the dendritic cells which are re-administered are autologous to the subject.

36. The method of claim 34 wherein the immature dendritic cells are exposed to a dendritic cell along with a self antigen or antigen which is associated with the immune-mediated disease to be treated.

37. A composition for the treatment of multiple sclerosis, the composition comprising a dendritic cell which has been primed with FHA.

38. A method of treating multiple sclerosis comprising the steps of:

- administering the modulated dendritic cells to a subject in need of treatment.

39. A method as claimed in claim 38 wherein the administration of the modified dendritic cells results in the down regulation of IL-17 production.

40. A method of preventing the generation of neutralising antibodies against a biologic used in therapy, the method comprising the steps of:

- isolating and generating immature dendritic cells from a subject,

- exposing the isolated dendritic cells to FHA along with the biologic, - culturing the dendritic cells under conditions such that they undergo maturation, and - re-administering the FHA/biologic stimulated mature dendritic cells to a subject.

41. Use of a dendritic cell which has been primed with an agent comprising filamentous haemagglutinin

(FHA) or a derivative or mutant or fragment or variant or peptide thereof in the preparation of a medicament for the treatment of an autoimmune disease or chronic inflammatory condition.

42. Dendritic cells which have been exposed ex vivo to FHA for use in medicine/for use in the treatment of an immune-mediated disorder.

43. The use of dendritic cells which have been exposed ex vivo to an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof in the preparation of a medicament for the treatment of an autoimmune disease.